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Expert Opinion on Biological Therapy
ISSN: 1471-2598 (Print) 1744-7682 (Online) Journal homepage: http://www.tandfonline.com/loi/iebt20
Using glyco-engineering to produce therapeutic
proteins
Martina Dicker & Richard Strasser
To cite this article: Martina Dicker & Richard Strasser (2015) Using glyco-engineering to
produce therapeutic proteins, Expert Opinion on Biological Therapy, 15:10, 1501-1516, DOI:
10.1517/14712598.2015.1069271
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Date: 17 May 2016, At: 09:13
Review
Using glyco-engineering to
produce therapeutic proteins
Martina Dicker & Richard Strasser†
1.
Introduction
2.
N-glycosylation of proteins
3.
What are the targets for
N-glycan-engineering?
4.
O-glycosylation of proteins
5.
What are the targets for
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O-glycan-engineering?
6.
O-glycan engineering
7.
Glyco-engineered therapeutic
glycoproteins
8.
Conclusion
9.
Expert opinion
10.
So what are the challenges
for the future?
University of Natural Resources and Life Sciences, Department of Applied Genetics and Cell
Biology, Vienna, Austria
Introduction: Glycans are increasingly important in the development of
new biopharmaceuticals with optimized efficacy, half-life, and antigenicity.
Current expression platforms for recombinant glycoprotein therapeutics
typically do not produce homogeneous glycans and frequently display nonhuman glycans which may cause unwanted side effects. To circumvent these
issues, glyco-engineering has been applied to different expression systems
including mammalian cells, insect cells, yeast, and plants.
Areas covered: This review summarizes recent developments in glycoengineering focusing mainly on in vivo expression systems for recombinant
proteins. The highlighted strategies aim at producing glycoproteins with
homogeneous N- and O-linked glycans of defined composition.
Expert opinion: Glyco-engineering of expression platforms is increasingly recognized as an important strategy to improve biopharmaceuticals. A better
understanding and control of the factors leading to glycan heterogeneity
will allow simplified production of recombinant glycoprotein therapeutics
with less variation in terms of glycosylation. Further technological advances
will have a major impact on manufacturing processes and may provide a
completely new class of glycoprotein therapeutics with customized functions.
Keywords: biopharmaceuticals, glycosyltransferases, N-glycosylation, O-glycosylation,
recombinant glycoprotein
Expert Opin. Biol. Ther. (2015) 15(10):1501-1516
1.
Introduction
Many protein drugs are glycosylated and the attached glycan structures often influence the therapeutic properties. The number and composition of the glycans play an
important role for protein folding, solubility, and intracellular trafficking. Glycans
can shield the protein backbone to prevent immunogenic reactions and distinct
cellular recognition events depend on the presence of specific glycan structures.
Thus, glycosylation has a huge impact on the biological activity of glycoproteins
and should be carefully controlled during manufacturing to achieve optimized therapeutic efficacy. Dependent on the species, cell-type, and physiological status of the
production host, the glycosylation pattern on recombinant glycoproteins can differ
significantly. Glycans on proteins are structurally quite diverse and consist of a set of
monosaccharides that are assembled by different linkages. The glycosylation processes in the ER and Golgi generate the majority of rather heterogeneous glycan
structures found on recombinant glycoproteins. Due to the recognized importance
in therapy, substantial efforts have been made in recent years to overcome glycan
heterogeneity and establish in vivo and in vitro glyco-engineering technologies for
efficient production of homogeneous therapeutic glycoproteins. Such recombinant
proteins with custom-made glycosylation are essential to understand glycanmediated functions of glycoprotein therapeutics and represent a new class of nextgeneration drugs with enhanced biological activity.
10.1517/14712598.2015.1069271 © 2015 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682
All rights reserved: reproduction in whole or in part not permitted
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M. Dicker & R. Strasser
.
.
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.
.
.
Glyco-engineering offers great potential for the
generation of glycoprotein therapeutics with reduced
side effects and enhanced activity.
Analysis of recombinant glycoproteins from different
mammalian and non-mammalian-based expression
systems reveals the presence of unwanted glycan
modifications that mark targets for glyco-engineering.
Recent developments in modulation of N-glycosylation
and N-glycan processing demonstrate that recombinant
glycoproteins with defined homogeneous N-glycans can
be efficiently produced in mammalian and nonmammalian expression systems including insect cells,
yeast, and plants.
Compared to N-glycosylation, strategies and methods to
produce customized O-linked glycans are still in its
infancy. Nonetheless, future developments in this area
hold great promise for another class of improved
glycoprotein biopharmaceuticals.
The first glyco-engineered monoclonal antibodies have
already been approved in the US and in Japan.
Further biochemical and molecular understanding of
cellular process will be required to optimize current
glyco-engineering approaches and develop strategies for
so far elusive glycan modifications.
This box summarizes key points contained in the article.
2.
N-glycosylation of proteins
The most prominent and best characterized form of protein
glycosylation is the linkage of a glycan to the amide in
the side chain of an asparagine (N-glycosylation) on newly
synthetized proteins. N-glycosylation of proteins starts in
the lumen of the endoplasmic reticulum (ER) by transfer of
a conserved preassembled oligosaccharide precursor
(Glc3Man9GlcNAc2) (Figure 1A) to the consensus sequence
Asn-X-Ser/Thr (where X is any amino acid except proline)
exposed on nascent polypeptide chains. This initial glycan
transfer reaction is catalyzed by the heteromeric oligosaccharyltransferase (OST) complex and is supposed to precede
folding of the protein in the ER [1]. Immediately after the
oligosaccharide transfer, the two terminal glucose residues
are cleaved off by a-glucosidase I and II and the resulting
polypeptide with mono-glucosylated glycan structures
(Glc1Man9GlcNAc2) can interact with the ER-resident
membrane-bound lectin calnexin or its soluble homolog
calreticulin. These lectins support protein folding in a
glycan-dependent protein quality control cycle. Secretory
glycoproteins that have acquired their native conformation
are released from the calnexin/calreticulin cycle and exit the
ER to the Golgi apparatus. In the Golgi, the ER-derived
oligomannosidic N-glycans on maturely folded glycoproteins
are subjected to further N-glycan processing which generates
the highly diverse complex N-glycans with different functional properties (Figure 1B).
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What are the targets for N-glycanengineering?
3.
Article highlights.
Avoidance of macroheterogeneity
Macroheterogeneity on recombinant glycoproteins arises from
variations in glycosylation site occupancy. These differences in
glycosylation efficiency are dependent on the manufacturing
system (e.g., organism/cell-type-specific) and protein intrinsic
features. The presence of the Asn-X-Ser/Thr sequon is necessary but not sufficient for N-glycosylation of mammalian
proteins. While all eukaryotic cells have an overall conserved
machinery for N-glycosylation and display similar structural
requirements for efficient N-glycosylation, there are minor differences in utilization of glycosylation sites between species [2,3].
Consequently, glycosylation sites may be skipped leading to
underglycosylation of recombinant proteins or additional
sites may be used leading to aberrant glycosylation. Protein
intrinsic factors like the surrounding amino-acid sequence
and secondary structure, the positioning of the consensus
sequence within the polypeptide as well as the presence of other
protein modifications like disulfide bond formation
contribute to N-glycosylation efficiency [4]. Recognition of
these glycoprotein-specific features depends on the presence
and function of the different OST subunits. Mammalian cells
contain two OST complexes that differ in their catalytic subunit as well as in accessory proteins. These OST complexes
display partially overlapping functions, but also preferences
for certain glycoprotein substrates [3]. Engineering of the
OST complex is one possible way to overcome differences
related to N-glycosylation site occupancy. Yet, the individual
roles of distinct OST catalytic subunits and their accessory proteins are still not fully understood in eukaryotes making rational approaches difficult. Moreover, a recent study has indicated
that overexpression of individual subunits is not sufficient to
restore N-glycosylation in mutant cells presumably due to the
inability to form functional heteromeric OST complexes [5].
However, in protists like Leishmania major, OST is composed
of a single subunit that can replace the whole Saccharomyces cerevisiae OST complex and can be used to overcome underglycosylation of proteins. Overexpression of the single-subunit OST
from L. major in the methylotrophic yeast Pichia pastoris
increased the N-glycosylation site occupancy of a monoclonal
antibody from 75 to 85% to greater than 99% [6].
Other engineering attempts to reduce macroheterogeneity
are based on mutation of the N-glycosylation consensus
sequence, for example, from Asn-X-Ser to Asn-X-Thr which
is more efficiently glycosylated in different eukaryotes [2].
Besides modifying the consensus sequence itself, adjacent
amino acids may also be altered to enhance N-glycosylation
efficacy. The major drawback of these strategies is an alteration of the primary amino acid sequence of the therapeutic
glycoprotein which can have an influence on protein properties and may create unwanted regulatory concerns. Nonetheless, novel N-glycosylation sites have been successfully
3.1
Expert Opin. Biol. Ther. (2015) 15(10)
Using glyco-engineering to produce therapeutic proteins
A.
G
M
S
F
G
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B.
Figure 1. Schematic presentation of N-glycan processing pathways in mammals. (A) N-glycosylation is initiated by OSTcatalyzed transfer of the lipid-linked preassembled oligosaccharide to Asn with the Asn-X-Ser/Thr consensus sequence.
N-glycan processing starts in the endoplasmic reticulum (ER) by the removal of glucose and mannose residues and
(B) continues in the different Golgi cisternae by numerous processing reactions. Only a selection of possible complex N-glycan
modifications is shown.
FUT8: Core a1,6-fucosyltransferase; GALT1: b1,4-galactosyltransferase; GCSI: a-glucosidase I; GCSII: a-glucosidase II; GMI: Golgi-a-mannosidase I; GMII: Golgi-amannosidase II; GnTI: N-acetylglucosaminyltransferase I; GnTII: N-acetylglucosaminyltransferase II; GnTIV: N-acetylglucosaminyltransferase IV; GnTV:
N-acetylglucosaminyltransferase V; MANI: ER-a-mannosidase I; ST: a2,6-sialyltransferase.
engineered into recombinant proteins. The most prominent
example for this approach is darboetin alfa. This hyperglycosylated recombinant erythropoietin (EPO) variant has been
engineered to carry five instead of three N-glycosylation
sites [7]. The additional two N-glycans increase the total sialic
acid content which affects the protein half-life and boost the
in vivo potency [8]. In another elegant study, introduction of
a novel N-glycosylation site into the light chain of a monoclonal antibody against the HIV-1 receptor CD4 substantially
improved the virus neutralization activity [9]. Collectively,
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A.
B.
C.
D.
E.
F.
Figure 2. (A) Illustrations of representative N-glycans from human cells/tissues and characteristic structures from (B) CHO cells,
(C) mouse myeloma cells, (D) P. pastoris, (E) insect cells and (F) plants.
these examples illustrate the potential for protein engineering
toward incorporation of additional glycosylation sites.
Reduction of microheterogeneity
The commonly used expression systems for recombinant
glycoproteins typically produce a mixture of different glycans
on the same protein. This means that different glycans are found
at the same glycosylation site and that individual sites on the
same protein can be furnished with glycans of high structural
diversity. The N-glycome of mammalian cells shows a wide
range of N-glycan compositions from oligomannosidic to
branched complex-type N-glycans (Figure 2A) [10,11]. Microheterogeneity arises from variations in N-glycan processing which
depends on numerous cellular factors including tissue/celltype-specific expression of processing enzymes, their concentrations and enzyme kinetic parameters, availability of the nucleotide sugar donors and the glycoprotein residence, and contact
time in the reaction compartment (e.g., Golgi apparatus). In
addition, intrinsic protein structural properties can have a strong
effect on site-specific N-glycan processing. Systems’ glycobiology approaches have been used to correlate the transcript expression of glycosylation enzymes and the generation of N-glycan
structures on glycoproteins from mouse tissues or cancer
cells [10,12]. In mouse tissues, a positive correlation was found
between transfer of fucose residues and the transcript expression
3.2
1504
of corresponding fucosyltransferases. On the other hand, for
complex N-glycan formation or sialylation, this could not be
established highlighting a more complicated regulation for these
trimming and processing steps [10]. In addition, models have
been developed to predict the influence of enzyme expression,
subcellular localization, nucleotide sugar levels, and culture conditions on glycan processing [13]. While the described models
may already be useful to predict the rather homogeneous Fcglycosylation on a recombinant monoclonal antibody [14],
important key assumptions of these models have to be validated
by additional experimental data in the future. Moreover, the
robustness of current mathematical models needs to be tested
in host cells on recombinant glycoproteins which display a
more complicated and heterogeneous glycosylation pattern
including branching and sialylation of N-glycans [15].
Other factors that contribute to microheterogeneity are
the competition of different modifying enzymes for the
same oligosaccharide substrate and the presence of endogenous glycoproteins that may interfere with processing or
contact time in the Golgi. Although it is commonly accepted
that the Golgi processing enzymes are organized sequentially
in some kind of assembly line across the individual Golgi
cisternae [16], there is typically an overlapping distribution
of multiple enzymes. These enzymes may modify the same
glycan intermediate structure and block the action of
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Using glyco-engineering to produce therapeutic proteins
other glycosyltransferases and glycosidases. The addition of
the bisecting GlcNAc to complex N-glycans by
N-acetylglucosaminyltransferase III prevents, for example,
further modifications by other Golgi-resident glycosyltransferases. This substrate competition and inhibition of further
processing is the key factor of the GlycoMab technology that
was developed by Glycart Biotechnology (acquired by
Roche) and is used to produce glyco-engineered monoclonal
antibodies with reduced amounts of a1,6-linked core
fucose [17]. The glyco-engineered obinutuzumab, an antiCD20 monoclonal antibody produced by this platform,
was recently approved by the US FDA for the treatment of
patients with previously untreated chronic lymphocytic leukemia [18]. Galactosylation and branching of complex
N-glycans are other frequent modifications that use the
same substrates (Figure 1B) [15]. Apart from these competing
reactions that act in the same biosynthetic compartment, a
significant impact of enzymes involved in catabolic processes
cannot be excluded. Extracellular a-neuraminidases or
b-hexosaminidases can act on terminal sugar residues and
remove them from exposed N-glycans after secretion leading
to variation in glycan profiles on recombinant glycoproteins [10,19]. As a consequence, genetic inactivation or
inhibition of competing glycosyltransferases and (catabolic)
glycoside hydrolases are beneficial strategies to prevent
unwanted glycan modifications and reduce the complexity
of glycosylation. The successful generation of recombinant
glycoprotein therapeutics with homogeneous glycosylation
in yeast or plants [20,21] which have much less complicated
N-glycan processing machinery than mammalian cells demonstrates the capability of glyco-engineering. The generation
of defined glycosylation patterns in Chinese Hamster Ovary
(CHO) cells with defined defects in glycosylation shows that
‘simplifying’ strategies are also feasible in mammalian
cells [22]. Antibodies produced in Lec8 CHO cells which
lack UDP-galactose in the Golgi display mainly homogeneous biantennary N-glycans [23,24].
The residence time of the secreted recombinant glycoprotein in the Golgi and the contact time with the membraneanchored glycan-modifying enzymes are other factors that
may contribute significantly to microheterogeneity. Differences in residence times and transport kinetics through the
Golgi have been described for several proteins [25,26] and their
influence on glycan modifications is documented [27]. The
residence time distribution and the interaction with the glycosylation enzymes are dependent on the mode of intra-Golgi
cargo transport (vesicular transport/tubular connections,
cisternal maturation, rapid partitioning, or mixed models)
and are cell-type and organism-specific [28]. Furthermore,
recent studies suggest that different protein cargos (e.g., large
vs small cargo) are transported by varying mechanisms and
therefore very likely interact with different Golgi-resident
enzymes [29]. For some glycosylation processes, it has been
demonstrated that skipping of transit through individual
Golgi cisternae is an elegant way of regulating glycan
modifications [30]. Despite some progress in this field, fundamental experimental data on Golgi kinetics of different
recombinant glycoproteins and the impact on glycan processing are not available to rationally engineer transport and trafficking pathways toward more homogeneous glycan
structures. In other words, for many cell-based manufacturing
systems, we need a better understanding of cellular transport
processes in order to reduce glycan microheterogeneity related
to Golgi residence time or intra-Golgi transport.
Finally, a major contribution to site-specific modifications
comes from glycoprotein intrinsic features that are determined by the protein sequence and structure [31]. Certain protein conformations completely prevent or limit the access of
the processing enzymes to the glycans by steric hindrance [32].
Monoclonal antibodies like cetuximab with an additional
N-glycan in the variable region of the heavy chain are good
examples for the site-specific processing on the same molecule. While the Fc N-glycan of cetuximab is less modified,
the N-glycan in the variable region is more exposed and displays extensive processing [33,34]. Strategies to engineer the
protein intrinsic features without altering the protein function
are challenging, but the increasing availability of protein
structures together with powerful molecular dynamics simulations will open new possibilities for site-specific glycan engineering in the future [35]. Alternatively, the rational design
of glycan-modifying enzymes toward relaxed or novel
substrate specificities may allow more efficient processing of
partially accessible sites.
Elimination of non-human and potentially
immunogenic sugar residues
3.3
The majority of the currently used expression systems for
glycoprotein therapeutics produce N-glycans carrying nonhuman structures that can lead to potential side effects of
the drugs. These non-human epitopes may elicit unwanted
immune responses that neutralize the applied drug or
even worse cause a hypersensitivity reaction. Recombinant
glycoproteins produced in CHO cells carry the non-human
sialic acid N-glycolylneuraminic acid (Neu5Gc) (Figure 2B)
[36-38]. In addition, CHO cells attach N-acetylneuraminic
acid (Neu5Ac) in a2,3-linkage instead of a2,6-linkage that
is mainly found on N-glycans from human glycoproteins
because CHO cells lack the corresponding a2,6-sialyltransferase activity. A number of different approaches have been
proposed to eliminate the Neu5Gc incorporation [39]. The
most straightforward glyco-engineering strategy is the genetic
knockout of the gene coding for CMP-Neu5Ac hydroxylase
(Figure 3C), the enzyme which converts CMP-Neu5Ac into
CMP-Neu5Gc. Similarly, the a2,3-sialyltransferase could be
eliminated by genetic modification and ectopically expression
of the missing a2,6-sialyltransferase will lead to humanized
N-glycans.
NS0 and SP2/0 mouse myeloma or Baby Hamster
Kidney (BHK) cells generate glycoproteins with galactose-a
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A.
B.
C.
D.
E.
F.
G.
Figure 3. Strategies for N-glycan engineering. (A) Overexpression of the branching enzymes N-acetylglucosaminyltransferase
IV and V (GnTIV/V); (B) knockout of core a1,6-fucosyltransferase (FUT8); (C) knockout of CMP-Neu5Ac hydroxylase (CMAH);
(D) knockout of a1,3-galactosyltransferase (a-1,3-GalT); (E) knockout of a1,6-mannosyltransferase (Och1p) in yeast;
(F) knockout of core a1,3-fucosyltransferase (FUT) and b-N-acetylglucosaminidase (b-hex) in insect cells; (G) knockout of
core a1,3-fucosyltransferase (FUT) and b1,2-xylosyltransferase (XylT) in plants.
1,3-galactose (a-Gal epitope) (Figure 2C) that represent antigenic epitopes for humans. The responsible enzyme,
a1,3-galactosyltransferase, is inactive in humans, but present
in most non-human mammalian cell lines including
CHO [40]. BHK-derived recombinant human factor VIII
(rhFVIII) carries 3% of the a-Gal epitope, while it is not
detected on rhFVIII produced in human embryonic kidney
cells (HEK293) [37]. Significant amounts of the a-Gal epitope
are present in the N-glycan from the variable region of cetuximab [33,34] which is produced in SP2/0 mouse myeloma cells
and worldwide approved for treatment of different types of
cancer. A hypersensitive reaction to the drug in a number of
cetuximab-treated patients in the US has been linked to the
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presence of the a-Gal epitope [41]. This example highlights
the importance of glyco-engineering for the generation of
bio-better therapeutic proteins (Figure 3D).
Glycosylation of recombinant proteins derived from
human cell lines such as HEK293 can be highly heterogeneous including different biantennary and branched structures with variable terminal sugar residues and thus do
not necessarily resemble N-glycosylation of serum-derived
glycoproteins. Despite the fact that human cell lines deficient
in distinct N-glycan processing steps (e.g., GnTI-deficient)
have been reported [42] and companies like the German-based
Glycotope offer the production of recombinant glycoproteins
in glyco-engineered human cell lines [43], an extensive
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Using glyco-engineering to produce therapeutic proteins
re-modelling of the N-glycosylation pathway toward homogeneous complex N-glycans has not been described in human
cells. Comparison of several different proteins expressed
in CHO and HEK293 cells indicated significant differences
in N-glycosylation with reduced sialic acid content in
HEK293 cell produced recombinant glycoproteins [44].
Besides reducing heterogeneity, potential targets for glycoengineering of human cells are elimination of core-fucose
(Figure 3B) and knockout of N-acetylglucosaminyltransferase
III which generates the bisecting GlcNAc and thus prevents
further processing and overexpression of enzymes for branching or sialylation.
The hypermannosylated N-glycan structures of yeast
differ significantly from human N-glycans (Figure 2D) and
may cause immunogenic reactions that affect the biological
activity of drugs [45]. Pioneering work in P. pastoris demonstrated that humanized complex N-glycans can be generated
in yeast by elimination of deleterious mannosyltransferases
(Figure 3E) and controlled expression of mannosidases and
N-acetylglucosaminyltransferases from other species [46].
The success of this early work from GlycoFi Inc. (now a
division of Merck) was based on high throughput screening
of a combinatorial library of glycosylation enzymes designed
for differential subcellular localization and efficient
production of secreted recombinant proteins with defined
N-glycans. More recently, similar but more rationally
designed approaches were used to eliminate hypermannosylation and engineer P. pastoris [47], Saccharomyces cerevisiae
[48], Yarrowia lipolytica [49], and Hansenula polymorpha [50]
toward the production of humanized N-glycans on recombinant glycoproteins.
Insect cells typically produce truncated (‘paucimannosidic’)
N-glycans due to the action of a processing b-hexosaminidase
[51]. Moreover, a potentially immunogenic core a1,3-fucose
residue is present on recombinant glycoproteins expressed in
some insect cell lines (Figure 2E) [52]. Different methods
have been developed to overcome these shortcomings and
glyco-engineered insect cells that produce galactosylated
N-glycans on monoclonal antibodies and fucose-deficient
N-glycans have been described recently (Figure 3F) [53,54].
Plants typically generate rather simple biantennary complex
N-glycans with attached b1,2-xylose and core a1,3-fucose
residues (Figure 2F) that are not found on N-glycans in
mammals and therefore represent potentially immunogenic
residues [55]. Genetic knockout as well as knockdown
approaches have successfully been used to eliminate
b1,2-xylose and core a1,3-fucose from recombinant glycoprotein therapeutics produced in different plants (Figure 3G)
[56-59]. Compared to conventional CHO-produced recombinant monoclonal antibodies the N-glycans from plant-derived
IgGs were highly homogeneous and displayed mainly the
GlcNAc2Man3GlcNAc2 glycan which is the optimal substrate
for further modifications like branching or galactosylation.
Notably, the Nicotiana benthamiana-based plant expression
platform also enables the production of functionally active
recombinant IgM variants with defined N-glycosylation [60]
and is currently used to manufacture the monoclonal antibody cocktail against Ebola virus [61].
Similar to insect cells, plants also contain
b-hexosaminidases that remove GlcNAc residues from some
recombinant glycoproteins and expose terminal mannose on
truncated N-glycans. While this post-Golgi processing event
can be prevented by genetic knockout of the corresponding
b-hexosaminidases [62], N-glycans with terminal mannose
residues are beneficial structures for recombinant glycoproteins used for enzyme replacement therapy to treat lysosomal
storage diseases. Plant-produced recombinant glucocerebrosidase (taliglucerase alfa) has been approved by the FDA in
2013 to treat Gaucher disease. This drug is expressed in genetically modified carrot cells and contains mainly Man3XylFucGlcNAc2 N-glycans [63] which are efficiently internalized
by mannose receptors in humans. In contrast to other
commercially available recombinant glucocerebrosidases,
taliglucerase alfa does not require any in vitro exoglycosidase
digestions or a-mannosidase inhibitors during the production
process to generate N-glycans with terminal mannose residues.
Optimization of pharmacokinetics and biological
activity by incorporation or removal of specific
monosaccharides
3.4
Beneficial properties can be engineered by introduction of
additional sugar residues that form novel recognition sites or
mask binding sites for receptors involved in clearance. For
instance, optimized pharmacokinetic behavior is achieved by
increasing the sialic acid content of recombinant glycoproteins. Highly sialylated EPO variants have been produced in
mammalian cells (darboetin alfa) [7] as well as in nonmammalian systems including yeast [20], insect cells [64], and
plants [65]. Moreover, sialylation of antibodies is thought to
play an important role for anti-inflammatory properties [66].
However, the potential use of sialylated IgGs in this context
is still controversial because the used preparations to perform
studies are often heterogeneous in terms of glycosylation [67].
In glyco-engineering approaches towards optimizing activity, unwanted glycosyltransferases are typically inactivated to
prevent the transfer of single monosaccharides to glycan structures. Knockout of core a1,6-fucosyltransferases (FUT8) has
been used to generate CHO cells lacking N-glycans with
core fucose (Figure 3B) [68]. In another approach, the concentration of specific nucleotide sugars can be altered, for example, by inactivation or overexpression of the corresponding
nucleotide sugar transporter or metabolic control of nucleotide sugar flux. It has been shown that branching of
N-glycans is sensitive toward changes in UDP-GlcNAc concentrations [69] which is used as donor substrate by enzymes
that are involved in complex N-glycan formation and initiation of N-glycan branching. Since UDP-GlcNAc levels are
dependent on the hexosamine pathway, metabolic control
may be used to modify the branching of N-glycans which
Expert Opin. Biol. Ther. (2015) 15(10)
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M. Dicker & R. Strasser
can lead to the production of therapeutic proteins that have
increased amounts of sialic acid and consequently an
increased half-life (Figure 3A).
system displayed interesting features that need to be further
tested to fully assess the potential of this novel glycoengineering approach.
Other recent N-glycan engineering approaches
Mammalian-type N-glycosylation pathways have been engineered into E. coli and N-glycosylation of recombinant glycoproteins has been shown [70]. Such glyco-engineering attempts
are very promising, but currently limited by the restricted
acceptor site specificity of the used bacterial OST [71].
Sequence-guided protein evolution may help to overcome
such bottlenecks [72].
Synthetic and semi-synthetic approaches have been
applied to remodel N-glycans on proteins and generate
homogeneous glycoproteins with defined glycan structures [73].
In vitro approaches include total chemical synthesis of glycoproteins like sialylated EPO [74] or involve cell-based production followed by chemical or enzymatic post-production
modifications to obtain homogeneous glycoproteins.
Recently, in vitro incubation of b1,4-galactosyltransferase
and a2,6-sialyltransferase together with their corresponding
nucleotide sugars (UDP-Gal, CMP-Neu5Ac) was used to
increase the disialylated glycan content and homogeneity of
commercially available intravenous immunoglobulin (IVIg)
[75]. In vitro enzymatic processing steps using a-neuraminidase, b-galactosidase, and b-N-acetylglucosaminidase have
been applied to generate N-glycans with exposed mannose
residues on recombinant glucocerebrosidase (imiglucerase)
[76]. In addition to these stepwise modifications of existing
N-glycans, enzymatic removal of heterogeneous glycans and
in vitro enzymatic transfer of a preassembled oligosaccharide
has been used to engineer homogeneous disialylated IgG Fc
fragments in order to investigate the anti-inflammatory role
of sialylation [77,78]. The recombinant glycoproteins are produced in eukaryotic cells or even in genetically engineered
bacteria that can perform distinct N-glycosylation reactions [70]. The cell-type specific heterogeneous N-glycans are
enzymatically removed by specific endoglycosidases leaving
glycoproteins with a single N-linked GlcNAc (Figure 4A).
Enzymatic transglycosylation adds a structurally homogeneous pre-synthesized oligosaccharide to the N-linked
GlcNAc resulting in a glycoprotein with defined N-glycans.
While it is possible to generate homogeneous glycans on glycoproteins by this chemoenzymatic remodeling strategy, the
industrial scale production of semi-synthetic glycoprotein
therapeutics remains to be shown.
The Callewaert group recently developed a similar in vivo
remodeling system for N-glycans in human cells (Figure 4B)
[79]. In the GlycoDelete strategy, the endoglycosidasemediated enzymatic removal of N-glycans was engineered
into GnTI-deficient HEK293 cells to generate N-GlcNAcmodified glycoproteins in the Golgi. Endogenous galactosyltransferase and sialyltransferase can further elongate the
N-linked GlcNAc leading to three truncated N-linked glycans. Recombinant antibodies produced in the GlycoDelete
4.
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3.5
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O-glycosylation of proteins
Different types of O-glycosylation (e.g., O-linked fucose,
glucose, mannose, xylose, or GalNAc) have been described
on secretory proteins in mammals [80]. The formation of the
highly abundant mucin-type O-glycans is initiated by the
polypeptide GalNAc-transferase family. In this common
mammalian O-glycosylation pathway, Golgi-resident polypeptide GalNAc-transferases transfer a single GalNAc residue
to Ser/Thr sites of fully folded proteins which do not display a
clear consensus sequence. Subsequent stepwise elongations are
typically carried out by different glycosyltransferases resulting
in the formation of highly diverse core O-glycan structures
(Figure 5A).
What are the targets for O-glycanengineering?
5.
Remarkably, the contribution of distinct O-glycans to therapeutic properties (e.g., for EPO which contains a single
O-glycan) is still not very well investigated. As a consequence,
the specific targets for O-glycan engineering are less obvious
than for N-glycan engineering. The development of systems
capable of producing defined O-glycans is vital to improve
our understanding of O-glycan function for glycoprotein
therapeutics. IgA antibodies have, for example, high potential
as a new class of drugs to combat infections or kill tumor
cells [81]. The engineering of O-glycan residues in the hinge
region of IgA1 antibodies toward highly sialylated structures
represents an interesting target to optimize the stability and
pharmacokinetic behavior of recombinant IgA1 therapeutics.
Mucin-type O-glycosylation in mammalian cells typically
varies in the frequency of site occupancy and display a mixture
of different structures [82]. In human cells, the generation of
homogeneous mucin-type O-glycans is hampered by the large
repertoire of competing glycosyltransferases (Figure 5A and B).
Insect cells can generate some mucin-type modifications, but
also produce a number of structurally diverse non-human
O-glycans including the incorporation of fucose and hexuronic acid as well as further substitutions with phosphocholine
or sulfate [83]. In contrast to animal cells, yeast and plants do
not contain the typical mucin-type O-glycosylation machinery, which allows the de novo synthesis of these mammaliantype O-glycans without any interference from the endogenous
glycosyltransferases. However, recombinant EPO derived
from P. pastoris contained two mannose residues linked to
the single O-glycosylation site at Ser126 (Figure 5C) [84].
These O-mannose structures differ from mammalian
a-dystroglycan-type O-mannosylation and may cause
unwanted side effects when present on recombinant glycoproteins. Plants can convert exposed proline residues adjacent to
Expert Opin. Biol. Ther. (2015) 15(10)
Using glyco-engineering to produce therapeutic proteins
A.
B.
Endo S
α-fucosidase
Endo S mutant
Deglycosylation
Transglycosylation
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Golgi
Endo T
GaIT
ST
Deglycosylation
Mannose
GlcNAc
N-acetylneuraminic acid
Galactose
Fucose
Figure 4. (A) In vitro chemoenzymatic synthesis of homogeneous glycoproteins [73]. (B) In vivo GlycoDelete system [79]:
Recombinant glycoproteins are expressed in mammalian cells lacking N-acetylglucosaminyltransferase I. Man5GlcNAc1 is
cleaved off by a specific endo-b-N-acetylglucosaminidase (Endo T) in the Golgi and the resulting N-linked GlcNAc may be
further elongated by endogenous galactosyl- (GalT) and sialyltransferases (ST).
O-glycosylation sites of recombinant proteins into hydroxyproline residues [85], which can be further modified by arabinosyltransferases leading to the presence of small arabinose
chains (Figure 5D) [86].
6.
O-glycan engineering
In mammals, control of mucin-type O-glycosylation initiation and elongation are quite complex as a defined consensus
sequence for attachment of the first monosaccharide has not
been established and the factors that contribute to site-specific
O-glycosylation are not well understood [80]. The human
polypeptide GalNAc-transferase family consists of 20 members which are differentially expressed and display distinct as
well as overlapping peptide acceptor substrate specificities [87].
CHO cells produce mainly core 1 structures that can be engineered for the production of defined and elongated structures [88,89]. Strategies to eliminate O-glycan heterogeneity
include expression of the recombinant glycoprotein in
non-mammalian hosts that cannot perform mucin-type
O-glycosylation [90-92] or systematic elimination of the
enzymes that initiate or elongate O-glycans in mammalian
cells (Figure 5B) [35].
De novo O-glycan engineering has been achieved in yeast
and plants. The mucin-type core 1 O-glycan (T-antigen)
was generated on a peptide derived from human mucin in
S. cerevisiae [90]. The machinery for O-linked GalNAc
formation has been successfully expressed in plants [92] and
di-sialylated core 1 O-glycans (Figure 5A) have been efficiently
generated on N. benthamiana-derived human EPO-Fc [91].
A strategy to avoid the non-desired O-mannosylation of
yeast involves genetic engineering with expression of
an a-mannosidase. This approach will generate a single
O-linked mannose that can either be further elongated with
mammalian-type modifications such as the generation of adystroglycan-type O-glycans [93]. Alternatively, the mannose
residues can be removed by in vitro digestion of P. pastorisproduced glycoproteins with recombinant lysosomal mannosidases [94] or the generation of O-linked mannose chains
may be prevented by inhibition of the involved protein
O-mannosyltransferases (Figure 5C). In a semi-synthetic
O-glycan engineering approach, a polypeptide GalNActransferase was expressed in E. coli to initiate mucin-type
O-glycosylation on a recombinant protein [95]. The GalNAccontaining proteins were further modified in vitro by a
glycan-based PEGylation technique to generate proteins
with increased half-life. A similar strategy has been used to
attach PEG-modified sialic acid to the O-glycan of recombinant blood factor FVIII produced in mammalian cells [96].
Glyco-engineered therapeutic
glycoproteins
7.
In recent years, the pharmaceutical industry has put considerable effort into the development of novel glycoprotein therapeutics. Table 1 provides an overview of glyco-engineered
monoclonal antibodies that are approved or in clinical trials
and have been generated using different technologies.
Expert Opin. Biol. Ther. (2015) 15(10)
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M. Dicker & R. Strasser
A.
M
G
A
Golgi
C
C
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C
C
E
O
B.
C.
D.
Figure 5. (A) Human mucin-type O-glycan biosynthesis pathway. Only the enzymes involved in the first biosynthesis steps are
shown [80]. (B) Strategies for O-glycan engineering in mammalian cells: to produce homogeneous mucin-type O-glycans
consisting of O-linked GalNAc without any further extensions (Tn antigen) a knockout of core 1 b1,3-galactosyltransferase
(C1GALT1) or its chaperone COSMC can be performed; knockout of individual polypeptide GalNAc-transferases may
completely prevent mucin-type O-glycan formation. (C) In yeast transfer and elongation of O-linked mannose can be avoided
by knockout or inhibition of protein O-mannosyltransferases (PMTs). (D) In plants, hydroxyproline formation and subsequent
transfer of arabinose residues (Hyp) may be abolished by inactivation of the respective prolyl-4-hydroxylases (P4H).
Glyco-engineering of cells for IgG1 expression has focused
mainly on the elimination of core-fucose from the N-glycan
at Asn297 in the Fc region of the heavy chain. The absence
of core-fucose increases the affinity for FcgRIII receptor binding leading to improved antibody-dependent cellular cytotoxicity on natural killer cells [17,97]. A similar glycosylationdependent mechanism has an impact on antibody-dependent
cellular phagocytosis by macrophages [98] and influences the
receptor-mediated effector function of virus-neutralizing antibodies [99]. The ZMAPP antibody cocktail for treatment of
Ebola virus infections and reversion of the disease [61] contains
three plant-produced antibodies that lack core-fucose residues. In mice, the fucose-free monoclonal antibody
13F6 which is one of the ZMAPP components displayed
clearly enhanced potency against Ebola virus compared to
13F6 variants with core fucose [100]. These examples clearly
1510
demonstrate the impact of glycosylation and highlight the
potential of glyco-engineered mAbs for different applications
(Table 1) in humans. As a consequence, the number of
glyco-engineered mAbs approved for different treatments is
expected to increase in the near future [43]. Apart from
modulating effector functions, future glycosylation remodeling strategies will also focus on the engineering of mAbs
for enhanced anti-inflammatory properties [66,67]. IgG
glycoforms with high amounts of terminal sialic acid can
display increased affinity for lectin-type receptors and may
represent another emerging field for the development of
next-generation antibodies. Apart from the engineered antibody therapeutics, recombinant glycoprotein drugs with
enhanced half-life (similar to darboetin alfa), entirely humanized glycosylation (e.g., follicle-stimulating hormone) as well
as different recombinant products for enzyme replacement
Expert Opin. Biol. Ther. (2015) 15(10)
Using glyco-engineering to produce therapeutic proteins
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Table 1. Examples of glyco-engineered antibodies that are approved or in clinical studies.
Product
Company and production
platform
Glyco-engineered modification
Status
Mogamulizumab
(anti-CCR4)
Obinutuzumab
(anti-CD20)
ZMAPP
(anti-Ebola
virus)
Ublituximab
(anti-CD20)
Kyowa Hakko Kirin
Potteligent technology
Glycart-Roche
GlycoMab technology
MAPP Biopharmaceutical
Core fucose-deficient CHO cells
FUT8 knockout
Core fucose-reduced CHO cells
GnTIII overexpression
Core fucose and b1,2-xylose-deficient Nicotiana benthamiana
RNAi of xylosyl-/fucosyltransferases
Approved
TG Therapeutics
LFB Biotechnologies
EMABling technology
LFB Biotechnologies
EMABling technology
Seattle Genetics
SEA technology
Kyowa Hakko Kirin
MedImmune
Potteligent technology
Kyowa Hakko Kirin
MedImmune
Potteligent technology
Kyowa Hakko Kirin Potteligent
technology
Kyowa Hakko Kirin
Teva
Potteligent technology
Kyowa Hakko Kirin Potteligent
technology
Kyowa Hakko Kirin Potteligent
technology
Kyowa Hakko Kirin Potteligent
technology
Kyowa Hakko Kirin
Life Science Pharmaceuticals
Potteligent technology
Glycart-Roche
GlycoMab technology
Glycotope
GlycoExpress technology
Glycotope
GlycoExpress technology
Glycotope
GlycoExpress technology
Expression in YB2/0 rat hybridoma cells (low amount of FUT8)
In clinical
trials
Expression in YB2/0 rat hybridoma cells (low amount of FUT8)
Inhibition of core fucose incorporation by addition of modified
sugars to the culture medium
Core fucose-deficient CHO cells
FUT8 knockout
In clinical
trials
In clinical
trials
In clinical
trials
Core fucose-deficient CHO cells
FUT8 knockout
In clinical
trials
Core fucose-deficient CHO cells
FUT8 knockout
Core fucose-deficient CHO cells
FUT8 knockout
In clinical
trials
In clinical
trials
Core fucose-deficient
FUT8 knockout
Core fucose-deficient
FUT8 knockout
Core fucose-deficient
FUT8 knockout
Core fucose-deficient
FUT8 knockout
In clinical
trials
In clinical
trials
In clinical
trials
In clinical
trials
Roledumab
(anti-RhD)
SEA-CD40
(anti-CD40)
Benralizumab
(anti-IL-5Ra)
MEDI-551
(anti-CD19)
BIW-8962
(anti-GM2)
KHK2804
(anti-tumor specific
antigen)
KHK2823
(anti-CD123)
KHK2898
(anti-CD98)
KHK4083
(immunomodulator)
Ecromeximab
(anti-GD3)
Lumretuzumab
(anti-HER3)
PankoMab
(anti-TA-MUC1)
TrasGEX
(anti-HER2)
CetuGEX
(anti-EGFR)
CHO cells
CHO cells
CHO cells
CHO cells
Core fucose-reduced CHO cells
GnTIII overexpression
Human cell lines
low or no core fucose
Human cell lines
low or no core fucose
Human cell lines
Low or no core fucose
Approved
In clinical
trials
In clinical
trials
In clinical
trials
In clinical
trials
In clinical
trials
CCR4: CC chemokine receptor 4; FUT8: Core a1,6-fucosyltransferase; GnTIII: N-acetylglucosaminyltransferase III; HER: Human epidermal growth factor;
IL-5Ra: IL-5 receptor a; MUC1: Mucin 1; RhD: Rhesus D antigen.
therapy (like a-galactosidase A) are in the pipelines of
companies.
8.
Conclusion
Despite the fact that we still do not understand all factors that
influence and control glycosylation in eukaryotic cells, major
advances in engineering of expression hosts towards homogeneous glycosylation patterns have been achieved in the past.
The use of genome editing tools, extensive integration of analytical data, and a better understanding of cellular processes
will facilitate the development of next-generation expression
hosts for custom-made expression of recombinant glycoprotein pharmaceuticals. These glyco-engineering approaches
will allow easier production of biosimilar drugs (same homogeneous glycosylation) and lead to the generation of bio-better
pharmaceuticals with altered glycosylation and optimized
efficacy and safety.
9.
Expert opinion
The importance of glycosylation for recombinant glycoprotein biopharmaceuticals is more and more recognized and
enormous efforts are currently undertaken in academic groups
Expert Opin. Biol. Ther. (2015) 15(10)
1511
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M. Dicker & R. Strasser
and industry to provide robust solutions for controlled glycosylation. Recent advances in engineering of cell-based expression platforms demonstrate that rather uniform and
customized N-glycan structures can be made in different systems including mammalian cells (human and non-human),
yeast, insect cells, and plants. By employing genome editing
tools like CRISPR/Cas [35], existing differences and shortcomings of species and cell-types can be eliminated and numerous
remodeling tools for the generation of defined N-glycans on
specific glycoproteins (e.g., monoclonal antibodies, EPO)
are already available in different expression platforms. The
ultimate goal will be the establishment of manufacturing platforms for the production of recombinant glycoprotein therapeutics with homogeneous custom-made N-glycan structures
that are designed for desired activities. Currently, such ‘on
demand’ N-glycans include highly sialylated tetraantennary
complex N-glycans (Figure 3A), terminally sialylated biantennary N-glycans (Figure 3C and D), and fucose-deficient biantennary N-glycans (Figure 3B, F and G). In addition, the
potential to produce homogeneous oligomannosidic structures like Man8GlcNAc2 (Figure 3E) and defined O-linked
glycans (Figure 5A) is of interest. The means to produce glycoproteins with defined glycans will drive progress in the understanding of a glycan’s function leading to novel targets for
glyco-engineering of glycoprotein therapeutics.
So what are the challenges for the
future?
10.
We still do not understand all pathways leading to glycan
diversity as seen in most eukaryotic cells and our knowledge
of essential enzymes involved in initiation of N- and
O-glycosylation (OST complex and polypeptide GalNActransferases) is limited. In addition, it is still difficult to
produce N-glycans with long terminal elongations like
polysialylation which might significantly contribute to halflife extension. Such extensive modifications might depend
on so far unknown factors that regulate the retention or
contact time in the Golgi. A better characterization of cellular
factors like Golgi organization and cargo transport processes
will be essential to overcome some of the current limitations.
Moreover, site-specific N-glycosylation and N-glycan processing will be difficult to achieve using available in vivo production systems. More research is needed to understand the
protein intrinsic factors that influence the relationship of a
particular glycan with the polypeptide sequence and structure
as well as the contribution of other still unknown aspects of
site-specific glycosylation. Together with other experimental
techniques, molecular dynamics simulations will be highly
suitable to examine carbohydrate--protein interactions and
1512
may allow the development of strategies for site-specific
remodeling of N-glycans. Structure-guided protein engineering to alter the substrate specificity of glycosylation enzymes
like the OST catalytic subunit or glycan processing enzymes
may provide additional tools required for site-specific engineering in cell-based production systems [72]. Alternatively,
site-specific modifications may be accomplished by advanced
chemoenzymatic remodeling or de novo synthesis of glycoproteins with precise control of attached glycan structures. In the
latter case, it remains to be shown whether such sophisticated
and cost-intensive approaches will be of interest for the biopharmaceutical industry or remain the field of specialized
laboratories.
Another challenge for the future will be the coordination of
N- and O-glycan engineering attempts. So far there are only
proof-of-concept studies that have shown the feasibility of
extensive remodeling of both glycosylation pathways. Due to
the requirement for the simultaneous engineering of two
pathways which act in the same subcellular compartment
and utilize the same nucleotide sugar donors, the control
over these modifications under manufacturing conditions
will be a great challenge. A better understanding of cell biology like cargo transport processes through the Golgi and
development of tools to channel nucleotide sugar substrates
and processing enzymes will be fundamental for more complex approaches that interfere with different cellular processes.
Ultimately, the experimentally obtained parameters need to
be incorporated into robust models to accurately predict the
N- and O-glycan profiles of recombinant glycoprotein biopharmaceuticals. Given the great potential that glycoprotein
therapeutics with defined glycans offers, the biopharmaceutical industry should extend their efforts in glyco-engineering
to spur the development of next-generation drugs with
tailored properties and function.
Declaration of interest
M Dicker and glyco-engineering work in the Strasser group
were supported by a grant from the Austrian Federal Ministry
of Transport, Innovation, and Technology (bmvit) and
Austrian Science Fund (FWF): TRP 242-B20. M Dicker
and R Strasser are employees of University of Natural Resources and Life Sciences, Vienna, Austria. The authors have no
other relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the
manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants
or patents received or pending, or royalties.
Expert Opin. Biol. Ther. (2015) 15(10)
Using glyco-engineering to produce therapeutic proteins
Bibliography
Papers of special note have been highlighted as
either of interest () or of considerable interest
() to readers.
1.
Aebi M. N-linked protein glycosylation
in the ER. Biochim Biophys Acta
2013;1833:2430-7
2.
Zielinska DF, Gnad F, Schropp K, et al.
Mapping N-glycosylation sites across
seven evolutionarily distant species reveals
a divergent substrate proteome despite a
common core machinery. Mol Cell
2012;46:542-8
Downloaded by [University of California, San Diego] at 09:13 17 May 2016
3.
4.
5.
6.
7.
Shrimal S, Gilmore R. Glycosylation of
closely spaced acceptor sites in human
glycoproteins. J Cell Sci
2013;126:5513-23
Shrimal S, Cherepanova NA, Gilmore R.
Cotranslational and posttranslocational
N-glycosylation of proteins in the
endoplasmic reticulum. Semin Cell
Dev Biol 2015;41:71-8
Shrimal S, Gilmore R. Reduced
expression of the oligosaccharyltransferase
exacerbates protein hypoglycosylation in
cells lacking the fully assembled
oligosaccharide donor. Glycobiology
2015;25(7):774-83
Choi BK, Warburton S, Lin H, et al.
Improvement of N-glycan site occupancy
of therapeutic glycoproteins produced in
Pichia pastoris.
Appl Microbiol Biotechnol
2012;95:671-82
Elliott S, Lorenzini T, Asher S, et al.
Enhancement of therapeutic protein
in vivo activities through
glycoengineering. Nat Biotechnol
2003;21:414-21
8.
Egrie JC, Browne JK. Development and
characterization of novel erythropoiesis
stimulating protein (NESP). Br J Cancer
2001;84:3-10
9.
Song R, Oren DA, Franco D, et al.
Strategic addition of an N-linked glycan
to a monoclonal antibody improves its
HIV-1-neutralizing activity.
Nat Biotechnol 2013;31:1047-52
This elegant study demonstrates the
power of protein glyco-engineering.
.
10.
Nairn AV, York WS, Harris K, et al.
Regulation of glycan structures in animal
tissues: transcript profiling of glycanrelated genes. J Biol Chem
2008;283:17298-313
11.
12.
13.
.
14.
15.
North SJ, Huang HH, Sundaram S,
et al. Glycomics profiling of Chinese
hamster ovary cell glycosylation mutants
reveals N-glycans of a novel size and
complexity. J Biol Chem
2010;285:5759-75
Bennun SV, Yarema KJ, Betenbaugh MJ,
et al. Integration of the transcriptome
and glycome for identification of glycan
cell signatures. PLoS Comput Biol
2013;9:e1002813
Spahn PN, Lewis NE. Systems
glycobiology for glycoengineering.
Curr Opin Biotechnol 2014;30:218-24
Comprehensive summary of systems
glycobiology approaches aiming at
prediction of product glycosylation.
Jedrzejewski PM, del Val IJ,
Constantinou A, et al. Towards
controlling the glycoform: a model
framework linking extracellular
metabolites to antibody glycosylation.
Int J Mol Sci 2014;15:4492-522
Hossler P, Mulukutla BC, Hu WS.
Systems analysis of N-glycan processing
in mammalian cells. PLoS ONE
2007;2:e713
16.
Moremen KW, Tiemeyer M, Nairn AV.
Vertebrate protein glycosylation:
diversity, synthesis and function. Nat Rev
Mol Cell Biol 2012;13:448-62
17.
Umaña P, Jean-Mairet J, Moudry R,
et al. Engineered glycoforms of an
antineuroblastoma IgG1 with optimized
antibody-dependent cellular cytotoxic
activity. Nat Biotechnol 1999;17:176-80
Describes the GlycArt technology for
the reduction of core fucose
on antibodies.
.
18.
19.
Goede V, Fischer K, Busch R, et al.
Obinutuzumab plus chlorambucil in
patients with CLL and coexisting
conditions. N Engl J Med
2014;370:1101-10
Zhang M, Koskie K, Ross JS, et al.
Enhancing glycoprotein sialylation by
targeted gene silencing in mammalian
cells. Biotechnol Bioeng
2010;105:1094-105
20.
Hamilton SR, Davidson RC,
Sethuraman N, et al. Humanization of
yeast to produce complex terminally
sialylated glycoproteins. Science
2006;313:1441-3
21.
Strasser R, Altmann F, Steinkellner H.
Controlled glycosylation of plant-
Expert Opin. Biol. Ther. (2015) 15(10)
produced recombinant proteins.
Curr Opin Biotechnol 2014;30C:95-100
22.
Stanley P. Chinese hamster ovary cell
mutants with multiple glycosylation
defects for production of glycoproteins
with minimal carbohydrate heterogeneity.
Mol Cell Biol 1989;9:377-83
23.
Wright A, Sato Y, Okada T, et al. In
vivo trafficking and catabolism of
IgG1 antibodies with Fc associated
carbohydrates of differing structure.
Glycobiology 2000;10:1347-55
24.
Yoo EM, Yu LJ, Wims LA, et al.
Differences in N-glycan structures found
on recombinant IgA1 and IgA2 produced
in murine myeloma and CHO cell lines.
MAbs 2010;2:320-34
Comparison of N-glycan profiles found
on IgA subtypes expressed in murine
myeloma and CHO cells.
.
25.
Patterson G, Hirschberg K,
Polishchuk R, et al. Transport through
the Golgi apparatus by rapid partitioning
within a two-phase membrane system.
Cell 2008;133:1055-67
26.
Beznoussenko GV, Parashuraman S,
Rizzo R, et al. Transport of soluble
proteins through the Golgi occurs by
diffusion via continuities across cisternae.
Elife 2014;3:doi: 10.7554/eLife.02009
27.
Nabi IR, Dennis JW. The extent of
polylactosamine glycosylation of MDCK
LAMP-2 is determined by its Golgi
residence time. Glycobiology
1998;8:947-53
28.
Glick BS, Luini A. Models for Golgi
traffic: a critical assessment. Cold Spring
Harb Perspect Biol 2011;3:a005215
29.
Lavieu G, Dunlop MH, Lerich A, et al.
The Golgi ribbon structure facilitates
anterograde transport of large cargoes.
Mol Biol Cell 2014;25:3028-36
30.
D’Angelo G, Uemura T, Chuang CC,
et al. Vesicular and non-vesicular
transport feed distinct glycosylation
pathways in the Golgi. Nature
2013;501:116-20
31.
Thaysen-Andersen M, Packer NH.
Site-specific glycoproteomics confirms
that protein structure dictates formation
of N-glycan type, core fucosylation and
branching. Glycobiology
2012;22:1440-52
This study analyzed N-glycan profiles
from 117 puplished papers to
investigate how the individual protein
..
1513
M. Dicker & R. Strasser
specific for galactose-alpha-1,3-galactose.
N Engl J Med 2008;358:1109-17
structures affect the N-glycan type,
core fucosylation, and branching.
32.
Wormald MR, Dwek RA. Glycoproteins:
glycan presentation and protein-fold
stability. Structure 1999;7:R155-60
33.
Stadlmann J, Pabst M, Kolarich D, et al.
Analysis of immunoglobulin glycosylation
by LC-ESI-MS of glycopeptides and
oligosaccharides. Proteomics
2008;8:2858-71
Downloaded by [University of California, San Diego] at 09:13 17 May 2016
34.
Ayoub D, Jabs W, Resemann A, et al.
Correct primary structure assessment and
extensive glyco-profiling of cetuximab by
a combination of intact, middle-up,
middle-down and bottom-up ESI and
MALDI mass spectrometry techniques.
MAbs 2013;5:699-710
35.
Steentoft C, Bennett EP,
Schjoldager KT, et al. Precision genome
editing: a small revolution for
glycobiology. Glycobiology
2014;24:663-80
36.
Ghaderi D, Taylor RE,
Padler-Karavani V, et al. Implications of
the presence of N-glycolylneuraminic
acid in recombinant therapeutic
glycoproteins. Nat Biotechnol
2010;28:863-7
Demonstration of the presence of
covalently bound Neu5Gc in
cetuximab and generation of immune
complexes in vitro.
..
37.
Kannicht C, Ramstr€om M, Kohla G,
et al. Characterisation of the
post-translational modifications of a
novel, human cell line-derived
recombinant human factor VIII.
Thromb Res 2013;131:78-88
42.
Reeves PJ, Callewaert N, Contreras R,
et al. Structure and function in
rhodopsin: high-level expression of
rhodopsin with restricted and
homogeneous N-glycosylation by a
tetracycline-inducible
N-acetylglucosaminyltransferase
I-negative HEK293S stable mammalian
cell line. Proc Natl Acad Sci USA
2002;99:13419-24
43.
Beck A, Reichert JM. Marketing
approval of mogamulizumab: a triumph
for glyco-engineering. MAbs
2012;4:419-25
44.
Croset A, Delafosse L, Gaudry JP, et al.
Differences in the glycosylation of
recombinant proteins expressed in HEK
and CHO cells. J Biotechnol
2012;161:336-48
45.
Chiba Y, Suzuki M, Yoshida S, et al.
Production of human compatible high
mannose-type (Man5GlcNAc2) sugar
chains in Saccharomyces cerevisiae.
J Biol Chem 1998;273:26298-304
46.
..
Hamilton SR, Bobrowicz P,
Bobrowicz B, et al. Production of
complex human glycoproteins in yeast.
Science 2003;301:1244-6
Using a combinatorial library
approach, the first production of
complex N-glycans in yeast is shown.
47.
Jacobs PP, Geysens S, Vervecken W,
et al. Engineering complex-type
N-glycosylation in Pichia pastoris using
GlycoSwitch technology. Nat Protoc
2009;4:58-70
Shahrokh Z, Royle L, Saldova R, et al.
Erythropoietin produced in a human cell
line (Dynepo) has significant differences
in glycosylation compared with
erythropoietins produced in CHO cell
lines. Mol Pharm 2011;8:286-96
48.
Parsaie Nasab F, Aebi M, Bernhard G,
et al. A combined system for engineering
glycosylation efficiency and glycan
structure in Saccharomyces cerevisiae.
Appl Environ Microbiol
2013;79:997-1007
Ghaderi D, Zhang M, Hurtado-Ziola N,
et al. Production platforms for
biotherapeutic glycoproteins. Occurrence,
impact, and challenges of non-human
sialylation. Biotechnol Genet Eng Rev
2012;28:147-75
49.
40.
Bosques CJ, Collins BE, Meador JW,
et al. Chinese hamster ovary cells can
produce galactose-a-1,3-galactose
antigens on proteins. Nat Biotechnol
2010;28:1153-6
50.
41.
Chung CH, Mirakhur B, Chan E, et al.
Cetuximab-induced anaphylaxis and IgE
38.
39.
1514
De Pourcq K, Tiels P, Van Hecke A,
et al. Engineering Yarrowia lipolytica to
produce glycoproteins homogeneously
modified with the universal
Man3GlcNAc2 N-glycan core.
PLoS One 2012;7:e39976
Wang H, Song HL, Wang Q, et al.
Expression of glycoproteins bearing
complex human-like glycans with
galactose terminal in Hansenula
polymorpha. World J
Microbiol Biotechnol 2013;29:447-58
Expert Opin. Biol. Ther. (2015) 15(10)
51.
Altmann F, Schwihla H, Staudacher E,
et al. Insect cells contain an unusual,
membrane-bound
beta-N-acetylglucosaminidase probably
involved in the processing of protein
N-glycans. J Biol Chem
1995;270:17344-9
52.
Shi X, Jarvis DL. Protein N-glycosylation
in the baculovirus-insect cell system.
Curr Drug Targets 2007;8:1116-25
53.
Palmberger D, Wilson IB, Berger I, et al.
SweetBac: a new approach for the
production of mammalianised
glycoproteins in insect cells. PLoS One
2012;7:e34226
54.
Mabashi-Asazuma H, Kuo CW,
Khoo KH, et al. A novel baculovirus
vector for the production of
nonfucosylated recombinant
glycoproteins in insect cells. Glycobiology
2014;24:325-40
55.
Altmann F. The role of protein
glycosylation in allergy. Int Arch
Allergy Immunol 2007;142:99-115
56.
Cox K, Sterling J, Regan J, et al. Glycan
optimization of a human monoclonal
antibody in the aquatic plant Lemna
minor. Nat Biotechnol 2006;24:1591-7
57.
Schähs M, Strasser R, Stadlmann J, et al.
Production of a monoclonal antibody in
plants with a humanized N-glycosylation
pattern. Plant Biotechnol J
2007;5:657-63
58.
Strasser R, Stadlmann J, Schähs M, et al.
Generation of glyco-engineered Nicotiana
benthamiana for the production of
monoclonal antibodies with a
homogeneous human-like N-glycan
structure. Plant Biotechnol J
2008;6:392-402
59.
Koprivova A, Stemmer C, Altmann F,
et al. Targeted knockouts of
Physcomitrella lacking plant-specific
immunogenic N-glycans.
Plant Biotechnol J 2004;2:517-23
60.
Loos A, Gruber C, Altmann F, et al.
Expression and glycoengineering of
functionally active heteromultimeric IgM
in plants. Proc Natl Acad Sci USA
2014;111:6263-8
The assembly of pentameric and
hexameric glyco-engineered forms of
IgM is demonstrated using a Nicotiana
benthamiana plant system.
..
61.
Qiu X, Wong G, Audet J, et al.
Reversion of advanced Ebola virus disease
Using glyco-engineering to produce therapeutic proteins
.
62.
Downloaded by [University of California, San Diego] at 09:13 17 May 2016
63.
64.
65.
66.
67.
68.
.
69.
70.
71.
in nonhuman primates with ZMapp.
Nature 2014;514:47-53
ZMappTM is composed of three
“humanized” monoclonal antibodies
manufactured in glyco-engineered
Nicotiana benthamiana plants.
Castilho A, Windwarder M, Gattinger P,
et al. Proteolytic and N-Glycan
Processing of Human a1-Antitrypsin
Expressed in Nicotiana benthamiana.
Plant Physiol 2014;166:1839-51
Tekoah Y, Tzaban S, Kizhner T, et al.
Glycosylation and functionality of
recombinant b-glucocerebrosidase from
various production systems. Biosci Rep
2013;33:doi: 10.1042/BSR20130081
Mabashi-Asazuma H, Shi X, Geisler C,
et al. Impact of a human CMP-sialic
acid transporter on recombinant
glycoprotein sialylation in
glycoengineered insect cells. Glycobiology
2013;23:199-210
Castilho A, Neumann L, Gattinger P,
et al. Generation of biologically active
multi-sialylated recombinant human
EPOFc in plants. PLoS One
2013;8:e54836
Kaneko Y, Nimmerjahn F, Ravetch JV.
Anti-inflammatory activity of
immunoglobulin G resulting from Fc
sialylation. Science 2006;313:670-3
Raju TS, Lang SE. Diversity in structure
and functions of antibody sialylation in
the Fc. Curr Opin Biotechnol
2014;30:147-52
Yamane-Ohnuki N, Kinoshita S,
Inoue-Urakubo M, et al. Establishment
of FUT8 knockout Chinese hamster
ovary cells: an ideal host cell line for
producing completely defucosylated
antibodies with enhanced antibodydependent cellular cytotoxicity.
Biotechnol Bioeng 2004;87:614-22
Provides a description of the
Potteligent technology.
Lau KS, Partridge EA, Grigorian A, et al.
Complex N-glycan number and degree
of branching cooperate to regulate cell
proliferation and differentiation. Cell
2007;129:123-34
Baker JL, Çelik E, DeLisa MP.
Expanding the glycoengineering toolbox:
the rise of bacterial N-linked protein
glycosylation. Trends Biotechnol
2013;31:313-23
Valderrama-Rincon JD, Fisher AC,
Merritt JH, et al. An engineered
83.
Gaunitz S, Jin C, Nilsson A, et al.
Mucin-type proteins produced in the
Trichoplusia ni and Spodoptera
frugiperda insect cell lines carry novel
O-glycans with phosphocholine and
sulfate substitutions. Glycobiology
2013;23:778-96
84.
Gong B, Burnina I, Stadheim TA, et al.
Glycosylation characterization of
recombinant human erythropoietin
produced in glycoengineered Pichia
pastoris by mass spectrometry.
J Mass Spectrom 2013;48:1308-17
85.
Parsons J, Altmann F, Graf M, et al.
A gene responsible for prolylhydroxylation of moss-produced
recombinant human erythropoietin.
Sci Rep 2013;3:3019
86.
Zimran A, Elstein D, Levy-Lahad E,
et al. Replacement therapy with
imiglucerase for type 1 Gaucher’s disease.
Lancet 1995;345:1479-80
Karnoup AS, Turkelson V,
Anderson WH. O-linked glycosylation in
maize-expressed human IgA1.
Glycobiology 2005;15:965-81
87.
Huang W, Giddens J, Fan SQ, et al.
Chemoenzymatic glycoengineering of
intact IgG antibodies for gain of
functions. J Am Chem Soc
2012;134:12308-18
Kong Y, Joshi HJ, Schjoldager KT, et al.
Probing polypeptide GalNAc-transferase
isoform substrate specificities by in vitro
analysis. Glycobiology 2015;25:55-65
88.
Lindberg L, Liu J, Gaunitz S, et al.
Mucin-type fusion proteins with blood
group A or B determinants on defined
O-glycan core chains produced in
glycoengineered Chinese hamster ovary
cells and their use as immunoaffinity
matrices. Glycobiology 2013;23:720-35
89.
Liu J, Jin C, Cherian RM, et al.
O-glycan repertoires on a mucin-type
reporter protein expressed in CHO cell
pools transiently transfected with
O-glycan core enzyme cDNAs.
J Biotechnol 2015;199:77-89
Describes the effect of transiently
expressed O-glycan core chain
glycosyltransferases on O-glycan
biosynthesis pathways in CHO cells.
eukaryotic protein glycosylation pathway
in Escherichia coli. Nat Chem Biol
2012;8:434-6
72.
Ollis AA, Zhang S, Fisher AC, et al.
Engineered oligosaccharyltransferases with
greatly relaxed acceptor-site specificity.
Nat Chem Biol 2014;10:816-22
73.
Wang LX, Amin MN. Chemical and
chemoenzymatic synthesis of
glycoproteins for deciphering functions.
Chem Biol 2014;21:51-66
74.
Wang P, Dong S, Shieh JH, et al.
Erythropoietin derived by chemical
synthesis. Science 2013;342:1357-60
75.
76.
77.
Washburn N, Schwab I, Ortiz D, et al.
Controlled tetra-Fc sialylation of IVIg
results in a drug candidate with
consistent enhanced anti-inflammatory
activity. Proc Natl Acad Sci USA
2015;112:E1297-306
78.
Ahmed AA, Giddens J, Pincetic A, et al.
Structural characterization of antiinflammatory immunoglobulin G Fc
proteins. J Mol Biol 2014;426:3166-79
79.
Meuris L, Santens F, Elson G, et al.
GlycoDelete engineering of mammalian
cells simplifies N-glycosylation of
recombinant proteins. Nat Biotechnol
2014;32:485-9
This study describes a novel approach
to reduce the glycan heterogeneity of
recombinant proteins by simplification
of N-glycan processing steps.
..
80.
Bennett EP, Mandel U, Clausen H, et al.
Control of mucin-type O-glycosylation:
A classification of the polypeptide
GalNAc-transferase gene family.
Glycobiology 2012;22:736-56
81.
Bakema JE, van Egmond M.
Immunoglobulin A: A next generation of
therapeutic antibodies? MAbs
2011;3:352-61
82.
Taschwer M, Hackl M,
Hernández Bort JA, et al. Growth,
productivity and protein glycosylation in
a CHO EpoFc producer cell line adapted
to glutamine-free growth. J Biotechnol
2012;157:295-303
Expert Opin. Biol. Ther. (2015) 15(10)
.
90.
Amano K, Chiba Y, Kasahara Y, et al.
Engineering of mucin-type human
glycoproteins in yeast cells. Proc Natl
Acad Sci USA 2008;105:3232-7
91.
Castilho A, Neumann L, Daskalova S,
et al. Engineering of Sialylated Mucintype O-Glycosylation in Plants.
J Biol Chem 2012;287:36518-26
Demonstrates the de novo generation
of sialylated core 1 O-glycans on
recombinant EPO-Fc produced
in plants.
.
92.
Yang Z, Drew DP, Jørgensen B, et al.
Engineering mammalian mucin-type
1515
M. Dicker & R. Strasser
demonstrates full efficacy and prolonged
effect in hemophilic mice models. Blood
2013;121:2108-16
O-glycosylation in plants. J Biol Chem
2012;287:11911-23
93.
.
Downloaded by [University of California, San Diego] at 09:13 17 May 2016
94.
Hamilton SR, Cook WJ,
Gomathinayagam S, et al. Production of
sialylated O-linked glycans in Pichia
pastoris. Glycobiology 2013;23:1192-203
Generation of a-dystroglycan-type
O-glycans in yeast.
Hopkins D, Gomathinayagam S,
Hamilton SR. A practical approach for
O-linked mannose removal: the use of
recombinant lysosomal mannosidase.
Appl Microbiol Biotechnol
2015;99:3913-27
95.
Henderson GE, Isett KD, Gerngross TU.
Site-specific modification of recombinant
proteins: a novel platform for modifying
glycoproteins expressed in E.
coli. Bioconjug Chem 2011;22:903-12
96.
Stennicke HR, Kjalke M, Karpf DM,
et al. A novel B-domain
O-glycoPEGylated FVIII (N8-GP)
1516
97.
..
Ferrara C, Grau S, Jäger C, et al. Unique
carbohydrate-carbohydrate interactions
are required for high affinity binding
between FcgammaRIII and antibodies
lacking core fucose. Proc Natl Acad
Sci USA 2011;108:12669-74
Insight into the role of specific glycans
in receptor-mediated antibodydependent cytotoxocity using
X-ray crystallography.
98.
Golay J, Da Roit F, Bologna L, et al.
Glycoengineered CD20 antibody
obinutuzumab activates neutrophils and
mediates phagocytosis through CD16B
more efficiently than rituximab. Blood
2013;122:3482-9
99.
Forthal DN, Gach JS, Landucci G, et al.
Fc-glycosylation influences Fcg receptor
binding and cell-mediated anti-HIV
Expert Opin. Biol. Ther. (2015) 15(10)
activity of monoclonal antibody 2G12.
J Immunol 2010;185:6876-82
100. Zeitlin L, Pettitt J, Scully C, et al.
Enhanced potency of a fucose-free
monoclonal antibody being developed as
an Ebola virus immunoprotectant.
Proc Natl Acad Sci 2011;108:20690-4
Affiliation
Martina Dicker1 & Richard Strasser†2
†
Author for correspondence
1
PhD candidate,
University of Natural Resources and Life
Sciences, Department of Applied Genetics and
Cell Biology, Muthgasse 18, Vienna, Austria
2
Associate Professor, Group Leader,
University of Natural Resources and Life
Sciences, Department of Applied Genetics and
Cell Biology, Muthgasse 18, Vienna, Austria
Tel: +43 1 47654 6705;
Fax: +43 1 47654 6392;
E-mail: [email protected]
Advancesintheproductionofhumantherapeuticproteinsin
yeastsandfilamentousfungiGerngrossTU,Nat.Biotech,22:1409
(2004)
© 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology
REVIEW
Advances in the production of human
therapeutic proteins in yeasts and
filamentous fungi
Tillman U Gerngross
Yeast and fungal protein expression systems are used for the production of many industrially relevant enzymes, and are widely
used by the research community to produce proteins that cannot be actively expressed in Escherichia coli or require glycosylation
for proper folding and biological activity. However, for the production of therapeutic glycoproteins intended for use in humans,
yeasts have been less useful because of their inability to modify proteins with human glycosylation structures. Yeast glycosylation
is of the high-mannose type, which confers a short in vivo half-life to the protein and may render it less efficacious or even
immunogenic. Several ways of humanizing yeast-derived glycoproteins have been tried, including enzymatically modifying
proteins in vitro and modulating host glycosylation pathways in vivo. Recent advances in the glycoengineering of yeasts and the
expression of therapeutic glycoproteins in humanized yeasts have shown significant promise, and are challenging the current
dominance of therapeutic protein production based on mammalian cell culture.
Protein-based therapeutics are emerging as the largest class of new
chemical entities being developed by the drug industry1. Unlike
small molecules, which typically are synthesized by chemical means,
most proteins are sufficiently complex to necessitate their production in living systems, mostly by recombinant DNA technology.
As such, the choice of recombinant expression hosts has been the
subject of ongoing evaluation and much effort has been directed at
developing novel protein expression systems with improved characteristics. The five primary metrics for evaluating such novel
protein expression hosts are: (i) the cost of manufacturing and purification (ii) the ability to control the final product including its posttranslational processing (iii) the time required from gene to purified
protein (iv), the regulatory path to approve a drug produced on a
given expression platform and (v) the overall royalties associated
with the production of a recombinant product in a given host.
Yeasts and filamentous fungi have been extensively used in the
industrial enzyme industry for the production of recombinant proteins—and most companies (DSM (Heerlen, The Netherlands),
Genencor (Palo Alto, CA, USA), Novozymes (Bagsveard, Denmark)
and others) have developed large-scale fermentation capacity
around yeasts and/or fungi. The ability of these organisms to grow
in chemically defined medium in the absence of animal-derived
growth factors (e.g., calf serum) and to secrete large amounts of
Thayer School of Engineering, the Department of Biological Sciences and the
Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755,
USA and GlycoFi Inc., 21 Lafayette Street, Lebanon, New Hampshire 03766,
USA. Correspondence should be addressed to T.U.G.
([email protected]).
Published online 4 November 2004; doi:10.1038/nbt1028
recombinant protein, together with the ease of scale-up, have made
these hosts the system of choice for producing many industrial
enzymes, where cost of goods is a primary concern2. The research
community has relied on yeasts for the production of recombinant
proteins that cannot be obtained from E. coli because of folding
problems or the requirement for glycosylation. Yeasts have been
used to express proteins at very high protein titers, including
mammalian proteins, as exemplified by the production of 14.8 g/l of
gelatin in Pichia pastoris3. Expression kits based on yeasts are available from almost all major research tool companies and their ease
of use has made them an attractive solution for many protein
expression needs.
However, the choice of protein expression system is most often dictated by the ultimate use of the product and not necessarily the ease of
initial production. Proteins intended for use in humans impose the
most significant challenges in this context, and the aberrant nature or
absence of post-translational processing associated with a given host,
often becomes an overriding consideration. In this article, we review
the challenges involved in the production of therapeutic glycoproteins and the current state of development of engineered yeast strains
that are able to produce humanized glycoproteins.
Glycosylation of therapeutic proteins
There are approximately 140 therapeutic proteins approved in the
United States and Europe, and an additional 500 in clinical trials1;
with an even larger number in preclinical development. The therapeutic protein market can be roughly divided into two segments—
proteins that are not post-translationally modified and those that
require post-translational processing (mostly N-glycosylation) to
attain full biological function. Nonglycosylated proteins are typi-
NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 11 NOVEMBER 2004
1409
REVIEW
© 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology
a
b
Figure 1 Protein structure of interferon-β illustrates the impact of
glycosylation on hydrodynamic volume; glycosylated interferon-β (top) and
glycosylated interferon-β with glycosylation highlighted in blue (bottom).
Source: http://www.dkfz.de/spec/glycosciences.de/modeling/glyprot/php/
main.php
cally expressed in either E. coli or the yeast Saccharomyces cerevisiae
and currently constitute about 40% of the therapeutic protein market with an annual growth rate of about 12%4. Some aglycosylated
proteins are expressed in both systems, for example, recombinant
insulin is produced in E. coli (Humulin, Eli Lilly, Indianapolis, IN,
USA) and yeast (Novolog, Novo Nordisk, Bagsvaerd, Denmark).
The production of recombinant proteins that are N-glycosylated
in their native state has in most cases required mammalian expression hosts that have the ability to mimic human glycosylation.
Prokaryotic hosts such as E. coli do not glycosylate proteins, and
lower eukaryotic expression systems such as yeast and insect cells
are typically unable to provide mammalian glycosylation. Aglycosylated forms of glycoproteins tend to be misfolded, biologically
inactive or rapidly cleared from circulation5. Yeasts are able to glycosylate proteins, yet the nonhuman nature of yeast glycans negatively
impacts protein half-life because of the affinity of these glycans to
high-mannose receptors present on macrophages and endothelial
cells6. For these reasons, mammalian cells, in particular Chinese
hamster ovary (CHO) cells, have emerged as the most widely used
expression system for the production of complex human glycoproteins—currently about 60% of the therapeutic protein market are
glycoproteins with an annual growth rate of approximately 26%4.
CHO cells are typically able to express glycoproteins with humanlike glycosylation patterns, although it is commonly recognized that
glycan structures produced from CHO cell lines differ from those
produced in human cells and sometimes have to be modified to
meet therapeutic efficacy7,8. A few natively glycosylated proteins are
produced in nonglycosylating hosts (e.g. Betaferon (interferon-β1b); Schering-Plough, Kenilworth, NJ, USA) yet the lack of glycosylation can compromise half-life and other clinically relevant
properties of the molecule such as renal clearance, which is affected
by the hydrodynamic volume of the protein (Fig. 1).
1410
It has to be recognized that glycoproteins produced by mammalian cell culture are inherently heterogenous mixtures of glycoforms, and that scale-up and regulatory approval have been a matter
of maintaining a reproducible composition and ratio of glycoforms.
Because it is well understood that certain glycoforms are more active
than others9, the relative distribution of individual glycoforms has
to be kept constant during preclinical manufacturing and postapproval production—often representing a major bioprocessing
challenge. A purified protein preparation derived from a mammalian cell culture process is essentially a mixture of individual
drugs, each with its own pharmacokinetic, pharmacodynamic properties and efficacy profile10. For example, human erythropoietin
(EPO), a glycoprotein that plays a major role in regulating the level
of circulating erythrocytes, has found wide use in the treatment of
anemia. Natural EPO contains three N-glycans (glycans linked to
asparagine residues of the glycoprotein), which are critical to its
therapeutic activity, and one O-glycosylation site which has no
known function; removal of the N-glycans from EPO results in a
protein with a very short half-life and virtually no erythropoietic
function in vivo10. Whereas glycosylation of EPO appears to be
essential, it is not sufficient to ensure full biological activity. Recent
work has shown that the production of recombinant EPO is highly
sensitive to the production process. Different process conditions
have been shown to yield material that has a fivefold difference in
erythropoietic function in vivo, and a detailed analysis of this effect
demonstrated that variations in the glycosylation are responsible for
these differences11. Recent work by Erbayraktar and colleagues has
shown that by modifying the glycosylation of EPO, one can create a
molecule that entirely lacks erythropoietic function while retaining
the neuroprotective function of the protein12.
Some therapeutic proteins require specific glycosylation to ensure
proper cellular targeting. For example, glucocerebrosidase, the
enzyme used for replacement therapy in patients with Gaucher disease, is based on a recombinant enzyme produced in mammalian
cells7. However, the therapeutically active form of glucocerebrosidase requires terminal mannose sugars, which are responsible for
delivery of the protein to macrophages and subsequent incorporation into lysozomes13. As discussed, glycoproteins from CHO cells
contain complex N-glycans which contain: N-acetyl glucosamine
(GlcNAc), galactose (Gal) and terminal sialic acid (NANA) (Fig. 2).
It is therefore necessary to remove all terminal nonmannose sugars
after isolation of the glycoprotein. To do so, in vitro treatment with
neuraminidase (to remove sialic acid), galactosidase (to remove
galactose) and hexosaminidase (to remove GlcNAc) is required. The
process thus involves a mammalian cell culture step to produce the
protein of interest and a purification step followed by three enzymatic processing steps to yield a glycan structure that is appropriate
for therapeutic use.
Collectively, these examples are intended to support the view that
glycosylation plays an essential role in the function of many therapeutically relevant proteins and that controlling glycosylation can
be used to modulate and improve therapeutic function, or even
eliminate certain undesirable functions.
Because mammalian cells typically express a heterogenous mixture of glycoforms, several approaches have been developed to overcome this shortcoming including modulating glycosylation
pathways in mammalian host cells or using in vitro enzymatic methods to modify glycosylation after purification of the protein. For
example, Umana and coworkers at the Institute of Biotechnology
(ETH, Zurich) showed that CHO cells that overexpress UDPglucosaminyl transferase III, produce IgGs with increased bisecting
VOLUME 22 NUMBER 11 NOVEMBER 2004 NATURE BIOTECHNOLOGY
REVIEW
© 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology
GlcNAc and elevated antibody-dependent cell-mediated cytotoxicity (ADCC) activity14. Methods to engineer glycosylation in mammalian cells have been reviewed elsewhere15,16.
Yeast and fungal expression systems
The growing demand for improved biopharmaceutical expression
hosts has led several companies to return to yeast and fungal expression systems, which provide high protein titers (>1 g/l) in fermentation processes that last a few days, are scalable to the 100 m3 scale,
allow for rapid turnaround from gene to protein and offer an array
of genetic tools to manipulate the host organism. Decades of
research and the production of many industrial enzymes in both
yeast and fungi have resulted in robust, scalable processes that allow
the low-cost production of many recombinant enzymes. In addition, about a sixth of all currently approved therapeutics are made
in yeasts1, and thus fewer regulatory hurdles are expected when
comparing yeast-based production to entirely new expression platforms, such as transgenic animals or plants.
All yeast-based therapeutic proteins are currently produced in the
Baker’s yeast S. cerevisiae (Table 1), but other yeasts have been developed to make therapeutic proteins. The yeast P. pastoris was originally developed as a single-cell protein-production system by
Philips Petroleum (Bartlesville, OK, USA) but was subsequently
adapted for heterologous protein expression. More than 120 recombinant proteins have been expressed in this host—many of which
are of human or mammalian origin17. More recently, P. pastoris
has been used to express therapeutic proteins that have entered clinical trials (Table 1), although some proteins expressed in this host
have been shown to contain O-mannosylation not present on the
native protein18.
Genencor (Palo Alto, CA, USA) has used the filamentous fungi
Aspergillus niger and Trichoderma reesei for the large-scale production of recombinant industrial enzymes. Recent efforts to express
therapeutically relevant proteins have focused on the expression of
full-length IgGs in A. niger19. This antibody is produced at titers just
under 1 g/l, is correctly assembled and binds antigen. Ogunjimi
et al.20 have had similar success with the expression of intact antibody in P. pastoris; however, titers were below 40 mg/l. The structure
of the glycans added to the antibodies by A. niger and P. pastoris
are of the high-mannose type, as expected from fungi and yeasts.
Ward and colleagues19 demonstrated that the antibody produced
in A. niger had similar pharmacokinetic behavior and ADCC activity when compared with a mammalian cell–derived antibody,
although the glycan structures differed from mammalian glycosylation and not all glycosylation sites were occupied with glycans.
Although this is promising, concern remains that these nonhuman
aberrant sugar structures may be immunogenic in humans upon
long-term administration.
Berna Biotech, formerly Rhein Biotech (Bern, Switzerland), has
developed a proprietary protein expression platform based on the
methylotrophic yeast Hansenula polymorpha that is currently being
further developed for the production of recombinant vaccines.
Although yeast systems (e.g., H. polymorpha, S. cerevisiae and P. pastoris) are often ideal for producing high titers of recombinant proteins, their nonhuman glycosylation patterns have in the past
precluded them from consideration for the production of glycoproteins intended for therapeutic use in humans.
Humanizing glycosylation pathways in yeasts and fungi
Given the otherwise attractive attributes of yeasts and filamentous
fungi, several groups have investigated the possibility of humanizing
ER
(human, P. pastoris)
Golgi (human)
Man8B
Mns
IA, IB and IC
Golgi (P. pastoris)
1,6 MnT (Och1p)
High-mannose
glycans
Man5
GnTI
1,2 MnTs
1,6 MnTs
GlcNAcMan5
Man9
Hybrid
glycans
MnsII
Man9-14
GlcNAcMan3
β-1,N-GlcNAc
GnTII
β-1,4-GlcNAc
β-1,2-GlcNAc
β-1,4-Man
α-1,6-Man
α-1,2-Man
GlcNAc2Man3
α-1,3-Man
GalT
Complex
glycans
β-1,4-Gal
α-2,3-NANA/α-2,6-NANA
Gal2GlcNAc2Man3
ST
NANA2Gal2GlcNAc2Man3
Figure 2 Glycosylation pathways in humans and yeast. Mns, α-1,2mannosidase; MnsII, mannosidase II; GnT1, β-1,2-N-acetylglucosaminyltransferase I; GnTII, β-1,2-N-acetylglucosaminyltransferase II; GalT, β-1,
4-galactosyltransferase; ST, sialyltransferase; MnT, mannosyltransferase.
glycosylation pathways in these hosts to produce human-like glycoproteins. Early attempts to replace mammalian cell lines with fungal
expression hosts were based on the observation that the filamentous
fungus T. reesei was able to secrete glycoproteins containing intermediates of human glycosylation that were amenable to in vitro processing by mammalian glycosyltransferases21. In this approach, a
fungal precursor protein serves as a substrate for one or several
chemoenzymatic glycosylation steps that result in hybrid glycoproteins, albeit it at low yield22. Although encouraging, this approach
requires several in vitro glycosylation reactions, which are costly and
cumbersome, and it became apparent that a more attractive solution for high-titer production of human glycoproteins in yeast and
filamentous fungi would be to genetically engineer the human glycosylation pathway into the host organism itself.
Although conceptually straightforward, the technical complexity
of replicating human glycosylation pathways in fungal hosts is
daunting. First, any attempt to reengineer a protein expression host
NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 11 NOVEMBER 2004
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© 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology
REVIEW
to perform human glycosylation requires a
Table 1 Therapeutic proteins produced in the yeasts S. cerevisiae and P. pastoris
complete inventory of all glycosylation reactions known to occur in a given host, includProducts on the market
ing O-glycosylation (i.e., the glycome). By
Commercial name
Recombinant protein
Company
Expression system
the early 1990s, much was known about the
principal glycosylation pathways in humans
Actrapid
Insulin
NovoNordisk
S. cerevisiae
and several yeasts23,24. Whereas human
Ambirix
Hepatitis B surface antigen
GlaxoSmithKline
S. cerevisiae
N-glycosylation is of the complex type, built
Comvax
Hepatitis B surface antigen
Merck
S. cerevisiae
on a tri-mannose core extended with
GlcNAc, galactose, and sialic acid (Fig. 2),
Elitex
Urate oxidase
Sanofi-Synthelabo
S. cerevisiae
yeast N-glycosylation is of the highGlucagen
Glucagon
Novo Nordisk
S. cerevisiae
mannose type containing up to 100 or more
HBVAXPRO
Hepatitis B surface antigen
Aventis Pharma
S. cerevisiae
mannose sugars (hypermannosylation)25,26.
Hexavac
Hepatitis B surface antigen
Aventis Pasteur
S. cerevisiae
Early N-glycan processing, involving (i)
the assembly of the core oligosaccharide, (ii)
Infanrix-Penta
Hepatitis B surface antigen
GlaxoSmithKline
S. cerevisiae
its site-specific transfer onto the protein,
Leukine
Granulocyte-macrophage colony
Berlex
S. cerevisiae
and (iii) its trimming by glucosidase I, II
stimulating factor
and an endoplasmic reticulum (ER) resiNovolog
Insulin
Novo Nordisk
S. cerevisiae
dent α-1,2-mannosidase, is highly conPediarix
Hepatitis
B
surface
antigen
GlaxoSmithKline
S. cerevisiae
served in mammals and yeast. In fact, up to
the formation of the Man8 glycan intermeProcomvax
Hepatitis B surface antigen
Aventis Pasteur
S. cerevisiae
diate (Man8B) in the ER both pathways are
Refuldan
Hirudin/lepirudin
Hoechst
S. cerevisiae
identical (Fig. 2). However, following transRegranex
rh
Platelet-derived
growth
factor
Ortho-McNeil
Phama
S. cerevisiae
port of the protein to the Golgi apparatus,
(US), Janssen-Cilag (EU)
the two pathways diverge significantly. It is
Revasc
Hirudin/desirudin
Aventis
S. cerevisiae
at this juncture that mammalian cells rely
on additional α-1,2-mannosidases to trim
Twinrix
Hepatitis B surface antigen
GlaxoSmithKline
S. cerevisiae
mannose residues27, whereas yeasts initiate
Products under development
a series of mannosyltransferase reactions to
Protein
Indication
Company
Expression system
yield the above-mentioned hypermannosylated glycan structures. To date, all successAngiostatin
Antiangiogenic factor
EntreMed
P. pastoris
ful efforts to humanize yeast glycosylation
Elastase inhibitor
Cystic fibrosis
Dyax
P. pastoris
pathways have focused on the deletion of
specific yeast genes involved in hypermanEndostatin
Antiangiogenic factor
EntreMed
P. pastoris
nosylation, and the introduction of genes
Epidermal growth
Diabetes
Transition Therapeutics
P. pastoris
catalyzing the synthesis, transport, and
factor analog
addition of human sugars.
Insulin-like growth Insulin-like growth factor-1
Cephalon
P. pastoris
Schachter’s group28 at the University of
factor-1
deficiency
Toronto was the first to report an attempt
Human serum
Stabilizing blood volume in
Mitsubishi Pharma
P. pastoris
to introduce a mammalian glycosylation
albumin
burns/shock
(formerly Welfide)
step in the filamentous fungus A. nidulans
Kallikrein inhibitor Hereditary angiodema
Dyax
P. pastoris
by expressing β-1,4-N-acetylglucosaminyl1
Includes approved products and those in clinical development. Sources: (G. Walsh, 2003) or personal
transferase I (GnTI), the key enzyme
communication.
responsible for the formation of hybrid glycans in mammals. However, although high
transcription levels of the enzyme were
observed, and active enzyme could be assayed in cell-free extracts, the ER, some Man5 N-glycans could be generated on an intracellular
no impact on the N-glycans was observed28.
reporter protein30. However, the paucity of Man5 obtained (less
One of the critical steps in obtaining complex glycoproteins in a than 30% of total glycans), despite the expression of an α-1,2fungal host requires the generation of Man5 in high yields, because mannosidase from a multicopy plasmid (yeast 2µ) and a strong conthis substrate is the precursor for all subsequent downstream stitutive promotor (glyceraldehyde-3-phosphate, GAPDH), was of
processing steps (Fig. 2). Early attempts to engineer active α-1,2- concern. These initial attempts in fungi and yeasts showed some
mannosidases into yeasts were rather discouraging, mostly because promise; however, they also revealed that genetically humanizing
P. pastoris appeared to compensate for mannose trimming by upreg- glycosylation pathways in these hosts was not trivial and that merely
ulating mannosyltransferase activity leading to even higher molecu- expressing enzymes involved in mammalian glycosylation pathways
lar weight glycan structures29. Jigami’s group30 at Japan’s National was unlikely to be successful.
Institute of Bioscience and Human Technology (Tsukuba, Ibaraki,
Japan), in collaboration with researchers at Kirin (Yokohama, The secretory pathway is a cellular assembly line
Japan), showed that after systematically eliminating yeast-specific In yeasts and humans, host-specific glycosyltransferases and glyglycosylation reactions by deleting och1, mnn1 and mnn4 in the cosidases line the luminal surface of the ER and Golgi apparatus and
yeast S. cerevisiae, and expressing an α-1,2-mannosidase targeted to thereby provide catalytic surfaces that allow the sequential process-
1412
VOLUME 22 NUMBER 11 NOVEMBER 2004 NATURE BIOTECHNOLOGY
Intensity (%)
Intensity (%)
Intensity (%)
ing of glycoproteins as they proceed from
β-1,N-GlcNAc
β-1,4-Man
α-1,6-Man
α-1,3-Man
β-1,4-Gal
the ER through the Golgi network. As a glycoprotein proceeds from the ER through the
a
b 100
secretory pathway, it is sequentially exposed
100
to different mannosidases and glycosyl75
75
transferases. An essential step in recreating
50
human glycosylation pathways in lower
50
eukaryotes is recreating the sequential
25
Man5
25
nature of complex N-glycan processing. In
0
2000, I initiated a research program with
0
850
1,350
1,850
2,350
2,850
850
1,350
1,850
2,350
2,850
3,350
three main focal points: (i) overcoming the
Mass (m/z)
Mass (m/z)
uncertainty related to the targeting of hetc
d
erologous glycosylation enzymes across dif100
100
ferent species and developing methods to
75
75
target enzymes to predetermined locations
in the secretory pathway of yeast and fila50
50
mentous fungi, (ii) developing methods to
GlcNAcMan3
GlcNAc2Man3
25
25
identify enzymes that are in fact active at the
site of localization (since the different
0
0
850
1,350
1,850
2,350
2,850
850
1,350
1,850
2,350
2,850
organelles of the yeast secretory pathway
Mass (m/z)
Mass (m/z)
provide different processing environments
with different pHs and sugar nucleotide
pools, one has to consider how a recombi- Figure 3 MALDI-TOF mass spectrometry of oligosaccharides released from purified kringle 3 protein
nant enzyme targeted to a particular produced in wild-type P. pastoris and glycoengineered strains of P. pastoris. (a–d) MALDI-TOF mass
organelle will behave in that nonnative envi- spectra positive mode: Wild type (a), and increasingly humanized glycan structures (see ref. 31, (b);
ref. 34, (c); and ref. 35, (d)).
ronment), and (iii) develop efficient screens
to identify cell lines that have acquired the
ability to process recombinant glycoproteins
in a human-like fashion.
by the elimination of two genes associated with the transfer of this
By 2003, my team had shown that many of the problems previ- sugar32. We have expressed several recombinant proteins in these
ously encountered by other researchers could be overcome by gener- strains and have found expression levels to be similar to wild-type
ating large-scale combinatorial genetic libraries of glycosylation yeast. Recent work by Contreras’ group33 at Ghent University
enzymes fused to libraries of yeast-targeting peptides31. By screen- (Belgium) has also shown the in vivo production of nonsialylated
ing a library of over 600 fusion constructs consisting of catalytic hybrid N-linked glycans in a glycoengineered strain of P. pastoris.
After the in vivo production of hybrid N-glycans, we have gone on
domains of α-1,2-mannosidases, and yeast type II leader peptides,
we were able to find several specific combinations of catalytic to further humanize the glycosylation of P. pastoris to perform
domains and leader peptides that displayed efficient mannosidase glycosylation of the complex type34. By successfully introducing
activity in vivo in P. pastoris. Screening was done by purifying a α-mannosidase II and GlcNAc-transferase II (GnTII) activities, folrecombinant reporter protein secreted by each strain, and analyzing lowing the combinatorial library approach discussed above, we were
its glycans by digestion with protein N-glycanase followed by able to generate yeast strains that perform essentially homogenous
matrix-assisted laser desorption ionization time-of-flight (MALDI- glycosylation containing GlcNAc2Man3 (Fig. 3c).
TOF) mass spectrometry. The entire procedure of purification and
More recently, we have demonstrated the proper localization of
analysis was carried out by an automated liquid handler allowing active β-1,4-galactosyltransferase (GalT) and UDP-galactose-4the processing of several thousand samples per week.
epimerase in P. pastoris conferring the transfer of galactose onto
Glycoproteins expressed in these engineered strains contain over both terminal GlcNAc residues of a GlcNAc2Man3 glycan, resulting
90% Man5 and thus provided the basis for the further humanization in the production of Gal2GlcNAc2Man3 in vivo35 (Fig. 3d). This has
of glycosylation pathways in P. pastoris. After the successful genera- rendered the humanization of P. pastoris one step away from comtion of Man5, we showed that by introducing an active GnTI gene pletion. Unfortunately, the final transfer of sialic acid will be most
and a UDP-GlcNAc transporter, we were able to add a terminal challenging, as unlike GlcNAc and galactose, a source of endogeGlcNAc residue to the 1,3 arm of the Man5 glycan, thereby generat- nous sialic acid is not known to exist in yeasts or other fungal organing the first genetically engineered yeast that efficiently produces isms. Thus, in addition to the requirement for a sialyltransferase
hybrid N-glycans. As in the mannosidase case, active GnTI was enzyme, several other genes involved in the synthesis of CMP-sialic
identified by introducing a GnTI catalytic domain/Golgi leader acid must also be genetically engineered into the host. We have
library into the yeast and screening for transformants that acquired recently shown that the precursor CMP-sialic acid, can be produced
the ability to produce GlcNAcMan5 at high efficiency. After knock- in P. pastoris and used to transfer sialic acid onto a secreted reporter
ing out the initiating α-1,6-mannosyltransferase activity, and intro- protein (Gerngross, T.U., unpublished data).
Unlike proteins obtained from mammalian cell culture, which are
ducing α-1,2-mannosidase, GnT1 and the UDP-GlcNAc transporter
into P. pastoris we were able to show the secretion of a reporter heterogenous, we have found that yeast cells can be engineered to
protein with over 90% of the N-glycans have the desired secrete glycoproteins with exceptional glycan uniformity31,34,35.
GlcNAcMan5 structure (Fig. 3b)31. The remaining glycans contain Although yeast-based manufacturing is expected to reduce the
mostly mannosylphosphate, which can be eliminated in P. pastoris production cost of protein-based therapeutics, we believe the main
Intensity (%)
© 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology
REVIEW
NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 11 NOVEMBER 2004
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REVIEW
© 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology
benefit of using glycoengineered yeast lies in the ability to rigorously
control glycosylation of the final product and thereby improve clinically relevant characteristics such as half-life or tissue distribution.
Using different yeast strains, a protein can be expressed in its various glycoforms (Fig. 3) and glycosylation-mediated functions can
be elucidated, provided an appropriate assay can be developed. Most
importantly, once a given glycoform has been identified, scaling-up
and manufacturing are done on the same platform allowing the
large-scale production of specific glycoforms.
The future of humanized yeast
With the advent of humanized yeast strains and their ability to exercise direct control over glycosylation, we see the advantages of
mammalian cell culture fading, and expect a significant number of
biopharmaceuticals to be switched to yeast-based expression platforms. The short turn-around time to obtain gram quantities of a
therapeutic protein (typically 4–6 months for a purified protein),
combined with the demonstrated scalability of yeast offer an attractive alternative to current mammalian cell culture processes.
Although this is expected to reduce the cost of biomanufacturing,
we believe that the main benefit of using glycoengineered yeast
strains lies in the ability to rigorously control glycosylation of the
final product and thereby improve clinically relevant properties of
the molecule. Instead of glycoform mixtures produced by mammalian cell culture processes, it is now possible to use glycoengineered yeast strains to produce a glycoprotein with a single type of
N-glycosylation (Fig. 3b–d). Most cost-of-goods analyses focus on
the cost required to produce a given amount of recombinant protein. What is often absent in this analysis is the quality of the final
product, which can have an impact on dosage requirements and
therapeutic effect and ultimately market share. Considering the
ability of yeast systems to express full antibodies20, cytokines, serum
proteins and other therapeutically relevant proteins, we expect
yeast-based therapeutic protein expression to become a serious contender in any manufacturing strategy.
COMPETING INTERESTS STATEMENT
The authors declare competeing financial interests (see the Nature Biotechnology
website for details).
Published online at http://www.nature.com/naturebiotechnology/
1. Walsh, G. Biopharmaceutical Benchmarks—2003. Nat. Biotechnol. 21, 865–870
(2003).
2. Punt, P.J., van Biezen, N. Conesa, A., Albers, A., Mangnus, J. & van den Hondel, C.
Filamentous fungi as cell factories for heterologous protein production. Trends
Biotechnol. 20, 200–206 (2002)
3. Werten, M.W.T., van den Bosch, T. J., Wind, R.D., Mooibroek, H. & De Wolf, F.A.
High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15,
1087–1096 (1999).
4. Humphreys, A. & Boersig, C. Cholesterol drugs dominate. MedAdNews 22, 42–57
(2003).
5. Coloma, M.J., Clift, A., Wims, L. & Morrison, S.L. The role of carbohydrate in the
assembly and function of polymeric IgG. Mol Immunol. 37, 1081–1090 (2000).
6. Mistry, P.K., Wraight, E.P. & Cox, T.M. Therapeutic delivery of proteins to
macrophages: implications for treatment of Gaucher’s disease. The Lancet, 348,
1555–1559 (1996).
7. Grabowski, G.A. et al. Enzyme therapy in type 1 Gaucher disease: comparative efficacy of mannose-terminated glycocerebrosidase from natural and recombinant
sources. Ann. Intern. Med. 122, 33–39 (1995).
8. Grabenhorst, E., Schlenke, P., Pohl, S., Nimtz, M. & Conradt, H.S. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host
1414
cells. Glyconj. J. 16, 81–97 (1999).
9. Suzuki, T., Kitajima, K., Inoue, S. & Inoue, Y. N-glycosylation/deglycosylation as a
mechanism for the post-translational modification/remodification of proteins.
Glycoconj J. 12, 189–193 (1995).
10. Takeuchi, M. et al. Relationship between sugar chain structure and biological activity
of recombinant human erythropoietin produced in Chinese hamster ovary cells. Proc.
Natl. Acad. Sci. USA. 86, 7819–7822 (1989).
11. Yuen, C.T. et al. Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures. Br. J. Haematol. 121, 511–526 (2003).
12. Erbayraktar, S. et al. Asialoerythropoietin is a nonerythropoietic cytokine with broad
neuroprotective activity in vivo. Proc. Natl. Acad. Sci. USA 100, 6741–6746 (2003).
Erratum in: Proc. Natl. Acad. Sci. USA 100, 9102 (2003).
13. Friedman, B. et al. A comparison of the pharmacological properties of carbohydrate
remodeled recombinant and placental-derived beta-glucocerebrosidase: implications
for clinical efficacy in treatment of Gaucher disease. Blood 93, 2807–2816 (1999).
14. Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H. & Bailey, J.E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular
cytotoxic activity. Nat. Biotechnol. 17, 176–180 (1999).
15. Warner, T.G. Enhancing therapeutic glycoprotein production in Chinese hamster ovary
cells by metabolic engineering endogenous gene control with antisense DNA and
gene targeting. Glycobiology 9, 841–850 (1999).
16. Warner, T.G. Metabolic engineering glycosylation: biotechnology’s challenge to the
glycobiologist in the next millennium. in Part IV Biology of Saccharides. (eds. Ernst,
B., Hart, G. & Sinay, P.) 1043–1064 (VCH-Wiley, New York, 2001).
17. Cregg, J.M., Cereghino, J.L., Shi, J. & Higgins, D.R. Recombinant protein expression
in Pichia pastoris. Mol. Biotechnol. 16, 23–52 (2000).
18. Mochizuki, S. et al. Expression and characterization of recombinant human
antithrombin III in Pichia pastoris. Protein Expr. Purif. 23, 55–65 (2001).
19. Ward, M. et al. Characterization of humanized antibodies secreted by Aspergillus
niger. Appl. Environ. Microbiol. 70, 2567–2576 (2004).
20. Ogunjimi, A.A., Chandler, J.M., Gooding, C.M. Recinos, A. & Choudary, P.V. Highlevel secretory expression of immunologically active intact antibody from the yeast
P. pastoris. Biotechnol. Letters, 21, 561–567 (1999).
21. Maras, M. & Contreras, R. Methods for modifying carbohydrate moieties. US patent
5,834,251 (Dec. 30, 1994).
22. Maras, M. et al. In vitro conversion of the carbohydrate moiety of fungal glycoproteins
to mammalian-type oligosaccharides. Eur. J. Biochem. 249, 701–707 (1997).
23. Kornfeld, R. & Kornfeld, S. Assembly of asparagine-linked oligosaccharides. Annu.
Rev. Biochem. 54, 631–664 (1985).
24. Brockhausen, I., Carver, J.P. & Schachter, H. Control of glycoprotein synthesis.
The use of oligosaccharide substrates and HPLC to study the sequential pathway for
N-acetylglucosaminyltransferases I, II, III, IV, V and VI in the biosynthesis of highly
branched N-glycans by hen oviduct membranes. Biochem. Cell Biol. 66, 1134–1151
(1988).
25. Silberstein, S. & Gilmore, R. Biochemistry, molecular biology and genetics of
oligosaccharyltransferase. FASEB J. 10, 849–858 (1996).
26. Gemmill, T.R. & Trimble, R.B. Overview of N- and O-linked oligosaccharide structures
found in various yeast species. Biochim. Biophys. Acta 1426, 227–237 (1999).
27. Tulsiani, D.R., Hubbard, S.C., Robbins, P.W. & Touster, O. Alpha-D-mannosidases of
rat liver Golgi membranes. Mannosidase II is the GlcNAcMan5-cleaving enzyme in
glycoprotein biosynthesis and mannosidases IA and IB are the enzymes converting
Man9 precursors to Man5 intermediates. J. Biol. Chem. 257, 3660–3668 (1982).
28. Kalsner, I., Hintz, W., Reid, L.S. & Schachter, H. Insertion into Aspergillus nidulans
of functional UDP-GlcNAc: α3-D-mannoside β-1,2-N-acetyl-glucosaminyltransferase
I, the enzyme catalyzing the first committed step from oligomannose to hybrid and
complex N-glycans. Glycoconj. J. 12, 360–370 (1995).
29. Martinet, W., Maras, M., Saelens, X., Jou, W.M. & Contreras, R. Modification of the
protein glycosylation pathway in the methylotrophic yeast, Pichia pastoris.
Biotechnol. Letters 20, 1171–1177 (1998).
30. Chiba, Y. et al. Production of human compatible high mannose-type (Man5GlcNAc2)
sugar chains in Saccharomyces cerevisiae. J. Biol. Chem. 273, 26298–26304
(1998).
31. Choi, B.K. et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. USA 100, 5022–5027
(2003).
32. Stadheim, T.A., Bobrowicz, P., & Wildt, S. Methods for eliminating mannosylphosphorylation of glycans in the production of glycoproteins. US Patent Appl. No.
60/532,461 (December 23, 2003).
33. Vervecken, W. et al. In vivo synthesis of mammalian-like, hybrid-type N-glycans in
Pichia pastoris. Appl Environ Microbiol. 70, 2639–2646 (2004).
34. Hamilton, S.R. et al. Production of complex human glycoproteins in yeast. Science,
301, 1244–1246 (2003).
35. Bobrowicz, P. et al. Engineering of an artificial glycosylation pathway blocked in core
oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology 14, 757–766 (2004).
VOLUME 22 NUMBER 11 NOVEMBER 2004 NATURE BIOTECHNOLOGY
GlycanEngineeringforCellandDevelopmentalBiologyGriffinME
andHsieh-WilsonLCCellChemBiol,23:108(2016)
Please cite this article as: Griffin and Hsieh-Wilson, Glycan Engineering for Cell and Developmental Biology, Cell Chemical Biology (2016), http://
dx.doi.org/10.1016/j.chembiol.2015.12.007
Cell Chemical Biology
Review
Glycan Engineering for Cell
and Developmental Biology
Matthew E. Griffin1 and Linda C. Hsieh-Wilson1,*
1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.chembiol.2015.12.007
Cell-surface glycans are a diverse class of macromolecules that participate in many key biological processes, including cell-cell communication, development, and disease progression. Thus, the ability to
modulate the structures of glycans on cell surfaces provides a powerful means not only to understand
fundamental processes but also to direct activity and elicit desired cellular responses. Here, we describe
methods to sculpt glycans on cell surfaces and highlight recent successes in which artificially engineered
glycans have been employed to control biological outcomes such as the immune response and stem
cell fate.
Introduction
Cell-surface glycans participate in many important processes
throughout the lifespan of an organism, ranging from cell migration and tissue patterning to the immune response, disease progression, and cell death (Fuster and Esko, 2005; Häcker et al.,
2005; Lichtenstein and Rabinovich, 2013; van Kooyk and Rabinovich, 2008). At a molecular level, glycans are often the first
points of contact between cells, and they function by facilitating
a variety of interactions both in cis (on the same cell) and in trans
(on different cells). The glycan covering that surrounds the cell
surface, termed the glycocalyx, can both promote and hinder
the binding of canonical protein ligands to their cell-surface receptors, as well as mediate ligand-independent receptor clustering and activation (Bishop et al., 2007; Coles et al., 2011;
Haines and Irvine, 2003; Rogers et al., 2011). Indeed, the integral
roles of cell-surface glycans in regulating cellular signaling
events are only beginning to be understood.
The ability to modulate and re-engineer the diverse structures
of glycans at the cell surface provides a powerful means to elucidate the molecular mechanisms that underlie glycan-mediated
signaling events and their downstream cellular consequences.
From a mechanistic standpoint, systematically altering glycan
structures provides insights into structure-function relationships
and the importance of individual structures in glycan-mediated
processes. From an engineering standpoint, the ability to
remodel glycan architectures on cell surfaces offers a novel
approach to manipulate cellular physiology and phenotypic outcomes. Here, we will describe the methods available to tailor the
structures of glycans on cells and provide notable examples of
how remodeling of cell-surface glycans have led to both new
biological insights and novel cellular functions.
Genetic Approaches
Genetic manipulation of glycosyltransferases (GTs) or other
genes involved in glycan biosynthesis provides a powerful
method to perturb specific glycan subpopulations in cells and
organisms. A variety of approaches have been developed,
including gene deletion or knockout, gene knockdown by
RNAi, and gene overexpression (Figure 1). Genetic methods
offer excellent spatial and temporal control, enabling the pre-
cise manipulation of specific genes in a cell-specific and inducible manner. However, as GTs typically operate on multiple protein substrates and assemble a variety of glycan structures, it is
difficult to study the impact of a single glycan on an individual
protein of interest. Moreover, genetic approaches usually subtract from existing glycan structures rather than add new
chemical functionalities. Finally, knocking out GTs can lead to
developmental defects or embryonic lethality, which can hinder
the identification of functions in the adult organism. Nonetheless, genetic approaches have provided invaluable information
on the functional importance of GTs and protein glycosylation
in vivo.
The power of genetic approaches is exemplified by elegant
studies on the Notch signaling pathway. Notch signaling is
essential for proper development, and dysregulation of the
pathway leads to various human diseases, including congenital
disorders and cancer (Andersson et al., 2011; Kopan and Ilagan, 2009). Glycosylation of the extracellular domain of the
Notch receptor has emerged as an important mechanism for
the regulation of Notch activity (Figure 2A). Early studies identified a b-1,3-N-acetylglucosaminyltransferase called Fringe
that modifies O-linked Fuc residues on Notch (Brückner
et al., 2000; Moloney et al., 2000). Ectopic expression of a catalytically inactive, mutant form of Fringe in Drosophila demonstrated that the GT activity of Fringe was required for certain
Notch ligand-receptor interactions and proper wing formation
in vivo (Moloney et al., 2000). Other GTs have also been shown
to be critical for Notch signaling. For example, protein O-fucosyltransferase 1 (Pofut1 in mammals or Ofut1 in Drosophila)
catalyzes the addition of O-Fuc to Notch, generating the
acceptor substrate for Fringe (Wang et al., 2001). RNAi-mediated knockdown of Ofut1 showed that the enzyme is required
for both Fringe-dependent and Fringe-independent Notch function in Drosophila (Okajima and Irvine, 2002). In mice, genetic
deletion of Pofut1 results in embryonic lethality with phenotypic
defects in cardiovascular and neurologic development, consistent with loss of all Notch paralog signaling (Shi and Stanley,
2003). In addition, inducible inactivation of Pofut1 in the immune-responsive cells and hematopoietic tissues of adult
mice revealed a key role for O-Fuc glycans on Notch in the
108 Cell Chemical Biology 23, January 21, 2016 ª2016 Elsevier Ltd All rights reserved
Please cite this article as: Griffin and Hsieh-Wilson, Glycan Engineering for Cell and Developmental Biology, Cell Chemical Biology (2016), http://
dx.doi.org/10.1016/j.chembiol.2015.12.007
Cell Chemical Biology
Review
A
RNAi
x
GT
GT
GT
GT
protein
expression
GT
GT
GT cDNA
GT
GT
B
offspring
breeding
mutated
stem cells
blastocyst
injection
pregnant
mouse
stable
knockout line
Figure 1. Genetic Approaches
(A) Transient transfection of cells in vitro with either
shRNA or siRNA (RNAi) targeting a gene causes a
decrease in expression of the protein. Transfection
with the cDNA of a protein leads to its overexpression.
(B) Mouse knockout models are traditionally produced by first stably disrupting gene expression in
mouse embryonic stem cells by homologous
recombination. Mutated stem cells are injected into
blastocysts, which are then implanted into mice to
produce mosaic offspring. Mice are further bred to
produce a stable knockout line.
(C) To generate stable CHO knockout cell lines,
parental cells were treated with a mutagenic agent
(e.g., ionizing radiation) and then selected for
resistance to cytotoxic lectins, which bind to specific carbohydrate epitopes now missing on the
mutated cells.
C
mutagenesis
parental cells
regulation of hematopoietic homeostasis (Yao et al., 2011).
Together, these genetic studies demonstrate the essential nature of glycosylation for Notch receptor function.
Mass spectrometry analysis indicated that the O-Fuc glycans
on mammalian Notch exist as a mixture of di-, tri-, and tetrasaccharides of NeuAca(2–3)Galb(1–4)GlcNAcb(1–3)Fuc (Moloney et al., 2000). To decipher the minimum structure necessary
for activity, a library of mutant Chinese hamster ovary (CHO)
cell lines deficient in GTs and activated nucleotide (NXP)-sugar
transporters was employed (Figures 1C and 2B) (Chen et al.,
2001). Expression of Fringe in parental CHO cells led to a
decrease in Jagged1 stimulation of Notch, presumably due to
the loss of Jagged1-Notch complexation mediated by the
O-Fuc glycan. However, the Lec8 cell line, which contains mutations in the uridine diphosphate (UDP)-Gal Golgi transporter
that result in defective protein galactosylation, showed no
change in Notch activation upon Fringe expression, suggesting
that the Gal moiety was required to disrupt the Jagged1-Notch
interaction. Conversely, the Lec2 cell line containing an inactive
cytidine monophosphate (CMP)-NeuAc Golgi transporter that
prevents protein sialylation showed the same response to Jagged1 stimulation as the parental cell line, indicating that the
NeuAc residue was unnecessary. Of the six mammalian
b-1,4-galactosyltransferase (b4GalT) enzymes that transfer
Gal to GlcNAc in CHO cells, only b4GalT-1 was required for
Notch stimulation, providing the identity of a key enzyme
involved in synthesizing the minimum trisaccharide. Thus, genetic approaches not only illustrate the necessity of specific
glycans for activity in physiological systems but also can be
systematically used to determine their structure and the enzymes required for their biosynthesis.
Another example of the power of genetic
approaches to reveal important developselection
mental functions of glycans involves
studies on heparan sulfate (HS) glycosamimutated
noglycans (GAGs). HS binds to diffusible
cell line
chemical signals known as morphogens
and aids in establishing gradients of
these secreted proteins to relay positional
information in developing organisms
(Figure 3A) (Häcker et al., 2005). Phenotypic screens in Drosophila showed that mutations in tout-velu
(ttv), a homolog of the mammalian HS polymerase Ext1, caused
pattern defects in the wing imaginal disc similar to the loss of
Hedgehog (Hh) signaling (Bellaiche et al., 1998; The et al., 1999).
The morphogen Hh specifies anterior-posterior orientation during
wing development by diffusing from the posterior compartment of
the imaginal disc to anterior cells in a graded fashion (Tabata and
Kornberg, 1994). To elucidate the importance of ttv, mosaic
knockout clones were produced in which defined regions of the
Drosophila embryo lacked ttv (Bellaiche et al., 1998). Loss of ttv
in the posterior compartment where Hh is secreted showed no effect on the distance of Hh-mediated activation into the anterior
compartment. However, anterior ttv / clones showed a severe
spatial attenuation of active Hh signaling in the anterior compartment. Defective distribution of Hh signaling was also observed in
mutant clones of the Ext2 homolog sister-of-tout-velu (sotv)
and the Extl3 homolog brother-of-tout-velu (botv) (Han et al.,
2004), suggesting that the HS produced by these enzymes is
necessary for Hh binding, gradient formation, and signal transduction. Furthermore, wingless (Wg) and decapentaplegic (dpp)
morphogen signaling were shown to be disrupted by altered HS
biosynthesis and display (Baeg et al., 2004; Belenkaya et al.,
2004; Kirkpatrick et al., 2004), providing evidence for a general
mechanism by which HS mediates morphogen gradients.
Interestingly, ostensibly conflicting data were obtained from
mouse models (Koziel et al., 2004). Here, hypomorphic mutation
of Ext1, which severely limits HS production, led to an extended
tissue distribution of Indian hedgehog (Ihh), the mouse homolog
of Hh, during endochondral ossification (Figure 3B). Using haploinsufficiency studies in which one or both alleles of Ext1 were
replaced with the hypomorphic variant, lower production of HS
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Figure 2. Genetic Studies of Notch
A
B
correlated with higher rates of proliferation in a region of chondrocytes distal from Ihh secretion, implying increased Ihh
diffusion. In vitro growth of embryonic mouse forelimbs with
exogenous GAGs showed that Ihh diffusion was inversely proportional to HS concentration, suggesting that HS retarded the
diffusion of Ihh, unlike the results seen for ttv in Drosophila. It
has been proposed that the disparity in these results is due to differences in the genetic approaches used (Häcker et al., 2005). As
HS is required for ligand-receptor binding, the use of a null allele
for ttv prohibits all HS biosynthesis and Hh-mediated signaling.
In contrast, the hypomorphic Ext1 allele produces enough HS
to elicit Ihh-protein complexation, and the lower overall amount
of HS may facilitate further morphogen diffusion. Thus, complete
ablation of HS in Drosophila decreases Hh signaling due to a loss
of ligand-receptor binding, but the decrease of HS in mice increases Ihh signaling by allowing greater diffusion of Ihh. The
interpretation of these results highlights that careful consideration is necessary in selecting genetic models due to the multifaceted nature of glycan-protein interactions in vivo.
The examination of naturally occurring mutations in human
genes provides a complementary approach to establish the molecular determinants that underlie development and diseaserelated phenotypes. Human genetic mutations that lead to
abnormal glycosylation have been well documented and span
a wide clinical spectrum, impacting nearly every organ system
in the body. Advances in next-generation DNA sequencing technologies have helped to propel the discovery of >100 such
congenital disorders of glycosylation (CDGs), providing unique
opportunities to understand the significance of glycosylation in
human physiology. As CDGs have been well reviewed (Freeze,
2013; Hennet and Cabalzar, 2015), we highlight only a few representative examples.
(A) Structure of the O-fucose glycan found on
mammalian Notch.
(B) The O-fucose glycan of Notch causes a
decrease in Jagged1-mediated stimulation. By
eliminating specific monosaccharides on the Notch
glycan using genetic methods, it was found that a
trisaccharide was the minimum structure necessary to decrease Jagged1 activity.
Similar to classical forward genetics,
genetic mapping of disease-related mutations has led to the discovery of novel
genes responsible for a single set of pathological symptoms. Hereditary multiple
exostoses (HME), a condition characterized by abnormal growths at the ends of
long bones, has been associated with
loss-of-function mutations in the Ext1,
Ext2, and Ext3 genes by chromosomal
linkage analysis (Cook et al., 1993; Le
Merrer et al., 1994; Wu et al., 1994). These
genes were later determined to encode
GTs responsible for HS biosynthesis (McCormick et al., 1998). Missense mutations
that were mapped for Ext1 were shown to
cause a deficiency in HS production. As
described above, decreased levels of HS results in increased
activation of Ihh (Koziel et al., 2004). This signaling dysregulation
triggers uncontrolled cellular growth and leads to cartilaginous
abscesses and benign tumors in endochondrial bone. Thus,
this particular example highlights how the mutational profile of
a disease enhanced an understanding of the molecular basis
of exostoses formation.
Genetic mapping of distinct but related disease states identified multiple important genes that are components of the same
glycosylation pathway. Mutations in at least ten enzymes have
been linked to congenital muscular dystrophies (Wells, 2013),
which are commonly associated with defective O-glycosylation
of a-dystroglycan, an integral component of muscle fibers
(Figure 4). The O-glycans of a-dystroglycan mediate interactions
with extracellular matrix proteins such as laminin (Yoshida-Moriguchi et al., 2010) and facilitate the formation of compact basement membranes and mature neuromuscular junctions (Goddeeris et al., 2013). Loss of these glycans disrupts extracellular
matrix organization and predisposes muscular tissue to dystrophy. Walker-Warburg syndrome and muscle-eye-brain disease,
two similar types of muscular dystrophy typified by anomalies of
the brain and eye, were linked to a number of proteins that
produce O-mannosyl glycans, including protein O-mannosyltransferase 1 and 2 (POMT1/2) (Beltran-Valero de Bernabé
et al., 2002; van Reeuwijk et al., 2005), protein O-mannosyl N-acetylglucosaminyltransferase 1 and 2 (POMGnT1/2) (Manzini et al.,
2012; Yoshida et al., 2001), b-1,3-N-acetylgalactosaminyltransferase 2 (b3GalNT2) (Stevens et al., 2013), and protein O-mannosyl kinase (POMK) (Yoshida-Moriguchi et al., 2013). Merosin-deficient congenital muscular dystrophy 1D (MDC1D) was associated
with mutations in like-acetylglucosaminyltransferase (LARGE)
(Longman et al., 2003), which transfers GlcAb(1–3)Xyla(1–3)
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A
wild-type
HSPGs
posterior
anterior
ttv-/-
posterior
anterior
B
Figure 4. O-Mannosyl Glycan of a-Dystroglycan
The laminin-binding glycan of a-dystroglycan contains a core trisaccharide
produced by POMT1/2, POMGnT2, and b3GalNT-2. The 6-OH position of
mannose is phosphorylated by POMK, on which an unknown structure is
added. b4GAT-1 makes an acceptor substrate for LARGE, which adds
repeating GlcAb(1–3)Xyla(1–3) units. The required roles of other associated
enzymes such as FKTN are still unknown.
Ext1Gt/Gt
prehypertrophic
hypertrophic
Figure 3. Genetic Studies Provide Insights into How HS Glycans
Control Morphogen Gradients
(A) In wild-type Drosophila, Hedgehog ligands (Hh, orange) are secreted from
posterior cells of the imaginal wing disc and diffuse to anterior cells. HS chains
(light green) on HS proteoglycans (HSPGs, dark green) displayed on the cell
surface trap these ligands for cellular signaling and prevent further diffusion.
However, the lack of HS in ttv / Drosophila prevents Hh recruitment to the
cell surface and downstream signaling.
(B) Mice with a hypomorphic mutation in Ext1 (Ext1Gt/Gt) produced less HS on
the cell surface. When Indian hedgehog ligands (Ihh, blue) were secreted by
prehypertrophic chondrocytes, the reduced amount of cell-surface HS allowed Ihh to diffuse further into the hypertrophic region. Unlike full knockout as
seen in the ttv / Drosophila, chondrocytes with a hypomorphic mutation in
Ext1 maintained enough cell-surface HS to transduce signaling, but not
enough to impede diffusion.
repeating units onto the O-mannosyl glycan (Goddeeris et al.,
2013). Finally, patients with Fukuyama-type congenital muscular
dystrophy, a frequent disorder in Japan that presents as brain
micro-polygria due to improper neuronal migration, contained
mutations in a novel gene called fukutin (FKTN) (Kobayashi
et al., 1998). Although its exact function remains unknown,
FKTN was postulated to be a glycosyltransferase and was
found to be required for LARGE activity (Ohtsuka et al., 2015).
Thus, an examination of these and other a-dystroglycanopathies
converged on genes involved in the synthesis of the a-dystrogly-
can O-mannosyl glycan, linking the biosynthetic pathway of a
complex glycan to a suite of developmental disorders.
Next-generation sequencing has allowed for both targeted
and untargeted screens to identify genes involved in the etiology
of glycosylation-related diseases. Targeted sequencing of 25
known CDG-related genes uncovered no observable mutations
in a patient with widespread mental and physical developmental
delays (Jones et al., 2012). Subsequent untargeted wholeexome sequencing against roughly 22,000 genes revealed two
mutations that caused premature truncation of DDOST, a
subunit of the oligosaccharyltransferase (OST) complex. The
DDOST mutations impaired cell-surface expression of intracellular adhesion molecule 1 (ICAM-1), whose trafficking to the
cell surface depends on proper N-glycosylation. Complementation with wild-type DDOST partially restored ICAM-1 trafficking,
indicating that the DDOST mutations were indeed pathogenic
and that their clinical manifestations were likely due to widespread defects in N-glycosylation. Untargeted genetic approaches have also revealed unanticipated genes important for
proper glycosylation. Microarray and sequencing analysis of
genes within autozygous regions of two glycosylation-deficient
siblings with multiple forms of dysplasia uncovered mutations
in TMEM165 (Foulquier et al., 2012). This protein has no known
function but is implicated in maintaining calcium levels within
the Golgi (Demaegd et al., 2013), suggesting that proper glycosylation relies on not only GTs and transporters but also
homeostatic regulation of Golgi pH and ion concentrations.
The identification of DDOST and TMEM165 mutations by these
methods illustrates the ability of next-generation sequencing to
reveal pathologies caused by glycosylation defects and potentially expose novel regulatory mechanisms of glycosylation as
targets for therapeutic intervention.
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Figure 5. Metabolic Oligosaccharide Engineering
MOE requires the passage of peracetylated glycans across the cell membrane and into the cell, where they are deacetylated by endogenous esterases. The
monosaccharides are phosphorylated and activated with a nucleotide phosphate group. Glycosyltransferases then incorporate the non-natural sugars into
nascent glycans. The functional handle (X) installed by the biosynthetic machinery can be reacted with other groups (Y) via bioorthogonal chemistry (X + Y
produce Z) for detection, imaging, or introduction of novel biological activity.
Thus, genetic methods provide a powerful means to understand the importance of glycosylation in development and
disease. However, knockout, knockdown, and overexpression
approaches result in loss of glycan structures or enhanced
expression of only natural structures. A complementary
approach is the use of chemical biology methods that enable
expansion past the confines of naturally occurring glycans
through incorporation of non-natural sugars. These non-natural
labeling approaches have facilitated the imaging, tracking, and
identification of glycan structures. Beyond advancing the study
of glycans, such approaches provide a novel way to control
cellular physiology and function by altering the glycan structures
presented on the cell surface. Below, we highlight a few notable
examples whereby cell-surface glycan engineering has been exploited to interrogate and control glycan-mediated biological
processes.
Metabolic Oligosaccharide Engineering
Metabolic oligosaccharide engineering (MOE) is a widely used
method for modifying cellular glycans with non-natural sugars
(Dube and Bertozzi, 2003; Laughlin and Bertozzi, 2009a; Rouhanifard et al., 2013). In this method, non-natural monosaccharide analogs are taken up by cells into biosynthetic salvage
pathways, converted into activated nucleotide phosphate donors, and incorporated by GTs into specific glycan structures
(Figure 5). A range of non-natural sugar analogs based on
Fuc, NeuAc, GalNAc, and GlcNAc have been successfully
installed in a variety of cells and organisms such as zebrafish
and Caenorhabditis elegans. In principle, the analog is inserted
into all glycans containing the monosaccharide of interest,
which allows for the simultaneous detection of multiple glycan
structures. However, this also precludes the selective detection of specific structures, such as motifs that differ by neighboring glycans or by glycosidic linkage. As the non-natural analogs compete with natural substrates, their incorporation into
cellular glycans is sub-stoichiometric, which minimizes perturbation to the system but also reduces detection sensitivity. The
analogs must also contain relatively small non-natural functionalities to allow efficient recognition by enzymes and incorporation into glycans. Nonetheless, a number of chemical groups,
including inert alkyl chains (Keppler et al., 1995), as well as
reactive groups such as ketones, thiols, azides, alkynes, alkenes, isonitriles, and cyclopropenes have been installed
(Cole et al., 2013; Hang et al., 2003; Hsu et al., 2007; Laughlin
et al., 2008; Mahal et al., 1997; Sampathkumar et al., 2006;
Späte et al., 2014; Stairs et al., 2013). Reactive groups have
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enabled the covalent attachment of a variety of small-molecule
reporters (e.g., biotin, fluorescent dyes, crosslinking agents)
via bioorthogonal chemistry (McKay and Finn, 2014; Sletten
and Bertozzi, 2009). Consequently, MOE provides a facile platform for endowing glycan structures with new chemical functionalities.
A beautiful example is the application of MOE to image cellsurface glycans in developing zebrafish (Laughlin et al., 2008).
By incubation of embryos with peracetylated N-azidoacetylgalactosamine (Ac4GalNAz), followed by two different strained
cyclooctyne-conjugated fluorophores, newly synthesized glycans were distinguished from older glycans and tracked in vivo.
Interestingly, distinct regions of the embryo containing only
newly synthesized glycans were observed, suggesting that the
production of glycoproteins is highly choreographed during
differentiation. To image glycosylation at earlier stages of development, N-azidoacetylsialic acid (NeuAz/SiaNAz) was microinjected into zebrafish embryos to bypass diffusion into the cell
and label sialic acid-containing glycans (Dehnert et al., 2012).
Dual-labeling experiments were also conducted in C. elegans
with Ac4GalNAz to distinguish regions of active glycan production during both larval and adult stages of development (Laughlin
and Bertozzi, 2009b). Thus, MOE provides a powerful approach
to visualize the spatiotemporal dynamics of glycan biosynthesis
in live organisms.
In addition to imaging applications, MOE has been used to
identify the binding partners of lectins such as the sialic acidbinding immunoglobulin-like lectin (Siglec) CD22 (Han et al.,
2005). CD22 functions as a negative regulator of B cell receptor
activation by establishing a minimum signaling threshold to prevent aberrant immune reactions (Nitschke et al., 1997). Although
it was known that cis interactions of CD22 with glycoproteins on
the same cell modulated CD22 activity, the identity of the glycoprotein(s) involved was unknown. Cells were incubated with
NeuAc containing an aryl-azide moiety (9-AAz-NeuAc) to photochemically crosslink NeuAc-bearing glycoproteins to their cisbinding partners. Surprisingly, the only proteins pulled down
with CD22 were other CD22 receptors, which formed self-regulating, homomultimeric complexes. To find the trans-binding
partners of CD22, the soluble ectodomain of CD22 was photochemically crosslinked to cells displaying 9-AAz-NeuAc-containing glycans (Ramya et al., 2010). The B cell receptor IgM
was discovered to be the preferred trans-binding partner.
Notably, IgM did not appear to crosslink to CD22 when presented in cis, underscoring the ability of this MOE approach to
dissect components of different CD22 binding complexes.
The ability of MOE to remodel glycan structures at the cell surface provides a powerful means to modulate cellular responses.
For example, Fuca(1–2)Gal-containing glycans have been implicated in cognitive processes such as learning and memory. To
study these glycans, 2-deoxygalactose (2-dGal) was used to
prevent the attachment of Fuc to the 2-position of Gal in the
Fuca(1–2)Gal structure (Figure 6A). Intracerebral injection of
2-dGal into day-old chicks induced reversible amnesia and interfered with the maintenance of long-term potentiation (Bullock
et al., 1990; Krug et al., 1991; Rose and Jork, 1987), an electrophysiological model of learning and memory. The amnesic effect
was not observed with other monosaccharides, and memory formation was rescued by co-injection with Gal but not Fuc (Rose
and Jork, 1987). At the cellular level, 2-dGal decreased neurite
outgrowth of embryonic cortical neurons and induced collapse
of synapses, while 3-deoxygalactose (3-dGal) and 6-deoxygalactose (6-dGal) had no effect (Kalovidouris et al., 2005; Murrey
et al., 2006). Treatment with Gal reversed the inhibitory effects
of 2-dGal on neurite outgrowth, presumably by enabling re-synthesis of Fuca(1–2)Gal-containing glycans (Kalovidouris et al.,
2005). Interestingly, Fuca(1–2)Gal glycans were enriched on synaptic proteins such as synapsin Ia and Ib (Murrey et al., 2006),
which regulate neurotransmitter release and synapse formation.
2-dGal disrupted the fucosylation of synapsin and promoted its
degradation. Thus, the alteration of Fuca(1–2)Gal glycans on
cell surfaces using non-natural sugars has provided insights
into the functions of these glycans in the nervous system, suggesting that they play roles in the regulation of important synaptic
proteins and morphological changes that underlie synaptic
plasticity.
An alternative to inhibiting protein glycosylation with deoxy
sugars is the use of fluorinated sugar analogs, which can serve
as inhibitors of GTs. Peracetylated and fluorinated Fuc and
NeuAc analogs were used to block protein fucosylation and
sialylation, respectively, through a two-pronged mechanism
(Figure 6B) (Rillahan et al., 2012). First, the fluorinated monosaccharide inhibitors were converted by salvage pathways to
the corresponding activated nucleotide sugar donors. Once
activated, the inhibitors bound to GTs but were not transferred
to nascent glycan structures, preventing protein fucosylation or
sialylation. The resulting accumulation of sugar donors also
stimulated feedback inhibition loops that suppressed de novo
biosynthesis of Fuc or NeuAc. This approach was utilized to
inhibit the production of Lewis X (LeX) and sialyl LeX antigens,
resulting in reduced binding to E- and P-selectins and
enhanced leukocyte rolling, a process important for fighting
infections.
The remodeling of cell-surface glycans with reactive chemical
handles provides another powerful means to modulate cellular
responses. For example, the incorporation of a thiol group into
NeuAc-containing glycoproteins promoted spontaneous cellcell clustering of Jurkat T cell lymphoma cells, as well as lineage-specific differentiation of human embryoid body-derived
(hEBD) stem cells (Figure 6C) (Sampathkumar et al., 2006). The
hEBD cells showed a decrease in proliferation, along with an increase in b-catenin expression and morphological changes
consistent with differentiation to a neural cell type. Moreover, Jurkat cells decorated with thiol-containing NeuAc could be immobilized onto gold or maleimide-functionalized glass surfaces
without compromising cell viability. Thus, the remodeling of
cell surfaces with non-natural sugars can both change their
adhesive properties and artificially induce cellular signaling
processes.
In another notable example, MOE was used to trigger an artificial immune response against Helicobacter pylori, a bacterium
responsible for stomach ulcers (Kaewsapsak et al., 2013).
Although tetraacetyl N-azidoacetylglucosamine (Ac4GlcNAz) is
incorporated primarily into cytosolic glycoproteins in mammalian
cells, H. pylori robustly processed the sugar for cell-surface
display (Figure 6D). The azido-functionalized glycoproteins on
the cell surface were subsequently conjugated to an immunostimulant, 2,4-dinitrophenol (DNP), via Staudinger ligation. After
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A
H
HO
Figure 6. Non-Natural Monosaccharides
Used for MOE
H
OH
O
GDP
HO
X
OH
H
H
2-dGal
B
AcO
OAc
AcO
AcHN
OAc
OAc
F
CO2Me
O
CMP
F
F
Ac5-3Fax-NeuAc
C
O
AcO HN
AcO
AcO
SAc
O
HS
CMP
N3
UDP
OAc
AcS
Ac4ManNTGc
D
OAc
O
AcO
AcO
X
(A) 2-dGal was incorporated into cell-surface glycans to prevent attachment of Fuc in neurons. A
representative glycan containing terminal Fuca(1–
2)Gal is shown.
(B) Fluorinated analogs of Fuc and NeuAc were
metabolized to their respective nucleotide phosphate donors and used to inhibit native GTs and
affect leukocyte rolling velocity.
(C) Ac5ManNTGc was converted to a thiol-containing analog of NeuAc and incorporated into cellsurface glycans to alter cell-cell adhesion and stem
cell differentiation.
(D) Ac4GlcNAz was selectively incorporated into
cell-surface glycans of Helicobacter pylori, allowing
the installation of a functional handle into unknown
glycan structures to elicit an immune response
against the bacteria.
OAc
NH
O
N3
N3
Ac4GlcNAz
labeling, the bacteria were treated with anti-DNP antibodies,
leading to a nearly two-fold increase in bacterial cell death, as
measured by an antibody-dependent cell-mediated cytotoxicity
assay. This method highlights how the differential metabolic
utilization of glycans can potentially be exploited to selectively
target and elicit an immune response against pathogenic
bacteria.
Overall, MOE provides a versatile approach to control the
glycan architecture on cell surfaces through the incorporation
of non-natural monosaccharides. In addition to facilitating the
detection and study of glycoconjugates, MOE can be exploited
to modulate cellular processes and possibly engineer new activities through chain termination, installation of reactive groups,
and functionalization with bioactive compounds.
Chemoenzymatic Labeling
With MOE, the non-natural analog in principle can be incorporated into all glycans
containing the monosaccharide of interest. However, determining the precise
HS
SH glycan structures labeled by the non-natural sugar and demonstrating labeling
specificity can be challenging in practice
and can complicate efforts to study glycan
function (Boyce et al., 2011; Chuh et al.,
2014). The chemoenzymatic labeling (CL)
of glycans represents a complementary
strategy to remodel glycans with non-natural functionalities. In this approach, an
exogenous GT transfers a non-natural
sugar that contains a reactive group onto
the glycan structure of interest (Figure 7).
Subsequent reaction allows for the attachN3
ment of a variety of chemical reporters as
readouts for the glycan of interest. The
specificity of the exogenous GT can be
determined using glycan microarrays,
and this approach can be used to distinguish and selectively label disaccharide
or higher-order motifs of specific sugar
composition and glycosidic linkage, such
as Fuca(1–2)Gal and N-acetyllactosamine
(LacNAc). Another advantage of this approach is that the chemoenzymatic reactions often proceed quantitatively, which leads to
highly sensitive detection. Finally, this system can be employed
in biological contexts where feeding cells non-natural sugar analogs is not possible, such as the ex vivo detection of disease
biomarkers. Although identifying a suitable GT that recognizes
the motif of interest is no small task and may limit the overall
scope of the approach, CL has been used both to provide novel
insights into glycan function and to control cellular processes.
A powerful example is the application of CL to study the function of O-linked GlcNAc glycosylation on cAMP response
element-binding protein (CREB), a crucial transcription factor
involved in neuronal development, plasticity, and long-term
memory formation (Lonze and Ginty, 2002). Whereas traditional
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HO
OH
O
X
Z
X
Z
HO
NH
OUDP
X
O
Y
Exogenous
Glycosyltransferase
Figure 7. Chemoenzymatic Labeling
In CL, cells are treated with a purified, exogenous GT and an appropriate nucleotide phosphate donor to stoichiometrically attach non-natural sugars onto
glycans of defined structure. The functional handle (X) can be further elaborated using bioorthogonal chemistry to detect and study glycans or to endow cells with
new biological properties.
methods such as wheat germ agglutinin lectin and O-GlcNAc antibodies failed to detect O-GlcNAc glycosylated CREB, CL using
an engineered b-1,4-galactosyltransferase enzyme and UDP-2acetonyl-2-deoxygalactose (UDP-ketoGal) allowed for its biotinylation and subsequent rapid, sensitive detection (Figure 8A)
(Khidekel et al., 2003; Rexach et al., 2012). To determine the stoichiometry of glycosylation, O-GlcNAc glycosylated proteins
from neuronal lysates were labeled with a 2,000-Da polyethylene
glycol mass tag (Rexach et al., 2010), which shifted the molecular weight of the glycosylated proteins. Immunoblotting for
CREB allowed for quantification of the glycosylated species,
demonstrating that nearly 50% of the CREB population was
O-GlcNAc-modified in neurons (Rexach et al., 2012). Furthermore, expression of CREB mutants in which glycosylation sites
were mutated to alanine, followed by measurement of their
glycosylation stoichiometry, enabled determination of the major
glycosylation site in neurons, which was also found to be the site
induced by neuronal depolarization. Identification of this key site
facilitated in-depth functional studies and revealed that glycosylation at this site repressed both basal and activity-induced
CREB-mediated transcription, regulating both neuronal growth
and long-term memory.
In another example, a CL approach was developed to detect
the potential biomarker Fuca(1–2)Gal, which is upregulated in
inflammatory diseases and cancer (Figure 8B) (Chaubard et al.,
2012). Terminal Fuca(1–2)Gal residues on complex cell-surface
glycans were labeled using a bacterial homolog of the human
blood group A antigen GT (BgtA) and UDP-GalNAz or UDP-ketoGal. Notably, no mutagenesis of the BgtA active site was
required to transfer the non-natural sugars, highlighting the substrate promiscuity of prokaryotic GTs. Glycan microarray data
demonstrated that BgtA specifically labeled glycans containing
a terminal Fuca(1–2)Gal disaccharide motif, in contrast to
MOE, which would have labeled all fucosylated glycans. Moreover, BgtA tolerated a variety of distal architectures appended
to the reducing end of Gal, labeling diverse N- and O-glycan
structures with a terminal Fuca(1–2)Gal moiety, including Globo
H and Fuc-GM1. Fluorescent labeling of Fuca(1–2)Gal glycans
and flow cytometry analysis demonstrated a significant increase
in Fuca(1–2)Gal expression on prostate cancer cells compared
with normal prostate epithelial cells. Thus, this CL method may
provide a novel tool to discriminate cancerous cells from normal
cells and to study glycans associated with tumor pathogenesis.
CL has also been applied to detect and image LacNAc, a universal component of the antennae of hybrid- and complex-type
N-glycans (Zheng et al., 2011). Here, a-1,3-fucosyltransferase
(FucT) from H. pylori was combined with GDP-Fuc derivatives
to label LacNAc on the surface of cell lines, primary spleenocytes, and zebrafish embryos (Figure 8C). To demonstrate its efficacy at labeling cells ex vivo, T and B cells isolated from mice
were treated in situ with exogenous FucT and GDP-6-azidofucose (GDP-FucAz), followed by reaction with strained cyclooctyne-conjugated biotin and incubation with fluorescently labeled
streptavidin. Flow cytometry analysis of the labeled cell population revealed that activated immune cells produced higher fluorescence signal than their naı̈ve counterparts, suggesting that
immune cell activation increased cell-surface LacNAc. When
applied to zebrafish, only a short incubation with the non-natural
sugar analog and exogenous enzyme were needed to produce
robust labeling, thereby obviating the need for microinjection
and minimizing perturbation to the developing animal. Moreover,
FucT tolerated fluorescein-labeled GDP-Fuc as a substrate,
which avoided the subsequent dye functionalization step.
In addition to glycan detection, the modification of cell-surface
glycans by CL has been exploited to control biological activity.
Although the use of mesenchymal stem cells (MSCs) to regrow
tissue and cure skeletal diseases holds great potential (Horwitz
et al., 2002), injected MSCs often do not migrate efficiently and
selectively to the tissues of interest. To provide a homing target
for circulating MSCs, the CD44 glycan on MSCs was modified
ex vivo by human-derived a-1,3-fucosyltransferase (FTVI) to produce the SLeX glycan motif (Figure 8D) (Sackstein et al., 2008).
This motif binds to E-selectin receptors that are specifically expressed in bone marrow tissue. Engineered HCELL+ MSCs
showed much stronger sheer-resistant adhesive interactions
with E-selectin-expressing endothelial cells compared with unmodified MSCs, suggesting that the Fuc modification of CD44
glycans endowed cells with new adherent properties.
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A
B
C
D
Figure 8. Examples of CL
(A) The O-GlcNAc modification was detected and quantified by labeling with ketoGal, followed by installation of a biotin or PEG mass tag.
(B) The Fuca(1–2)Gal epitope was selectively labeled using BgtA to install GalNAc derivatives with functional handles for downstream detection.
(C) a1,3FucT from H. pylori modified LacNAc moieties with Fuc derivatives for flow cytometry and imaging analysis.
(D) Human FTVI was used to functionalize the N-glycans of CD44 on MSCs, thereby producing SLeX moieties to help target MSCs to bone marrow tissue.
Furthermore, HCELL+ MSCs that were injected intravenously
into mice exhibited much greater levels of homing to bone
marrow tissue, highlighting the ability of CL to engineer cellular
functions in vivo.
Overall, CL provides a complementary alternative to MOE for
labeling and modifying intracellular and cell-surface glycans.
Both methods have strengths in particular biological systems
and can alter the properties of cells. In the future, mining of prokaryotic genomes may reveal many novel GT candidates, under-
scoring the opportunity to expand the collection of GTs and
chemical diversity of glycan architectures remodeled by CL.
De novo Glycan Display
The methods described thus far involve the modification of natural glycans on cell surfaces by manipulating the biosynthetic
machinery used for glycan production. An alternative approach,
which we term de novo glycan display, focuses on inserting
defined glycan or glycomimetic structures directly into plasma
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Cell Chemical Biology
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A
B
C
Figure 9. De novo Display of Cell-Surface Glycans
(A) NeuAc-containing glycans and HS GAGs were chemically appended to a ketone-functionalized polymer and inserted into the plasma membrane using a
phospholipid tail.
(B) CS GAGs were displayed on the cell surface by functionalization onto ketone-containing liposomes.
(C) Genetic incorporation of the HaloTag protein allowed for the immobilization of HS GAGs modified with a chlorohexyl linker.
membranes using approaches such as lipid insertion, liposomal
fusion, or protein conjugation (Frame et al., 2007; Huang et al.,
2014; Hudak et al., 2013; Paszek et al., 2014; Pulsipher et al.,
2014, 2015). De novo display methods provide excellent control
over the glycan structure of interest and enable the presentation
of both small and large glycans at the cell surface. Moreover, this
approach is not constrained by the substrate specificity of natural enzymes, thus expanding the scope of glycan engineering to
complex structures unattainable by other methods. However,
exogenous sugars are typically displayed alongside the native
glycan population, which could obscure the biological effects
of the newly added carbohydrates. To address this complication, de novo glycan display methods can be used in combination with genetic depletion approaches to minimize the contributions of interfering endogenous glycans (Huang et al., 2014). The
versatility of the technique also allows for the display of a wide
range of carbohydrate-based structures, including glycomimetics such as synthetic glycopolymers, glycans appended to
simplified proteins, or even the glycan component of glycoproteins alone (Huang et al., 2014; Hudak et al., 2013; Paszek
et al., 2014; Pulsipher et al., 2014, 2015). Despite the use of minimalist structures and possible interference from endogenous
carbohydrates, de novo glycan display has provided remarkable
insights into a variety of biological systems.
One of the first examples of de novo glycan display examined
the effects of altering blood group antigens on erythrocyte cell
surfaces (Frame et al., 2007). Blood type is determined by the
presence of specific glycolipid structures, the O-, A- and Btype antigens, on red blood cells. Synthetic glycolipid mimics
that contained each antigen type were incubated with O-type
erythrocytes (which lack A- and B-type antigens) to allow for lipid
insertion of the glycomimetics in the membrane. The newly
remodeled blood cells displaying the artificial A- and B-type
glycolipids acquired a strong immune response to anti-A and
anti-B antibodies, respectively. This proof-of-concept approach
demonstrated that de novo glycan display through the passive
insertion of lipid-anchored glycans into membranes could be
used to control biological responses.
More recently, the immunoevasive properties of tumorigenic
cells were examined through membrane insertion of synthetic
glycopolymers. Many cancer cells develop mechanisms such
as increased surface sialylation to avoid detection by natural
killer (NK) cells in the innate immune response (Büll et al.,
2014; Fuster and Esko, 2005). Using glycan-functionalized polymers containing a terminal phospholipid, different carbohydrates were anchored to the surface of cancer cells (Figure 9A)
(Hudak et al., 2013). Cells were then co-incubated with NK cells
to determine if the exogenous glycans recapitulated the naturally
observed avoidance of NK-mediated cell death. Only sialylated
NeuAc-containing polymers significantly attenuated cytotoxicity, and the polymer-displaying cells also were found to prevent
NK degranulation, a key process in cell killing. Notably, the
NeuAc polymers protected bone marrow-derived hematopoietic
stem cells against NK-mediated cytotoxicity, suggesting that de
novo glycan display of such polymers could help prevent cellular
rejection during transplantation.
The remodeling of cell surfaces with lipid-anchored glycopolymers has also provided key insights into the mechanobiology of
integrin signaling. Integrins are cell-surface receptors that regulate cancer cell migration, invasion, and proliferation (Guo and
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Giancotti, 2004). Computational modeling studies predicted that
the glycocalyx promotes integrin receptor clustering and enhances integrin signaling through mechanical interactions
between bulky carbohydrates and transmembrane proteins
(Paszek et al., 2009). To test this hypothesis, synthetic mucinlike glycopolymers of different lengths were conjugated to a
phospholipid tail and inserted into the membranes of mammary
epithelial cells (Paszek et al., 2014). Cells displaying longer
glycopolymers (80 nm) showed segregated regions of clustered
integrins, whereas cells decorated with shorter glycopolymers
(3 and 30 nm) showed no effect. The larger glycopolymers
were found to expand the distance between the cell surface
and the extracellular matrix by 19 nm, as determined by scanning
angle interference microscopy. This larger spacing enhanced
the clustering of integrins into fewer, larger focal adhesions,
promoting the formation of active signaling nodes on the
plasma membrane by physically restructuring the cell surface.
Corroborating these data, overexpression of mucin glycoprotein
MUC1 recapitulated the overall heightened membrane and
isolated regions of focal adhesion formation. Thus, de novo
glycan display provided a facile method to modulate systematically the chemical composition and physical parameters of the
glycocalyx.
De novo glycan display has also provided a physiologically
relevant model for studying developing neurons in the hippocampus. Neuronal growth and synapse formation are highly
regulated processes in the hippocampus, a brain region critical
for the consolidation of short- and long-term memory. Studies
suggested that chondroitin sulfate glycosaminoglycans (CS
GAGs) containing certain sulfation motifs can stimulate the
outgrowth of embryonic hippocampal neurons by recruiting
growth factors to the cell surface and facilitating activation of
their cognate cell-surface receptors (Clement et al., 1999;
Gama et al., 2006; Rogers et al., 2011). However, evidence for
this mechanism was primarily based on the growth-promoting
effects of CS GAGs adhered to cell culture plates or other artificial substrata. A liposome-based approach was developed to
recapitulate the natural display of CS GAGs on neuronal cell surfaces and probe the role of sulfation (Pulsipher et al., 2014). Liposomes were formed from commercially available lipids in the
presence of 2-dodecanone, which provided a reactive ketone
handle for covalent attachment of aminooxy-functionalized CS
polysaccharides (Figure 9B). Membrane fusion led to presentation of CS GAGs enriched in specific sulfation motifs on the surface of embryonic hippocampal neurons. Notably, only neurons
displaying the disulfated CS-E motif showed significantly greater
neurite outgrowth in response to nerve growth factor (NGF)
compared with neurons lacking lipid-anchored CS GAGs. The
extent of neurite outgrowth could be finely tuned by controlling
the amount of CS-E delivered to the cell surface. As further support for the mechanism, the presence of CS-E led to enhanced
activation of NGF-mediated signaling pathways, suggesting
that CS-E facilitates surface recruitment of NGF. Thus, these
studies demonstrated the proof-of-principle that de novo glycan
display of CS GAGs can be used to control neuronal signaling
pathways and downstream cellular responses.
GAG-mediated cell signaling plays a critical role not only in
neurons but also in stem cell development. For example, previous reports have suggested that HS sulfation levels influence
stem cell fate by regulating growth factor-mediated signaling
events that determine both pluripotent self-renewal and differentiation (Forsberg et al., 2012; Kraushaar et al., 2012). To explore
whether artificial cell-surface glycans could be used to direct this
process, synthetic phospholipid-anchored ketone polymers
were functionalized with different aminooxy-tagged HS disaccharides produced by enzymatic digestion (Huang et al., 2014).
Passive insertion of glycomimetics containing both 2-O- and 6O-sulfation, but not other sulfation motifs, activated fibroblast
growth factor 2 (FGF2) signaling pathways involved in stem cell
differentiation. Furthermore, stem cells decorated with the active
HS glycopolymers formed neural rosettes, suggesting the formation of intermediate neuroprogenitor cells.
A major challenge of de novo glycan display methods is that
the presentation of glycans at the cell surface is often shortlived. Lipid-anchored glycans typically exhibit a membrane
half-life of about 6–8 hr (Huang et al., 2014; Hudak et al.,
2013; Pulsipher et al., 2014). To achieve long-lived display, a
genetically encoded, transmembrane-anchored HaloTag protein was exploited (Figure 9C) (Pulsipher et al., 2015). The
HaloTag protein is a bacterial alkyl dehalogenase that forms a
covalent bond with chloroalkyl ligands (Los et al., 2008), which
allows for attachment of HS GAGs and other molecules.
Notably, fluorescent probes anchored to HaloTag proteins persisted on the cell surface for over 8 days. Moreover, stem cells
decorated with highly sulfated HS on HaloTag proteins underwent an accelerated loss of pluripotency and differentiation
into mature neuronal cells. Homogeneous neuronal cell populations could be valuable as cell replacement therapies for neurodegenerative disorders such as Parkinson’s disease (Freed
et al., 2001; Mendez et al., 2008). This method constitutes the
first example of long-term glycan display, significantly broadening the potential application of de novo display to a wide
range of biological processes.
De novo glycan display provides a powerful, versatile means
to direct key signaling events and biological outcomes such as
the immune response, cancer proliferation, and stem cell differentiation. The technique is modular by design, providing a
general platform to test numerous glycan structures and presentation architectures that best suit the biological system of
interest. Moreover, the method allows for the controlled display
of large polysaccharides, which are inaccessible to other forms
of glycan engineering. De novo glycan display approaches
have provided novel insights into the roles of specific carbohydrates in biology and have the potential to direct biomedically
important processes such as tissue transplantations or stem
cell fate.
Outlook
Considerable progress has been made toward developing novel
methods to remodel the structures of glycans at cell surfaces.
These studies have broadly demonstrated the ability to modulate
signaling pathways and important downstream biological events
by controlling glycan-protein interactions. To date, the majority
of glycan engineering techniques have been applied in vitro.
The dearth of in vivo experiments underscores the need to
expand these approaches to complex living systems. The ability
to perturb glycans in a tissue-specific manner or at an organismwide level will present new opportunities to unravel the
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Cell Chemical Biology
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intricacies of glycan-mediated processes that cannot be fully
recapitulated in vitro.
Excitingly, recent advances in other fields promise to fuel the
future development and expansion of glycan engineering technologies. With the advent of genome editing methods such as
CRISPR/Cas9 (Hsu et al., 2014), scientists now can more rapidly
knock out genes and screen for enzymatic function, and perhaps
even more impressively, knock in exogenous or evolved enzymes
with new activities. These advances should greatly facilitate the
identification of still unknown GTs and glycosylation-associated
proteins critically involved in development and disease. Furthermore, the integration of facile genetic editing with tissue-specific
or temporally regulated expression should enable the production
of animal models that possess modified glycan structures during
specific stages of development, growth, and disease.
Such technologies should also accelerate the expansion of
MOE and CL to a wider range of non-natural glycan structures.
Genetic knockins or targeted delivery of agents could produce
specific cell populations and even entire organisms capable of
incorporating a diverse assortment of non-natural sugars inaccessible to the natural glycosylation machinery, possibly allowing multiple glycan populations to be monitored in real time using
orthogonal non-natural functionalities. Furthermore, tagged glycans could be purified from living tissues to ascertain the differences in individual glycoprotein structures over time. With the
increase in non-natural sugars that could be simultaneously
incorporated, a complementary wider set of functional handles
would provide further power to control cellular function and
endow cells with novel properties.
Ongoing developments in DNA sequencing and bioinformatics
have dramatically increased the number of sequenced and annotated microbial genomes (Markowitz et al., 2012), which could
provide novel enzymes involved in glycosylation and expand
the chemical diversity of glycan architectures remodeled by CL.
As CL is uniquely suited for biomarker detection, these new GTs
could also provide a suite of tools to monitor human pathology
and perhaps predict the onset and progression of disease states.
Finally, recent advances in de novo glycan display, particularly
the expansion of its temporal limits, have opened an exciting new
frontier to explore the structure-function relationships of carbohydrates. These methods now present unparalleled opportunities to engineer novel signaling pathways and functions into
cells. Liposomes decorated with tissue-specific ligands could
be used to deliver exogenous glycans to specific cell populations
in vivo. Similarly, protein anchors like the HaloTag protein could
be incorporated through genetic engineering to direct glycan
display in model organisms. The attachment of glycans to
various protein architectures could help mimic the natural presentation of sugars as glycoproteins and dissect the importance
of different protein-carbohydrate combinations. Together, the
successes in glycan engineering underscore the powerful integration of chemistry and biology and its ability to drive technological innovations and new discoveries.
ACKNOWLEDGMENTS
This research was supported by the NIH (R01-GM093627 and R01GM084724) and a National Science Foundation Graduate Research Fellowship (DGE-1144469).
REFERENCES
Andersson, E.R., Sandberg, R., and Lendahl, U. (2011). Notch signaling:
simplicity in design, versatility in function. Development 138, 3593–3612.
Baeg, G.H., Selva, E.M., Goodman, R.M., Dasgupta, R., and Perrimon, N. (2004).
The wingless morphogen gradient is established by the cooperative action of
Frizzled and heparan sulfate proteoglycan receptors. Dev. Biol. 276, 89–100.
Belenkaya, T.Y., Han, C., Yan, D., Opoka, R.J., Khodoun, M., Liu, H.Z., and Lin,
X.H. (2004). Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan
sulfate proteoglycans. Cell 119, 231–244.
Bellaiche, Y., The, I., and Perrimon, N. (1998). Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion.
Nature 394, 85–88.
Beltran-Valero de Bernabé, D., Currier, S., Steinbrecher, A., Celli, J., van Beusekom, E., van der Zwaag, B., Kayserili, H., Merlini, L., Chitayat, D., Dobyns,
W.B., et al. (2002). Mutations in the O-mannosyltransferase gene POMT1
give rise to the severe neuronal migration disorder Walker-Warburg syndrome.
Am. J. Hum. Genet. 71, 1033–1043.
Bishop, J.R., Schuksz, M., and Esko, J.D. (2007). Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030–1037.
Boyce, M., Carrico, I.S., Ganguli, A.S., Yu, S.-H., Hangauer, M.J., Hubbard,
S.C., Kohler, J.J., and Bertozzi, C.R. (2011). Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc. Natl. Acad. Sci. USA 108, 3141–3146.
Brückner, K., Perez, L., Clausen, H., and Cohen, S. (2000). Glycosyltransferase
activity of Fringe modulates Notch-Delta interactions. Nature 406, 411–415.
Büll, C., den Brok, M.H., and Adema, G.J. (2014). Sweet escape: sialic acids in
tumor immune evasion. Biochim. Biophys. Acta 1846, 238–246.
Bullock, S., Potter, J., and Rose, S.P.R. (1990). Effects of the amnesic agent
2-deoxygalactose on incorporation of fucose into chick brain glycoproteins.
J. Neurochem. 54, 135–142.
Chaubard, J.-L., Krishnamurthy, C., Yi, W., Smith, D.F., and Hsieh-Wilson, L.C.
(2012). Chemoenzymatic probes for detecting and imaging fucose-a(1-2)galactose glycan biomarkers. J. Am. Chem. Soc. 134, 4489–4492.
Chen, J., Moloney, D.J., and Stanley, P. (2001). Fringe modulation of Jagged1induced Notch signaling requires the action of b4galactosyltransferase-1.
Proc. Natl. Acad. Sci. USA 98, 13716–13721.
Chuh, K.N., Zaro, B.W., Piller, F., Piller, V., and Pratt, M.R. (2014). Changes in
metabolic chemical reporter structure yield a selective probe of O-GlcNAc
modification. J. Am. Chem. Soc. 136, 12283–12295.
Clement, A.M., Sugahara, K., and Faissner, A. (1999). Chondroitin sulfate E
promotes neurite outgrowth of rat embryonic day 18 hippocampal neurons.
Neurosci. Lett. 269, 125–128.
Cole, C.M., Yang, J., Seckute, J., and Devaraj, N.K. (2013). Fluorescent livecell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives. Chembiochem 14, 205–208.
Coles, C.H., Shen, Y., Tenney, A.P., Siebold, C., Sutton, G.C., Lu, W., Gallagher, J.T., Jones, E.Y., Flanagan, J.G., and Aricescu, A.R. (2011). Proteoglycan-specific molecular switch for RPTPs clustering and neuronal extension.
Science 332, 484–488.
Cook, A., Raskind, W., Blanton, S.H., Pauli, R.M., Gregg, R.G., Francomano,
C.A., Puffenberger, E., Conrad, E.U., Schmale, G., Schellenberg, G., et al.
(1993). Genetic heterogeneity in families with hereditary multiple exostoses.
Am. J. Hum. Genet. 53, 71–79.
Dehnert, K.W., Baskin, J.M., Laughlin, S.T., Beahm, B.J., Naidu, N.N.,
Amacher, S.L., and Bertozzi, C.R. (2012). Imaging the sialome during zebrafish
development with copper-free click chemistry. Chembiochem 13, 353–357.
Demaegd, D., Foulquier, F., Colinet, A.-S., Gremillon, L., Legrand, D., Mariot, P.,
Peiter, E., Van Schaftingen, E., Matthijs, G., and Morsomme, P. (2013). Newly
characterized Golgi-localized family of proteins is involved in calcium and pH homeostasis in yeast and human cells. Proc. Natl. Acad. Sci. USA 110, 6859–6864.
Dube, D.H., and Bertozzi, C.R. (2003). Metabolic oligosaccharide engineering
as a tool for glycobiology. Curr. Opin. Chem. Biol. 7, 616–625.
Cell Chemical Biology 23, January 21, 2016 ª2016 Elsevier Ltd All rights reserved 119
Please cite this article as: Griffin and Hsieh-Wilson, Glycan Engineering for Cell and Developmental Biology, Cell Chemical Biology (2016), http://
dx.doi.org/10.1016/j.chembiol.2015.12.007
Cell Chemical Biology
Review
Forsberg, M., Holmborn, K., Kundu, S., Dagalv, A., Kjellén, L., and ForsbergNilsson, K. (2012). Undersulfation of heparan sulfate restricts differentiation
potential of mouse embryonic stem cells. J. Biol. Chem. 287, 10853–10862.
Kaewsapsak, P., Esonu, O., and Dube, D.H. (2013). Recruiting the host’s immune system to target Helicobacter pylori’s surface glycans. Chembiochem
14, 721–726.
Foulquier, F., Amyere, M., Jaeken, J., Zeevaert, R., Schollen, E., Race, V.,
Bammens, R., Morelle, W., Rosnoblet, C., Legrand, D., et al. (2012).
TMEM165 deficiency causes a congenital disorder of glycosylation. Am. J.
Hum. Genet. 91, 15–26.
Kalovidouris, S.A., Gama, C.I., Lee, L.W., and Hsieh-Wilson, L.C. (2005). A role
for fucose-a(1 2)-galactose carbohydrates in neuronal growth. J. Am. Chem.
Soc. 127, 1340–1341.
Frame, T., Carroll, T., Korchagina, E., Bovin, N., and Henry, S. (2007). Synthetic
glycolipid modification of red blood cell membranes. Transfusion 47, 876–882.
Freed, C.R., Greene, P.E., Breeze, R.E., Tsai, W.-Y., DuMouchel, W., Kao, R.,
Dillon, S., Winfield, H., Culver, S., Trojanowski, J.Q., et al. (2001). Transplantation of embryonic dopamine neurons for severe Parkinson’s Disease. N. Engl.
J. Med. 344, 710–719.
Freeze, H.H. (2013). Understanding human glycosylation disorders: biochemistry leads the charge. J. Biol. Chem. 288, 6936–6945.
Fuster, M.M., and Esko, J.D. (2005). The sweet and sour of cancer: glycans as
novel therapeutic targets. Nat. Rev. Cancer 5, 526–542.
Gama, C.I., Tully, S.E., Sotogaku, N., Clark, P.M., Rawat, M., Vaidehi, N., Goddard, W.A., III, Nishi, A., and Hsieh-Wilson, L.C. (2006). Sulfation patterns of
glycosaminoglycans encode molecular recognition and activity. Nat. Chem.
Biol. 2, 467–473.
Goddeeris, M.M., Wu, B., Venzke, D., Yoshida-Moriguchi, T., Saito, F., Matsumura, K., Moore, S.A., and Campbell, K.P. (2013). LARGE glycans on dystroglycan function as a tunable matrix scaffold to prevent dystrophy. Nature 503,
136–140.
Guo, W., and Giancotti, F.G. (2004). Integrin signalling during tumour progression. Nat. Rev. Mol. Cell Biol. 5, 816–826.
Häcker, U., Nybakken, K., and Perrimon, N. (2005). Heparan sulphate proteoglycans: the sweet side of development. Nat. Rev. Mol. Cell Biol. 6, 530–541.
Keppler, O.T., Stehling, P., Herrmann, M., Kayser, H., Grunow, D., Reutter, W.,
and Pawlita, M. (1995). Biosynthetic modulation of sialic acid-dependent virusreceptor interactions of two primate polyoma viruses. J. Biol. Chem. 270,
1308–1314.
Khidekel, N., Arndt, S., Lamarre-Vincent, N., Lippert, A., Poulin-Kerstien, K.G.,
Ramakrishnan, B., Qasba, P.K., and Hsieh-Wilson, L.C. (2003). A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J. Am. Chem. Soc. 125, 16162–16163.
Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., and Selleck, S.B. (2004).
Spatial regulation of wingless morphogen distribution and signaling by dallylike protein. Dev. Cell 7, 513–523.
Kobayashi, K., Nakahori, Y., Miyake, M., Matsumura, K., Kondo-Iida, E., Nomura, Y., Segawa, M., Yoshioka, M., Saito, K., Osawa, K., et al. (1998). An
ancient retrotransposal insertion causes Fukuyama-type congenital muscular
dystrophy. Nature 394, 388–392.
Kopan, R., and Ilagan, M.X.G. (2009). The canonical Notch signaling pathway:
unfolding the activation mechanism. Cell 137, 216–233.
Koziel, L., Kunath, M., Kelly, O.G., and Vortkamp, A. (2004). Ext1-dependent
heparan sulfate regulates the range of Ihh signaling during endochondral ossification. Dev. Cell 6, 801–813.
Kraushaar, D.C., Rai, S., Condac, E., Nairn, A., Zhang, S., Yamaguchi, Y.,
Moremen, K., Dalton, S., and Wang, L. (2012). Heparan sulfate facilitates
FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic
stem cells. J. Biol. Chem. 287, 22691–22700.
Haines, N., and Irvine, K.D. (2003). Glycosylation regulates Notch signalling.
Nat. Rev. Mol. Cell Biol. 4, 786–797.
Krug, M., Jork, R., Reymann, K., Wagner, M., and Matthies, H. (1991). The
amnesic substance 2-deoxy-D-galactose suppresses the maintenance of hippocampal LTP. Brain Res. 540, 237–242.
Han, C., Belenkaya, T.Y., Khodoun, M., Tauchi, M., Lin, X., and Lin, X. (2004).
Distinct and collaborative roles of Drosophila EXT family proteins in
morphogen signalling and gradient formation. Development 131, 1563–1575.
Laughlin, S.T., and Bertozzi, C.R. (2009a). Imaging the glycome. Proc. Natl.
Acad. Sci. USA 106, 12–17.
Han, S., Collins, B.E., Bengtson, P., and Paulson, J.C. (2005). Homomultimeric
complexes of CD22 in B cells revealed by protein-glycan cross-linking. Nat.
Chem. Biol. 1, 93–97.
Hang, H.C., Yu, C., Kato, D.L., and Bertozzi, C.R. (2003). A metabolic labeling
approach toward proteomic analysis of mucin-type O-linked glycosylation.
Proc. Natl. Acad. Sci. USA 100, 14846–14851.
Hennet, T., and Cabalzar, J. (2015). Congenital disorders of glycosylation: a
concise chart of glycocalyx dysfunction. Trends Biochem. Sci. 40, 377–384.
Horwitz, E.M., Gordon, P.L., Koo, W.K.K., Marx, J.C., Neel, M.D., McNall, R.Y.,
Muul, L., and Hofmann, T. (2002). Isolated allogeneic bone marrow-derived
mesenchymal cells engraft and stimulate growth in children with osteogenesis
imperfecta: implications for cell therapy of bone. Proc. Natl. Acad. Sci. USA 99,
8932–8937.
Hsu, T.-L., Hanson, S.R., Kishikawa, K., Wang, S.-K., Sawa, M., and Wong, C.H. (2007). Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc. Natl. Acad. Sci. USA 104, 2614–2619.
Hsu, P.D., Lander, E.S., and Zhang, F. (2014). Development and applications
of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278.
Laughlin, S.T., and Bertozzi, C.R. (2009b). In vivo imaging of Caenorhabditis
elegans glycans. ACS Chem. Biol. 4, 1068–1072.
Laughlin, S.T., Baskin, J.M., Amacher, S.L., and Bertozzi, C.R. (2008). In vivo
imaging of membrane-associated glycans in developing zebrafish. Science
320, 664–667.
Le Merrer, M., Legeai-Mallet, L., Jeannin, P.M., Horsthemke, B., Schinzel, A.,
Plauchu, H., Toutain, A., Archard, F., Munnich, A., and Maroteaux, P. (1994). A
gene for hereditary multiple exostoses maps to chromosome 19p. Hum. Mol.
Genet. 3, 717–722.
Lichtenstein, R.G., and Rabinovich, G.A. (2013). Glycobiology of cell death:
when glycans and lectins govern cell fate. Cell Death Differ. 20, 976–986.
Longman, C., Brockington, M., Torelli, S., Jimenez-Mallebrera, C., Kennedy,
C., Khalil, N., Feng, L., Saran, R.K., Voit, T., Merlini, L., et al. (2003). Mutations
in the human LARGE gene cause MDC1D, a novel form of congenital muscular
dystrophy with severe mental retardation and abnormal glycosylation of
a-dystroglycan. Hum. Mol. Genet. 12, 2853–2861.
Lonze, B.E., and Ginty, D.D. (2002). Function and regulation of CREB family
transcription factors in the nervous system. Neuron 35, 605–623.
Huang, M.L., Smith, R.A.A., Trieger, G.W., and Godula, K. (2014). Glycocalyx
remodeling with proteoglycan mimetics promotes neural specification in embryonic stem cells. J. Am. Chem. Soc. 136, 10565–10568.
Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M., et al. (2008). HaloTag:
a novel protein labeling technology for cell imaging and protein analysis. ACS
Chem. Biol. 3, 373–382.
Hudak, J.E., Canham, S.M., and Bertozzi, C.R. (2013). Glycocalyx engineering
reveals a Siglec-based mechanism for NK cell immunoevasion. Nat. Chem.
Biol. 10, 69–75.
Mahal, L.K., Yarema, K.J., and Bertozzi, C.R. (1997). Engineering chemical
reactivity on cell surfaces through oligosaccharide biosynthesis. Science
276, 1125–1128.
Jones, M.A., Ng, B.G., Bhide, S., Chin, E., Rhodenizer, D., He, P., Losfeld, M.E., He, M., Raymond, K., Berry, G., et al. (2012). DDOST mutations identified by
whole-exome sequencing are implicated in congenital disorders of glycosylation. Am. J. Hum. Genet. 90, 363–368.
Manzini, M.C., Tambunan, D.E., Hill, R.S., Yu, T.W., Maynard, T.M., Heinzen,
E.L., Shianna, K.V., Stevens, C.R., Partlow, J.N., Barry, B.J., et al. (2012). Exome
sequencing and functional validation in zebrafish identify GTDC2 mutations as a
cause of Walker-Warburg syndrome. Am. J. Hum. Genet. 91, 541–547.
120 Cell Chemical Biology 23, January 21, 2016 ª2016 Elsevier Ltd All rights reserved
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Review
Markowitz, V.M., Chen, I.-M.A., Palaniappan, K., Chu, K., Szeto, E., Grechkin,
Y., Ratner, A., Jacob, B., Huang, J., Williams, P., et al. (2012). IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 40, D115–D122.
Sackstein, R., Merzaban, J.S., Cain, D.W., Dagia, N.M., Spencer, J.A., Lin,
C.P., and Wohlgemuth, R. (2008). Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone. Nat.
Med. 14, 181–187.
McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L.E., Dyer,
A.P., and Tufaro, F. (1998). The putative tumour suppressor EXT1 alters the
expression of cell-surface heparan sulfate. Nat. Genet. 19, 158–161.
Sampathkumar, S.-G., Li, A.V., Jones, M.B., Sun, Z., and Yarema, K.J. (2006).
Metabolic installation of thiols into sialic acid modulates adhesion and stem
cell biology. Nat. Chem. Biol. 2, 149–152.
McKay, C.S., and Finn, M.G. (2014). Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 21, 1075–1101.
Shi, S.L., and Stanley, P. (2003). Protein O-fucosyltransferase 1 is an essential
component of Notch signaling pathways. Proc. Natl. Acad. Sci. USA 100,
5234–5239.
Mendez, I., Viñuela, A., Astradsson, A., Mukhida, K., Hallett, P., Robertson, H.,
Tierney, T., Holness, R., Dagher, A., Trojanowski, J.Q., et al. (2008). Dopamine
neurons implanted into people with Parkinson’s disease survive without pathology for 14 years. Nat. Med. 14, 507–509.
Moloney, D.J., Panin, V.M., Johnston, S.H., Chen, J.H., Shao, L., Wilson, R.,
Wang, Y., Stanley, P., Irvine, K.D., Haltiwanger, R.S., et al. (2000). Fringe is a
glycosyltransferase that modifies Notch. Nature 406, 369–375.
Murrey, H.E., Gama, C.I., Kalovidouris, S.A., Luo, W.I., Driggers, E.M., Porton,
B., and Hsieh-Wilson, L.C. (2006). Protein fucosylation regulates synapsin la/lb
expression and neuronal morphology in primary hippocampal neurons. Proc.
Natl. Acad. Sci. USA 103, 21–26.
Nitschke, L., Carsetti, R., Ocker, B., Köhler, G., and Lamers, M.C. (1997). CD22
is a negative regulator of B-cell receptor signalling. Curr. Biol. 7, 133–143.
Ohtsuka, Y., Kanagawa, M., Yu, C.-C., Ito, C., Chiyo, T., Kobayashi, K., Okada,
T., Takeda, S., and Toda, T. (2015). Fukutin is prerequisite to ameliorate
muscular dystrophic phenotype by myofiber-selective LARGE expression.
Sci. Rep. 5, 8316.
Okajima, T., and Irvine, K.D. (2002). Regulation of Notch signaling by O-linked
fucose. Cell 111, 893–904.
Paszek, M.J., Boettiger, D., Weaver, V.M., and Hammer, D.A. (2009). Integrin
clustering is driven by mechanical resistance from the glycocalyx and the substrate. PLoS Comput. Biol. 5, e1000604.
Paszek, M.J., DuFort, C.C., Rossier, O., Bainer, R., Mouw, J.K., Godula, K.,
Hudak, J.E., Lakins, J.N., Wijekoon, A.C., Cassereau, L., et al. (2014). The cancer glycocalyx mechanically primes integrin-mediated growth and survival.
Nature 511, 319–325.
Pulsipher, A., Griffin, M.E., Stone, S.E., Brown, J.M., and Hsieh-Wilson, L.C.
(2014). Directing neuronal signaling through cell-surface glycan engineering.
J. Am. Chem. Soc. 136, 6794–6797.
Pulsipher, A., Griffin, M.E., Stone, S.E., and Hsieh-Wilson, L.C. (2015). Longlived engineering of glycans to direct stem cell fate. Angew. Chem. Int. Ed.
Engl. 54, 1466–1470.
Ramya, T.N.C., Weerapana, E., Liao, L., Zeng, Y., Tateno, H., Liao, L., Yates,
J.R., III, Cravatt, B.F., and Paulson, J.C. (2010). In situ trans ligands of CD22
identified by glycan-protein photocross-linking-enabled proteomics. Mol.
Cell Proteomics 9, 1339–1351.
Rexach, J.E., Rogers, C.J., Yu, S.-H., Tao, J., Sun, Y.E., and Hsieh-Wilson,
L.C. (2010). Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags. Nat. Chem. Biol. 6, 645–651.
Rexach, J.E., Clark, P.M., Mason, D.E., Neve, R.L., Peters, E.C., and HsiehWilson, L.C. (2012). Dynamic O-GlcNAc modification regulates CREB-mediated gene expression and memory formation. Nat. Chem. Biol. 8, 253–261.
Rillahan, C.D., Antonopoulos, A., Lefort, C.T., Sonon, R., Azadi, P., Ley, K., Dell,
A., Haslam, S.M., and Paulson, J.C. (2012). Global metabolic inhibitors of sialyland fucosyltransferases remodel the glycome. Nat. Chem. Biol. 8, 661–668.
Rogers, C.J., Clark, P.M., Tully, S.E., Abrol, R., Garcia, K.C., Goddard, W.A.,
III, and Hsieh-Wilson, L.C. (2011). Elucidating glycosaminoglycan-proteinprotein interactions using carbohydrate microarray and computational approaches. Proc. Natl. Acad. Sci. USA 108, 9747–9752.
Rose, S.P.R., and Jork, R. (1987). Long-term memory formation in chicks is
blocked by 2-deoxygalactose, a fucose analog. Behav. Neural Biol. 48, 246–258.
Rouhanifard, S.H., Nordstrøm, L.U., Zheng, T., and Wu, P. (2013). Chemical
probing of glycans in cells and organisms. Chem. Soc. Rev. 42, 4284–4296.
Sletten, E.M., and Bertozzi, C.R. (2009). Bioorthogonal chemistry: fishing for
selectivity in a sea of functionality. Angew. Chem. Int. Ed. Engl. 48, 6974–6998.
Späte, A.K., Schart, V.F., Schöllkopf, S., Niederwieser, A., and Wittmann, V.
(2014). Terminal alkenes as versatile chemical reporter groups for metabolic
oligosaccharide engineering. Chemistry 20, 16502–16508.
Stairs, S., Neves, A.A., Stöckman, H., Wainman, Y.A., Ireland-Zecchini, H.,
Brindle, K.M., and Leeper, F.J. (2013). Metabolic glycan imaging by isonitrile-tetrazine click chemistry. Chembiochem 14, 1063–1067.
Stevens, E., Carss, K.J., Cirak, S., Foley, A.R., Torelli, S., Willer, T., Tambunan,
D.E., Yau, S., Brodd, L., Sewry, C.A., et al. (2013). Mutations in B3GALNT2
cause congenital muscular dystrophy and hypoglycosylation of a-dystroglycan. Am. J. Hum. Genet. 92, 354–365.
Tabata, T., and Kornberg, T.B. (1994). Hedgehog is a signaling protein with a
key role in patterning Drosophila imaginal discs. Cell 76, 89–102.
The, I., Bellaiche, Y., and Perrimon, N. (1999). Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol. Cell 4, 633–639.
van Kooyk, Y., and Rabinovich, G.A. (2008). Protein-glycan interactions in the
control of innate and adaptive immune responses. Nat. Immunol. 9, 593–601.
van Reeuwijk, J., Janssen, M., van den Elzen, C., Beltran-Valero de Bernabé,
D., Sabatelli, P., Merlini, L., Boon, M., Scheffer, H., Brockington, M., Mutoni, F.,
et al. (2005). POMT2 mutations cause a-dystroglycan hypoglycosylation and
Walker-Warburg syndrome. J. Med. Genet. 42, 907–912.
Wang, Y., Shao, L., Shi, S., Harris, R.J., Spellman, M.W., Stanley, P., and Haltiwanger, R.S. (2001). Modification of epidermal growth factor-like repeats
with O-fucose: molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase. J. Biol. Chem. 276, 40338–40345.
Wells, L. (2013). The O-mannosylation pathway: glycosyltransferases and proteins implicated in congenital muscular dystrophy. J. Biol. Chem. 288, 6930–
6935.
Wu, Y.Q., Heutink, P., de Vries, B.B.A., Sandkuijl, L.A., van den Ouweland,
A.M.W., Neirmeijer, M.F., Galjaard, H., Reyniers, E., Willems, P.J., and Halley,
D.J.J. (1994). Assignment of a second locus for multiple exostoses to the pericentromeric region of chromosome 11. Hum. Mol. Genet. 3, 167–171.
Yao, D., Huang, Y., Huang, X., Wang, W., Yan, Q., Wei, L., Xin, W., Gerson, S.,
Stanley, P., Lowe, J.B., et al. (2011). Protein O-fucosyltransferase 1 (Pofut1)
regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions. Blood 117, 5652–5662.
Yoshida, A., Kobayashi, K., Manya, H., Taniguchi, K., Kano, H., Mizuno, M., Inazu, T., Mitsuhashi, H., Takahashi, S., Takeuchi, M., et al. (2001). Muscular
dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev. Cell 1, 717–724.
Yoshida-Moriguchi, T., Yu, L., Stalnaker, S.H., Davis, S., Kunz, S., Madson, M.,
Oldstone, M.B.A., Schachter, H., Wells, L., and Campbell, K.P. (2010). O-Mannosyl phosphorylation of alpha-dystroglycan is required for laminin binding.
Science 327, 88–92.
Yoshida-Moriguchi, T., Willer, T., Anderson, M.E., Venzke, D., Whyte, T., Muntoni, F., Lee, H., Nelson, S.F., Yu, L., and Campbell, K.P. (2013). SGK196 is a
glycosylation-specific O-mannose kinase required for dystroglycan function.
Science 341, 896–899.
Zheng, T., Jiang, H., Gros, M., Soriano del Amo, D., Sundaram, S., Lauvau, G.,
Marlow, F., Liu, Y., Stanley, P., and Wu, P. (2011). Tracking N-acetyllactosamine
on cell-surface glycans in vivo. Angew. Chem. Int. Ed. Engl. 50, 4113–4118.
Cell Chemical Biology 23, January 21, 2016 ª2016 Elsevier Ltd All rights reserved 121