GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

The Eukaryotic Cell SurfaceThe Eukaryotic Cell Surface

Lodish, et al. (1995) Molecular Cell Biology 3rd ed.Lodish, et al. (1995) Molecular Cell Biology 3rd ed.

lipid bilayerlipid bilayer

fibroblastfibroblast

extracellularextracellular

intracellularintracellular

Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969

HIV gp120HIV gp120HIV gp120HIV gp120

“Structure” of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunol 4, 199

Burton DR et al (2012) Cell Host & Microbe 4:396

HIV gp120HIV gp120HIV gp120HIV gp120

Doores K J et al. (2010) PNAS 107:17107

Proteins fold onto Proteins fold onto themselves… themselves…

glycans spread out glycans spread out in spacein space

Thy-1cell-cell interaction

protein

1.5 kDa GPI

anchor

3 kDa N-linked

glycans

18 kDa protein

membrane

Second Edition

Essentials of Glycobiology

Roles of protein glycosylationRoles of protein glycosylation

• Protein folding*Protein folding*• Protein conformationProtein conformation• Protein trafficking*Protein trafficking*• Cell surface glycoprotein lattice*Cell surface glycoprotein lattice*• Regulation of signaling pathways*Regulation of signaling pathways*• Cell-cell interactions*Cell-cell interactions*

• Protein folding*Protein folding*• Protein conformationProtein conformation• Protein trafficking*Protein trafficking*• Cell surface glycoprotein lattice*Cell surface glycoprotein lattice*• Regulation of signaling pathways*Regulation of signaling pathways*• Cell-cell interactions*Cell-cell interactions*

* via glycan recognition* via glycan recognition

Protein Folding: Protein Folding:

N-linked protein glycosylation is essential for chaperone-assisted N-linked protein glycosylation is essential for chaperone-assisted folding of glycoproteins in the endoplasmic reticulum folding of glycoproteins in the endoplasmic reticulum

Helenius, A. and Aebi, M. (2001) Science 291, 2364-2369

Asn

http://www.cdgs.com/

Joel

Zachary

Megan

Protein Folding: Protein Folding:

Children with altered protein N-glycosylation have diverse motor, Children with altered protein N-glycosylation have diverse motor, physiological and cognitive deficits physiological and cognitive deficits

Protein Conformation: Protein Conformation: Closely spaced short O-linked glycans on mucinsClosely spaced short O-linked glycans on mucins

Protein Conformation: Protein Conformation: Conserved glycosylation of IgG is Conserved glycosylation of IgG is requiredrequired for interaction with for interaction with

immune effectors (complement, Fc-receptors) immune effectors (complement, Fc-receptors)

Protein Trafficking: Protein Trafficking: Unique glycosylation of lysosomal enzymes “labels” them for Unique glycosylation of lysosomal enzymes “labels” them for

trafficking to the lysosome via the Man-P receptor(s)trafficking to the lysosome via the Man-P receptor(s)

https://wikispaces.psu.edu/v

Asn

Cell surface glycoprotein lattice: Cell surface glycoprotein lattice: Sugar binding proteins (galectins) bind to Sugar binding proteins (galectins) bind to

and stabilize cell surface glycoproteinsand stabilize cell surface glycoproteins

Lau et al (2007) Cell 129:123

Altered glycoprotein glycans result in Altered glycoprotein glycans result in internalization of key glycoproteinsinternalization of key glycoproteins

Pancreatic β cells stained for Glut-2 (green), insulin (red), and DNA (blue). Ohtsubo et al (2005) Cell 123:1307

Regulation of signaling pathways: Regulation of signaling pathways: Notch signaling during development is driven by glycosylation

Stanley (2007) Curr Opin Struct Biol 17:530

Sharon and Lis (1993) Scientific American

Carbohydrate binding determinant

Carbohydrate recognition domain (CRD)

Glycans in cell-cell interactionsGlycans in cell-cell interactions

Lectin

Glycan

Roles of protein glycosylationRoles of protein glycosylation

• Protein folding*Protein folding*• Protein conformationProtein conformation• Protein trafficking*Protein trafficking*• Cell surface glycoprotein lattice*Cell surface glycoprotein lattice*• Regulation of signaling pathways*Regulation of signaling pathways*• Cell-cell interactions*Cell-cell interactions*

• Protein folding*Protein folding*• Protein conformationProtein conformation• Protein trafficking*Protein trafficking*• Cell surface glycoprotein lattice*Cell surface glycoprotein lattice*• Regulation of signaling pathways*Regulation of signaling pathways*• Cell-cell interactions*Cell-cell interactions*

* via glycan recognition* via glycan recognition

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

Modes of glycosylationModes of glycosylation

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

““N-linked” glycan linkage to protein asparagine residuesN-linked” glycan linkage to protein asparagine residues

N-glycosidicbond

N-linked glycanN-linked glycan

Major classes of N-linked glycansMajor classes of N-linked glycans

High mannose

Examples of structures found as termini on the branches Examples of structures found as termini on the branches of complex N-linked glycoprotein glycans….of complex N-linked glycoprotein glycans….

Terminal Saccharide Diversity --- A key to recognitionTerminal Saccharide Diversity --- A key to recognition

NeuAc NeuAc 2-6 Gal 2-6 Gal 1-4 GlcNAc---1-4 GlcNAc---NeuAc NeuAc 2-3 Gal 2-3 Gal 1-4 GlcNAc---1-4 GlcNAc---Gal Gal 1-3 Gal 1-3 Gal 1-4 GlcNAc---1-4 GlcNAc---4-SO4-SO33-GalNAc -GalNAc 1-4 GlcNAc---1-4 GlcNAc---

(-Gal (-Gal 1-4 GlcNAc 1-4 GlcNAc 1-3-)1-3-)nn = poly-N-acetyllactosamine = poly-N-acetyllactosamine

(n may be >6)(n may be >6)

(-NeuAc (-NeuAc 2-8-) 2-8-) nn = polysialic acid = polysialic acid

(n may be >50)(n may be >50)

Fuc residues Fuc residues (e.g. Fuc (e.g. Fuc 1-21-2 Gal; Fuc Gal; Fuc 1-3 GlcNAc; Fuc 1-3 GlcNAc; Fuc 1-4 GlcNAc1-4 GlcNAc

Human spleen

Human kidney

Human spleen Human kidneyHuman spleen Human kidney

N-linked glycosylation consensus sequenceN-linked glycosylation consensus sequence

The N-linked amino acid consensus The N-linked amino acid consensus sequence is “Asn-[any AA]- [Ser or Thr]” sequence is “Asn-[any AA]- [Ser or Thr]” where the middle amino acid is not Prowhere the middle amino acid is not Pro

GlycoformsGlycoforms

Whereas a protein is defined by its unique amino acid Whereas a protein is defined by its unique amino acid sequence, sequence, the glycan structure at a single protein the glycan structure at a single protein glycosylation site variesglycosylation site varies. .

Although glycan structures vary at each glycosylation Although glycan structures vary at each glycosylation site, the profile is reproducible, protein-specific, site-site, the profile is reproducible, protein-specific, site-specific, and cell type-specific.specific, and cell type-specific.

Three glycoforms of CD59, a compliment regulatory proteinThree glycoforms of CD59, a compliment regulatory protein

Human erythropoietin glycosylationHuman erythropoietin glycosylation

““Humanization” of Humanization” of yeast to produce yeast to produce complex N-linked complex N-linked glycoproteinsglycoproteins

Hamilton, et al. Hamilton, et al. Science Science 313, 1441-1443 (8 September 2006)313, 1441-1443 (8 September 2006)

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

“O-linked” glycan linkage to protein serine residues

O-linked glycanO-linked glycan

O-linked glycan O-linked glycan classes & diversityclasses & diversity

Human spleen

O-GlcNAc

Note: No further sugar substitutions have been confirmed

O-GlcNAc is localized to the cytoplasm and nucleus.O-GlcNAc is localized to the cytoplasm and nucleus.

O-GlcNAc is present in all higher eukaryotes studied.O-GlcNAc is present in all higher eukaryotes studied.

O-GlcNAc is as abundant as phosphorylation.O-GlcNAc is as abundant as phosphorylation.

UDP-GlcNAc is as abundant as ATP in some cell types. UDP-GlcNAc is as abundant as ATP in some cell types.

O-GlcNAc containing proteins are phosphoproteins.O-GlcNAc containing proteins are phosphoproteins.

O-GlcNAc and phosphorylation are often reciprocal.O-GlcNAc and phosphorylation are often reciprocal.

O-GlcNAc is dynamic, implying a regulatory role.O-GlcNAc is dynamic, implying a regulatory role.

Key Features of O-linked GlcNAc

Three-state model: unmodified / O-GlcNAc / phosphate

O-GlcNAc

GlycoproteinGlycoproteinUnmodified protein

Unmodified proteinPhosphoprotein

OPO32-

PhosphatasePhosphatase

OGTOGT

OGTOGT

PhosphatasePhosphatase

KinaseKinase

KinaseKinase

O-GlcNAcaseO-GlcNAcase

O-GlcNAcaseO-GlcNAcase

Natasha Zachara

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

Glycosyltransferase reactionGlycosyltransferase reaction(UDP-Gal:glucose (UDP-Gal:glucose ββ4 galactosyltransferase)4 galactosyltransferase)

nucleotide sugar donor acceptor glycoside

+

Sugar donorsSugar donors

http://www.cazypedia.org/index.php/Glycosyltransferase http://www.cazypedia.org/index.php/Glycosyltransferase

http://www.cazypedia.org/index.php/Glycosyltransferase http://www.cazypedia.org/index.php/Glycosyltransferase

Additional donor intermediatesAdditional donor intermediates

Diversity in glycosyltransferases – Diversity in glycosyltransferases – Example: 20 human sialyltransfearse genesExample: 20 human sialyltransfearse genes

“St3Gal ” genes (6)

“St6GalNAc ” genes (6)

“St6Gal ” genes (2)

“St8Sia ” genes (6)

General rule: at least one distinct enzyme for every linkage

Glycoprotein biosynthesisGlycoprotein biosynthesis

• N-Linked – en-bloc preconstructed core, trimming, N-Linked – en-bloc preconstructed core, trimming, terminal elaborationterminal elaboration

• O-Linked - sugar by sugar additionO-Linked - sugar by sugar addition• O-GlcNAc - dynamic transferase/glycosidaseO-GlcNAc - dynamic transferase/glycosidase

Glycolipid biosynthesisGlycolipid biosynthesis

• Stepwise sugar-by-sugar additionStepwise sugar-by-sugar addition• GPI Anchor – en-bloc preconstructed core, elaborationGPI Anchor – en-bloc preconstructed core, elaboration

Proteoglycan biosynthesisProteoglycan biosynthesis

• Stepwise sugar-by-sugar addition (core and repeating Stepwise sugar-by-sugar addition (core and repeating disaccharide)disaccharide)

• Post-polymerization modificationsPost-polymerization modifications

N-linked glycosylation starts with a lipid-linked intermediate: N-linked glycosylation starts with a lipid-linked intermediate: GlcGlc33ManMan99GlcNAcGlcNAc22-P-P-Dol-P-P-Dol

N-linked glycosylation: Core biosynthesis and transferN-linked glycosylation: Core biosynthesis and transfer

Freeze & Aebi (2005) Curr Opin Struct Biol 15:490Freeze & Aebi (2005) Curr Opin Struct Biol 15:490

N-linked glycosylation: Oligosaccharyltransferase (OST)N-linked glycosylation: Oligosaccharyltransferase (OST)

Mohorko et al (2011) J Inherit Metab Dis 34:869Mohorko et al (2011) J Inherit Metab Dis 34:869

N-linked glycosylation: Trimming and terminal elaborationN-linked glycosylation: Trimming and terminal elaboration

Freeze & Aebi (2005) Curr Opin Struct Biol 15:490Freeze & Aebi (2005) Curr Opin Struct Biol 15:490

Courtesy Hudson H. Freeze, Burnham Institute, La Jolla, CA

Congenital Disorders of Glycosylation (CDG)

Congenital Disorders of Glycosylation (CDG)

Freeze & Aebi (2005) Curr Opin Struct Biol 15:490Freeze & Aebi (2005) Curr Opin Struct Biol 15:490

Congenital Disorders of Glycosylation (CDG)

Freeze & Aebi (2005) Curr Opin Struct Biol 15:490Freeze & Aebi (2005) Curr Opin Struct Biol 15:490

GlycoproteinsGlycoproteinsGlycoproteinsGlycoproteins

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis

• Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation• Modes of glycoprotein glycosylationModes of glycoprotein glycosylation• N-linked glycansN-linked glycans• O-linked glycansO-linked glycans

– O-GalNAc – “mucin” type glycosylationO-GalNAc – “mucin” type glycosylation

– O-Man, O-Fuc, O-Glc, O-Xyl – signaling moleculesO-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules

– O-GlcNAc – intracellular protein regulationO-GlcNAc – intracellular protein regulation

• Glycan BiosynthesisGlycan Biosynthesis– General PrinciplesGeneral Principles

– N-linked glycoprotein biosynthesisN-linked glycoprotein biosynthesis