Download Extreme sweetness: protein glycosylation in archaea

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Extreme sweetness: protein
glycosylation in archaea
Jerry Eichler
Abstract | Although N‑glycosylation was first reported in archaea almost 40 years
ago, detailed insights into this process have become possible only recently, with
the availability of complete genome sequences for almost 200 archaeal species
and the development of appropriate molecular tools. As a result of these advances,
recent efforts have not only succeeded in delineating the pathways involved in
archaeal N‑glycosylation, but also begun to reveal how such post-translational
protein modification helps archaea to survive in some of the harshest environments
on the planet.
The advent of high-throughput sequencing
has had an enormous impact on many areas of
the biological sciences, but perhaps nowhere
more so than in microbiology. At the same
time, it has become clear that deciphering the
genome sequence of a given microorganism
cannot in itself explain the complexity and
variability of the associated proteome. Instead,
several processes are responsible for the diversity seen at the protein level, including alternative RNA splicing, modulation of the rate at
which different proteins are synthesized and/
or degraded, and distinct processing leading to
any of a long list of possible post-translational
modifications, including phosphorylation,
acetylation, methylation and glycosylation.
Of the various post-translational
modifications that a protein can undergo,
N‑glycosylation is the most complex 1–3. First
described by Neuberger in 1938 (REF. 4),
N‑glycosylation — that is, the covalent
linkage of a glycan moiety to selected Asn
residues in a target protein — was long held
to be a trait exclusive to eukaryotes, in which
proteins residing in or passing through the
secretory pathway en route to the cell surface
or beyond the confines of the cell can be thus
modified. This dogma was dispelled in 1976,
when the surface layer (S-layer) glycoprotein
of Halobacterium salinarum, a halophilic
archaeon, was identified as the first noneukaryotic glycoprotein5. Today, it is clear
that bacteria, like archaea, are capable of
N‑glycosylation (as well as O‑glycosylation,
in which sugars are added to Ser or Thr residues in target proteins)6–8. Until recently, our
knowledge of archaeal N‑glycosylation was
limited, compared to our relatively detailed
understanding of this process in eukaryotes
and bacteria. In the past few years, however,
genome sequencing efforts have allowed
for comparative studies, and the growing
number of species amenable to genetic
manipulation has allowed the development
of gene deletion and induced protein overexpression methods, as well as biochemical
tools suitable for use with archaeal proteins
that require defined surroundings, such as
high salt concentrations. As a direct result
of these advancements, many of the details of
the archaeal version of N‑glycosylation have
been elucidated.
In this Progress article, I discuss recent
advances describing the diversity in archaeal
N‑linked glycan content and the multiple
pathways of archaeal N‑glycosylation that are
used to achieve such diversity. I also consider
how N‑glycosylation helps archaea cope with
the extreme environments that they inhabit.
The diverse world of N‑linked glycans
In eukaryotes, the process of N‑glycosylation
is best understood in Saccharomyces cerevisiae
and in vertebrates1–3. In these organisms,
seven nucleotide-activated sugars are sequentially added to dolichol pyrophosphate (the
polyprenol that serves as the lipid glycan
carrier in eukaryotic N‑glycosylation) on the
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cytoplasmic face of the ER membrane. The
lipid-linked heptasaccharide is then trans­
located to face the ER lumen, where it is further modified by the addition of seven more
sugars, derived from individual dolichol
phosphates that are charged on the cytoplasmic face of the ER and flipped to face the
lumen. The resulting 14‑subunit branched
oligosaccharide is added en bloc to a target
protein and subsequently undergoes trimming and remodelling through the addition
of other sugars, such as sialic acids, as the
glycoprotein travels through the ER and
Golgi; differential trimming and remodel­
ling of this oligosaccharide yields one of a
set of tissue-, development stage- or proteinspecific N‑linked glycans. In plants and
lower eukaryotes (for example, invertebrates
and protists)9, variants of this oligomer
— using the same N‑acetylglucosamine
(GlcNAc)- and mannose-based core but
lacking or including additional sugar residues from a limited roster at various positions — serve to expand the repertoire of
N‑linked glycans.
In bacteria, N‑glycosylation is thought
to be restricted to deltaproteobacteria and
epsilonproteobacteria6, and to date the
pathway involved has been analysed only
in Campylobacter jejuni 6,10. In this species, a
heptasaccharide is assembled by the ordered
addition of seven nucleotide-activated sugars
onto undecaprenol pyrophosphate, which is
found in the cytoplasm-facing leaflet of the
plasma membrane. After translocation of
the lipid-linked heptasaccharide across the
membrane, the glycan is delivered to select
Asn residues of the modified protein without
undergoing further processing.
An examination of the structures of
N‑linked glycans from almost 30 different
Campylobacter spp., along with Wolinella
succinogenes, Helicobacter pullorum and
Helicobacter winghamensis, revealed
some variability in glycan content and
in the identity of the Asn-linking sugar,
with di-N-acetylbacillosamine and
N‑acetylhexosamines fulfilling this role11,12.
This variety, however, pales in comparison to that found in the archaeal N‑linked
glycans. Although the process of archaeal
N‑glycosylation is generally reminiscent of
its eukaryotic and bacterial counterparts
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Table 1 | N‑glycosylation across the three domains of life
Feature
Eukarya
Bacteria
Archaea
Carrier molecules
Dolichol phosphate
Dolichol pyrophosphate
Undecaprenol pyrophosphate
Dolichol phosphate
Dolichol pyrophosphate
Degree of lipid saturation
Monosaturated
Not saturated
Disaturated
Polysaturated
Flippase or associated protein
RFT1
PglK
AglR
N‑glycan diversity
Limited
Limited
Extensive
Linking sugars
GlcNAc
diNacBac
HexNAc
Variable
Multibranched N‑glycan
Yes
No
Yes
N‑glycan modification
Yes
No
Yes
Distinct N-glycans on the same protein
No
No
Yes
Composition
Multimer or single subunit
Single subunit
Single subunit
Catalytic subunit
Stt3
PglB
AglB
Catalytic motifs*
WWDYG
DXXKXXX(M/I)
WWDYG
MXXIXXX(I/V/W)
WWDYG
DXXKXXX(M/I) or
MXXIXXX(I/V/W) or
EXnKXXX(M/I/P)
Sequons recognized‡
NX(S/T)
NX(S/T)
(D/E)XNX(S/T)
NX(S/T)
NX(N/V/L)
Lipid glycan carrier
N‑linked glycan
Oligosaccharyltransferase
Agl, archaeal glycosylation; diNAcBac, di-N-acetylbacillosamine; HexNAc, N‑acetylhexosamine; GlcNAc, N-acetylglucosamine; Pgl, protein glycosylation.
*X represents any amino acid. ‡X represents any amino acid except Pro.
(TABLE 1), analysis of the 13 archaeal N‑linked
glycans that have been characterized to date
(FIG. 1) reveals a diversity in size, architecture
and composition (even in the composition
of the different glycans that can be N‑linked
to the same protein) that is not paralleled in
the other two domains. Indeed, when one
considers the limited number of archaeal
species for which the N‑linked glycans have
been elucidated, it is likely that the diversity
reported thus far reflects only the tip of the
iceberg 13. It follows that such diversity would
reflect processing by an equal number of
different archaeal N‑glycosylation pathways,
in marked contrast to the relatively conserved systems that are present in the other
two domains. Recent studies of halophilic,
methanogenic and thermophilic archaea
are discussed below and show that this does
indeed seem to be the case.
Halophiles: two routes to the same glycan
The simple growth requirements of halo­
archaea, coupled with the large set of genetic
and biochemical tools available for working
with these organisms and their proteins,
despite the high salt requirements of these
species14, means that they currently offer
the most detailed insights into archaeal
N‑glycosylation. In Haloferax volcanii, a
series of Agl (archaeal glycosylation) proteins constitute the N‑glycosylation pathway
used to assemble the pentasaccharide that
modifies glycoproteins in this species; this
pentasaccharide is composed of a hexose,
two hexuronic acids, a methyl ester of
hexuronic acid and a mannose7,15 (FIGS 1,2a).
The pathway begins with nucleotideactivated versions of the first four N‑linked
pentasaccharide subunits being sequentially
added to a common dolichol phosphate via
the ordered actions of the glycosyltransferases AglJ, AglG, AglI and AglE16–19. At the
same time, AglD adds the final mannose
subunit of the pentasaccharide to a unique
dolichol phosphate20. Despite serving a similar function, Hfx. volcanii dolichol phosphate
can be distinguished from its eukaryotic
counterpart because the eukaryotic molecule
is saturated only at the α-isoprene, whereas
the haloarchaeal molecule is saturated at
both the α-isoprene and the ω-isoprene16,21.
Dolichol phosphate loaded with the tetrasaccharide (and its precursors) is translocated
across the haloarchaeal plasma membrane in
an as‑yet-unknown manner, at which point
the glycan moiety is transferred to a select
Asn residue in a target protein by the oligosaccharyltransferase (OST), AglB18. Dolichol
phosphate charged with the final mannose
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is flipped across the membrane in a process
involving AglR16,22. AglS then delivers the
terminal mannose subunit to the proteinbound tetrasaccharide23. In addition, AglF
(a glucose-1‑phosphate uridyltransferase),
AglP (a S-adenosylmethionine-dependent
methyltransferase) and AglM (a UDPglucose dehydrogenase) also contribute to
the pathway 24,25.
Like Hfx. volcanii, Haloarcula marismortui originates from the Dead Sea and
is surrounded by a protein shell based on
an S‑layer glycoprotein that is modified
by a similar N‑linked pentasaccharide to
that found on Hfx. volcanii glycoproteins26.
Despite this and the phylogenetic proximity
of the two species, each has a distinct strategy for glycan assembly. Rather than recruiting two different glycan-charged dolichol
phosphate carriers, as occurs in Hfx. volcanii,
Har. marismortui assembles the entire
N‑linked glycan on a single dolichol phosphate carrier 26. Thus, N‑glycosylation in
Hfx. volcanii, involving multiple lipid glycan
carriers in a multistep protein-processing
event, is reminiscent of the eukaryotic pathway 1,2, whereas the same post-translational
modification process in Har. marismortui
is similar to the bacterial pathway, as exemplified by C. jejuni, in which the complete
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Archaeoglobus fulgidus
Halobacterium salinarum
N
–
O3
–
O3
–
O3
OS
OS
N
–
H3
O3
OC
OS
N
10–15
N
H
Methanococcus maripaludis
CO
3
Methanococcus voltae
T
T
T
T
N
N
–
O3
OS OS OS
N
Haloferax volcanii
–
O3
N
N
Methanothermus fervidus
Sulfolobus acidocaldarius
–
O3
H3 H3
OC OC
OS
N
N
Pyrococcus furiosus
Thermoplasma acidophilum
N
N
Hexose
N-acetylhexosamine
Hexuronic acid
Deoxy-hexose
Galactose
N-acetylgalactosamine
Galacturonic acid
Galactofuranose
Glucose
N-acetylglucosamine
Glucuronic acid
Di-N-acetylglucuronic acid
Mannose
N-acetylmannuronic acid
3-acetamidino-N-acetylmannuronic acid
(5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose
Quinovose
6-deoxy-6-C-sulpho-D-galactose
α-D-glycero-D-galacto-heptose
Rhamnose
Pentose
220 kDa or 262 kDa moiety
N Asn
T Thr
Figure 1 | The diversity of N‑linked glycans decorating archaeal glycoproteins. The composition
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of the characterized N‑linked glycans discussed in the text is presented,Nature
along with
the composition
of
the N-linked glycans from Methanothermus fervidus62 and Thermoplasma acidophilum63. The Asn residue
of the target protein (to which the glycan is added) is shown for each glycan.
N‑linked glycan is assembled on a single
lipid carrier and then delivered to the protein
target as a complete unit 6,10.
N‑glycosylation and the methanogens
Methanogens have also provided substantial insights into the process of archaeal
N‑glycosylation. The first archaeal genes
involved in N‑glycosylation were reported in
an isolate of Methanococcus voltae str. PS27.
In this strain, glycoproteins are modified
by a N‑linked trisaccharide28, although in a
different isolate of the same strain this tri­
saccharide is extended by an additional
220 kDa or 262 kDa moiety that is apparently
carbo­hydrate in nature29 (FIG. 1). Today, many
of the steps in M. voltae N‑glycosylation are
known (FIG. 2b). AglH charges the phospho­
dolichol with GlcNAc, the first sugar of the
N‑linked glycan. This role for AglH is
supported by the ability of the aglH gene
to comple­ment a conditionally lethal mutation in S. cerevisiae alg7, the gene encoding
the enzyme responsible for adding GlcNAc
to the dolichol phosphate carrier in eukaryotic N-glycosylation30. To date, this is the
only example of a component of an archaeal
N‑glycosylation pathway that can function
across domain lines. AglC and AglK contribute to the biosynthesis or addition of
the second sugar subunit, a di‑N‑acetyl­
glucuronic acid, and AglA is involved in
adding the final sugar, N-acetylmannuronic
acid, of the N‑linked trimer 29. Finally, as in
other archaea, AglB is the OST that delivers
the presumably lipid-bound glycan to an
Asn residue in the target protein27.
Despite its phylogenetic proximity to
M. voltae, Methanococcus maripaludis produces glycoproteins that carry a distinct
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N‑linked glycan (FIGS 1,2c). Recent studies
have revealed that in M. maripaludis, modified proteins are decorated with a tetrasaccharide that can be distinguished from its
M. voltae counterpart through the use of
N‑acetylgalactosamine instead of GlcNAc
as the linking first sugar, by the presence
of a 3‑acetamidino moiety on the third
sugar subunit (which is otherwise common
to the N‑linked glycans in both species)
and by the novel sugar ((5S)-2‑acetamido2,4‑dideoxy-5-O-α‑l‑erythro-hexos‑5‑ulo1,5‑pyranose) found at the fourth and final
position of the M. maripaludis glycan31.
This novel sugar is the first example of a naturally occurring diglycoside of an aldulose.
It has also been shown that MMP1685, the
major structural subunit of M. maripaludis
pilin, carries the same tetrasaccharide
modified by a hexose branch attached to
the N‑acetylgalactosamine subunit 32 (FIG. 1).
M. maripaludis relies on a distinct set of
Agl enzymes for N‑glycosylation compared
with M. voltae, with the exception of AglA,
which adds a similar third sugar to the
di‑N‑acetylglucuronic acid at position two
in both organisms, and AglB, which serves
as the OST in both organisms27,33(FIG. 2b,c).
To date, only a limited number of the
species-specific M. maripaludis pathway
components have been identified —
namely, AglO, which is involved in adding
the second sugar subunit, and AglL, which
has been assigned a role in adding the final
sugar subunit and/or the Thr attached to
the third sugar of the glycan33 (the former
role is more likely, given the homology
of AglL to other glycosyltransferases).
Moreover, the absence of the Thr moiety
from the third N‑glycan subunit in cells
lacking AglL could mean that the Thr modification occurs only after addition of the
final sugar. Finally, it has been reported that
AglY generates ammonia, which is subsequently tunnelled through AglZ to AglX,
the amidotransferase that modifies the
third sugar residue for the M. maripaludis
N‑linked glycan with an acetamidino group
before addition of the modified sugar by
AglA34 (FIG. 2c).
In both M. voltae27 and M. maripaludis33
(and also in Hfx. volcanii 15), N‑glycosylation
of the archaeal flagellum, or archaellum35, is
important for its assembly and therefore for
cellular motility, underscoring the importance
of this post-translational modification in
archaea.
Branching out in a thermoacidophile
At present, the majority of insights into
archaeal N‑glycosylation have come
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a Haloferax volcanii
OCH3
OCH3
AglS
N
AglB
OCH3
N
AglR
AglJ
Mannose
AglD
Hexuronic acid
AglG
UDP
AglM
UDP AglE
UDP
Hexose
OCH3
AglI
N Asn
AglP
SAM S-adenosylmethionine
SAM
AglM
UDP
AglF
P
b Methanococcus voltae
T
T
N
AglB
AglH
N-acetylmannuronic acid
AglC
AglK
Di-N-acetylglucuronic acid
T
AglA
N-acetylglucosamine
T
T Thr
N Asn
c Methanococcus maripaludis
T
T
AglB
AglO
T
AglA
AglY
N-acetylgalactosamine
T
AglZ
Q + H2O
(5S)-2-acetamido-2,4-dideoxy-5-O-methylα-L-erythro-hexos-5-ulo-1,5-pyranose
3-acetamidino-N-acetylmannuronic acid
Di-N-acetylglucuronic acid
AglL
AglX
N
NH3
E
N-acetylmannuronic acid
T Thr
Q Gln
E Glu
N Asn
Figure 2 | N‑glycosylation pathways in archaea. Our current understanding
of the N‑glycosylation
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pathways of three archaeal species, on the basis of both in vitro and in vivo findings. In each case, various
Agl (archaeal glycosylation) proteins catalyse the assembly and attachment of the N‑linked glycan, as
well as the biosynthesis of the glycan sugar subunits. In each pathway, the glycans are assembled on
a dolichol phosphate molecule. a | Haloferax volcanii. b | Methanococcus voltae. The N‑linked glycan
can include an additional 220 kDa or 262 kDa sugar moiety as a fourth subunit. c | Methanococcus
maripaludis. In this species, pili are glycosylated with a variation on the shown glycan, in which an
additional hexose subunit branches from the N-acetylgalactosamine.
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from studies of members of the phylum
Euryarchaeota, a major archaeal phylum
that includes the halophiles and the
methano­gens. Recently, however, studies
on Sulfolobus acidocaldarius, a member
of a second major archaeal phylum,
Crenarchaeota, have also enhanced our
understanding of this post-translational
modification.
In S. acidocaldarius, an organism that
grows optimally at pH 2–3 and at 75 °C
–80 °C36, the S‑layer protein SlaA is highly
N‑glycosylated, particularly in the carboxy‑
terminal region, where a tri-branched hexasaccharide is detected every 30–40 residues37.
Previously, the same N‑linked glycan had
been shown to decorate cytochrome b558/566 in
this species38. This N‑linked hexasaccharide
has attracted attention for several reasons.
First, the glycan contains 6‑sulphoquinovose
(6‑deoxy‑6‑sulphoglucose) (FIG. 1), an
unusual sugar that is commonly found
only in the photosynthetic membranes of
plants and phototrophic bacteria39. Second,
like N‑linked oligosaccharides in higher
eukaryotes1,2, the S. acidocaldarius glycan
is N‑linked via a chitobiose core, namely a
pair of GlcNAc residues. Finally, the multibranched structure of this archaeal hexa­
saccharide is a property that is shared by the
N‑linked glycans that decorate eukaryotic
glycoproteins1–3.
With a well-stocked genetic toolbox
now available for manipulating S. acidocaldarius — including tools such as markers
for selection, expression vectors containing
inducible promoters and the ability to create
deletion strains40 — we have begun to define
the pathway responsible for the biogenesis
of the N‑linked tri-branched glycan41. Such
efforts have, however, been complicated by
the limited homology of S. acidocaldarius
glycosyltransferases to their counterparts in
other species. Moreover, although glycosyltransferase-encoding genes are clustered in
the vicinity of aglB in many euryarchaeotes,
this is not the case in S. acidocaldarius and
most other crenarchaeotes42. Nonetheless,
gene deletion and mass spectrometry have
been successfully used to identify at least
one gene involved in the S. acidocaldarius
N‑glycosylation pathway, namely agl3
(Saci_0423), which was shown to encode
a UDP-sulphoquinovose synthase41. The
same study predicted that the neighbouring
genes, Saci_0421, Saci_0422 and Saci_0424,
encode additional N‑glycosylation pathway
components, namely Agl1 (annotated as
a membrane-bound glycosyltransferase),
Agl2 (annotated as a dTDP-glucose pyro­
phosphorylase) and Agl4 (annotated as
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a glucokinase), respectively. In addition,
another study described a hexose-charged
dolichol phosphate that is possibly involved
in S. acidocaldarius N‑glycosylation; this
dolichol phosphate is saturated at the α-,
ω- and internal-position isoprenes and is
shorter than the dolichol phosphates seen in
other species43.
Structural insights into OST function
In archaea (as in bacteria and eukaryotes),
N‑glycosylation occurs at Asn residues
within an Asn‑Xaa‑Ser/Thr motif, known
as the sequon (in which Xaa is any residue
except Pro)44,45, although N‑glycosylation
of Asn‑Xaa‑Asn/Leu/Val has been reported
in Hbt. salinarum46. In some bacteria, such
as C. jejuni, the sequon can be extended to
Asp/Glu‑Xaa‑Asn‑Xaa‑Ser/Thr 47 (TABLE 1).
In all three domains, N‑linked glycans are
transferred from polyprenol carriers to target Asn residues by the actions of OSTs. In
S. cere­visiae and higher eukaryotes, the OST
is a multisubunit complex based on the catalytic subunit Stt348. In bacteria and archaea,
OSTs consist of a single subunit, the Stt3
homologues protein glycosylation B (PglB)
and AglB, respectively 6,27,49–51; Stt3‑based
single-subunit OSTs can also be found in
lower eukaryotes52 (TABLE 1). Archaeal OSTs
possess traits that are seemingly unique to
this domain of organisms. For instance,
although Hbt. salinarum encodes a single
AglB, this enzyme is apparently able to
catalyse the transfer of two distinct glycans
to the S‑layer glycoprotein, one from a dolichol phosphate carrier and another from a
dolichol pyrophosphate carrier 53. A comparable situation might occur in Hfx. volcanii,
in which the single AglB identified would
have to attach the two distinct N‑linked
glycans that decorate the S‑layer glycoprotein, despite the fact that different linking
sugars are used in each case (see below)54.
However, many archaeal species encode
multiple AglBs, although the physiological
significance of this observation remains
unclear 42. Future studies on Methanosarcina
spp., which encode multiple AglBs42 and for
which advanced genetic tools are available55,
could help elucidate this point.
Recently, the crystal structures were
solved for the catalytic carboxy‑terminal
domains of AglB proteins from the hyperthermophiles Pyrococcus furiosus and
Archaeoglobus fulgidus, and these structures
have shed new light on the workings of
these OSTs50,51, identifying novel sequence
motifs involved in OST catalytic activity
other than the evolutionarily conserved
Trp-Trp-Asp- Tyr-Gly consensus motif 42,56–58.
Despite the low sequence homology between
Stt3, PglB and AglB proteins, an alignment
based on the P. furiosus AglB and C. jejuni
PglB structures revealed that a so‑called
DK motif (DXXKXX(M/I), in which X is
any amino acid) is restricted to eukaryotic Stt3 and most archaeal AglB proteins,
whereas bacterial PglB and the remaining
AglB proteins contain a distinct MI motif
(MXXIXXX(I/V/W), in which X is any
amino acid) at this position56 (TABLE 1).
Further analysis of the crystal structure of
a bacterial PglB in complex with an acceptor substrate revealed that the DK and
MI motifs contribute to the recognition
pocket surrounding the Ser/Thr residue
at the +2 position upstream of the glycosylated Asn in the target protein, and that
this pocket also contains the invariable
Trp-Trp-Asp portion of the Trp-Trp-AspTyr-Gly consensus motif of Stt3–PglB–AglB
proteins58. The structural information
obtained about the carboxy‑terminal catalytic domain of one of the three A. fulgidus
AglB proteins, AF_0329 (which is the
shortest Stt3–PglB–AglB protein known),
served to further classify these proteins by
identifying AglB proteins presenting a variant of the DK motif 51 (TABLE 1). Finally, by
analysing the in vitro activity of P. furiosus
AglB, the transfer of a lipid-linked heptasaccharide to an acceptor peptide was
demonstrated. This glycan, comprising
two N‑acetylhexosamines, two hexoses, a
hexuronic acid and two pentose branches50,
represents yet another distinct archaeal
N‑linked glycan structure (FIG. 1).
Coping with extremes
Archaea are found in a broad range of
environ­ments that are often extreme in
nature59. It is thus possible that the enormous
diversity seen in archaeal N‑glycosylation
reflects species-specific means of coping
with the environmental challenges encountered. The observation that S. acidocaldarius
cells lacking Agl3 grow increasingly poorly
as the NaCl concentration of the growth
medium increases41 suggests a physiological
role for cell surface N‑glycosylation in this
species. As the mutant cells do not contain
sulphoquinovose, they lack the negative
charges that are normally provided by this
sugar, which are thought to contribute to
the hydrated shell that surrounds the cell.
Therefore, S‑layer stability in the mutant
strain becomes compromised in an increasingly saline environment, affecting growth.
Indeed, an enhanced surface charge in the
face of increased salinity was also offered
as the reason for the higher proportion of
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sulphated sugars in the N‑linked glycans
decorating the S‑layer glycoprotein of the
extreme halophile Hbt. salinarum, relative
to the proportion in the N-linked glycans
modifying the same protein in Hfx. volcanii,
a moderate halophile60. Moreover, disruption
of Hfx. volcanii N‑glycosylation renders the
S‑layer surrounding the cell more susceptible
to proteolytic degradation18–20.
However, recent studies have shown
that the interplay between N‑glycosylation
and environmental conditions is more
complex than previously imagined54. When
grown in medium containing 3.4 M NaCl,
the Hfx. volcanii S‑layer glycoprotein residues Asn13 and Asn83 are modified by an
N‑linked pentasaccharide. However, when
the same cells are grown in medium containing 1.75 M NaCl, significant changes in
the N‑linked glycan profile of the protein
are observed. Although Asn13 and Asn83 are
still modified by the same pentasaccharide,
such modification occurs on a substantially
smaller proportion of the glycoprotein.
More strikingly, Asn498, a position that is
not modified when the cells are grown at
the higher salinity, carries a novel tetra­
saccharide comprising a sulphated hexose,
two hexoses and a terminal rhamnose sub­
unit (FIG. 1). At present, it is not clear what
advantage is offered to Hfx. volcanii by
such differential N‑glycosylation in
response to changes in environmental
salinity. It is equally striking that the Hfx.
volcanii S‑layer glycoprotein can be simultaneously modified by two distinct N‑linked
glycans. Apart from for the Hbt. salinarum
S‑layer glycoprotein53, this phenomenon has
not been reported for any other species or
protein4.
Outlook
With more archaeal genome sequences
becoming readily available and as new and
improved molecular tools for working with
the different archaeal species start to accumulate, we are beginning to make major
advances in our understanding of archaeal
N‑glycosylation. Such efforts will elucidate
how protein glycosylation contributes to the
remarkable ability of archaea to overcome
the physical challenges associated with the
extreme environments that they inhabit.
These efforts will also provide novel insight
into the evolution of this universal posttranslational modification. In addition,
we can expect to see new applied uses for
extremo­philic enzymes that can be tailored to
meet specific needs via glyco-engineering 61.
Looking forward, the future certainly
looks sweet.
VOLUME 11 | MARCH 2013 | 155
© 2013 Macmillan Publishers Limited. All rights reserved
PROGRESS
Jerry Eichler is at the Department of Life Sciences,
Ben Gurion University of the Negev, P.O. Box 653,
Beersheva 84105, Israel.
e-mail: [email protected]
doi:10.1038/nrmicro2957
Published online 28 January 2013
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Acknowledgements
Work in the author’s laboratory is supported by the Israel
Science Foundation (grant 8/11) and the US Army Research
Office (W911NF-11‑1‑520).
Competing interests statement
The author declares no competing financial interests.
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