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Glycoproteins
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•
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Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
The Eukaryotic Cell Surface
extracellular
lipid bilayer
fibroblast
intracellular
Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969
Lodish, et al. (1995) Molecular Cell Biology 3rd ed.
HIV gp120
“Structure” of HIV gp120
Zolla-Pazner (2004)
Nature Reviews Immunol 4, 199
Burton DR et al (2012) Cell Host & Microbe 4:396
HIV gp120
Doores K J et al. (2010) PNAS 107:17107
3 kDa N-linked
glycans
Proteins fold onto
themselves…
glycans spread out
in space
1.5 kDa GPI
anchor
18 kDa protein
membrane
Thy-1
cell-cell interaction
protein
Essentials of Glycobiology
Second Edition
Roles of protein glycosylation
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Protein folding*
Protein conformation
Protein trafficking*
Cell surface glycoprotein lattice*
Regulation of signaling pathways*
Cell-cell interactions*
* via glycan recognition
Protein Folding:
N-linked protein glycosylation is essential for chaperone-assisted
folding of glycoproteins in the endoplasmic reticulum
Asn
Helenius, A. and Aebi, M. (2001) Science 291, 2364-2369
Protein Folding:
Children with altered protein N-glycosylation have diverse motor,
physiological and cognitive deficits
Joel
Zachary
http://www.cdgs.com/
Megan
Protein Conformation:
Closely spaced short O-linked glycans on mucins
Protein Conformation:
Conserved glycosylation of IgG is required for interaction with
immune effectors (complement, Fc-receptors)
Protein Trafficking:
Unique glycosylation of lysosomal enzymes “labels” them for
trafficking to the lysosome via the Man-P receptor(s)
I-cell disease
(GlcNAc
phosphotransferase)
https://wikispaces.psu.edu/v
Asn
Cell surface glycoprotein lattice:
Sugar binding proteins (galectins) bind to
and stabilize cell surface glycoproteins
Altered glycoprotein glycans result in
internalization of key glycoproteins
Lau et al (2007) Cell 129:123
Pancreatic β cells stained for Glut-2 (green), insulin (red), and DNA (blue).
Ohtsubo et al (2005) Cell 123:1307
Regulation of signaling pathways:
Notch signaling during development is driven by glycosylation
Stanley (2007)
Curr Opin Struct Biol 17:530
Stanley & Okajima (2010)
Curr Topics Devel Biol 92:131
Glycans in cell-cell interactions
Carbohydrate
binding
determinant
Carbohydrate
recognition
domain (CRD)
Lectin
Glycan
Sharon and Lis (1993) Scientific American
Roles of protein glycosylation
•
•
•
•
•
•
Protein folding*
Protein conformation
Protein trafficking*
Cell surface glycoprotein lattice*
Regulation of signaling pathways*
Cell-cell interactions*
* via glycan recognition
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
Modes of glycosylation
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
“N-linked” glycan linkage to protein asparagine residues
N-glycosidic
bond
N-linked glycan
Major classes of N-linked glycans
High mannose
Terminal Saccharide Diversity --- A key to recognition
Examples of structures found as termini on the branches
of complex N-linked glycoprotein glycans….
NeuAc 2-6 Gal 1-4 GlcNAc--NeuAc 2-3 Gal 1-4 GlcNAc--Gal 1-3 Gal 1-4 GlcNAc--4-SO3-GalNAc 1-4 GlcNAc--(-Gal 1-4 GlcNAc 1-3-)n = poly-N-acetyllactosamine
(n may be >6)
(-NeuAc 2-8-) n = polysialic acid
(n may be >50)
Fuc residues
(e.g. Fuc 1-2 Gal; Fuc 1-3 GlcNAc; Fuc 1-4 GlcNAc
Human spleen
Human
spleen
Human
spleen
Human kidney
Humankidney
kidney
Human
N-linked glycosylation consensus sequence
The N-linked amino acid consensus
sequence is “Asn-[any AA]- [Ser or Thr]”
where the middle amino acid is not Pro
Glycoforms
Whereas a protein is defined by its unique amino acid
sequence, the glycan structure at a single protein
glycosylation site varies.
Although glycan structures vary at each glycosylation
site, the profile is reproducible, protein-specific, sitespecific, and cell type-specific.
Three glycoforms of CD59, a compliment regulatory protein
Human erythropoietin glycosylation
“Humanization” of
yeast to produce
complex N-linked
glycoproteins
Hamilton, et al. Science 313, 1441-1443 (8 September 2006)
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
“O-linked” glycan linkage to protein serine residues
O-linked glycan
O-linked glycan
classes & diversity
Human spleen
O-GlcNAc
Note: No further sugar substitutions have been confirmed
Key Features of O-linked GlcNAc
 O-GlcNAc is localized to the cytoplasm and nucleus.
 O-GlcNAc is present in all higher eukaryotes studied.
 O-GlcNAc is as abundant as phosphorylation.
 UDP-GlcNAc is as abundant as ATP in some cell types.
 O-GlcNAc containing proteins are phosphoproteins.
 O-GlcNAc and phosphorylation are often reciprocal.
 O-GlcNAc is dynamic, implying a regulatory role.
Three-state model: unmodified / O-GlcNAc / phosphate
OGT
Phosphatase
Phosphatase
OGT
O-GlcNAc
OPO32Unmodified
protein
Phosphoprotein
Glycoprotein
O-GlcNAcase
Kinase
O-GlcNAcase
Kinase
Natasha Zachara
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis
Glycosyltransferase reaction
(UDP-Gal:glucose β4 galactosyltransferase)
+
nucleotide sugar donor
acceptor
glycoside
Sugar donors
http://www.cazypedia.org/index.php/Glycosyltransferase
Additional donor intermediates
http://www.cazypedia.org/index.php/Glycosyltransferase
Diversity in glycosyltransferases –
Example: 20 human sialyltransfearse genes
“St6Gal ” genes (2)
“St3Gal ” genes (6)
“St6GalNAc ” genes (6)
“St8Sia ” genes (6)
General rule: at least one distinct enzyme for every linkage
Glycoprotein biosynthesis
• N-Linked – en-bloc preconstructed core, trimming,
terminal elaboration
• O-Linked - sugar by sugar addition
• O-GlcNAc - dynamic transferase/glycosidase
Glycolipid biosynthesis
• Stepwise sugar-by-sugar addition
• GPI Anchor – en-bloc preconstructed core, elaboration
Proteoglycan biosynthesis
• Stepwise sugar-by-sugar addition (core and repeating
disaccharide)
• Post-polymerization modifications
N-linked glycosylation starts with a lipid-linked intermediate:
Glc3Man9GlcNAc2-P-P-Dol
N-linked glycosylation: Core biosynthesis and transfer
Freeze & Aebi (2005) Curr Opin Struct Biol 15:490
N-linked glycosylation: Oligosaccharyltransferase (OST)
Mohorko et al (2011) J Inherit Metab Dis 34:869
N-linked glycosylation: Trimming and terminal elaboration
Freeze & Aebi (2005) Curr Opin Struct Biol 15:490
Congenital Disorders of Glycosylation (CDG)
Courtesy Hudson H. Freeze, Burnham Institute, La Jolla, CA
Congenital Disorders of Glycosylation (CDG)
Freeze & Aebi (2005) Curr Opin Struct Biol 15:490
Congenital Disorders of Glycosylation (CDG)
Freeze & Aebi (2005) Curr Opin Struct Biol 15:490
Glycoproteins
•
•
•
•
Roles of glycoprotein glycosylation
Modes of glycoprotein glycosylation
N-linked glycans
O-linked glycans
– O-GalNAc – “mucin” type glycosylation
– O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules
– O-GlcNAc – intracellular protein regulation
• Glycan Biosynthesis
– General Principles
– N-linked glycoprotein biosynthesis