Primer

1.  The 4-epimer of Glucose once it is 2-N-acetylated is called?2.  If the SNFG shape of a monosaccharide is a diamond it is an ___?3.  Which of the 11 common mammalian monosaccharides is:

a) a pentose?b) a deoxyhexose?c) never incorporated as a sugar nucleotide?

4.  If a GT uses a one-step mechanism it is retaining or inverting?5.  Name:

6.  Shape/Color:

Gal GlcNAc Neu5Gc Xyl GlcA

O-Glycosylation

Ser/Thr

GalNAc

Mucins

Ser/Thr

Man*

Yeast mannoproteinsα-dystroglycan

cadherins

Ser/Thr

Fuc*

NotchCoagulation FactorsFibrinolytic Factors

Ser/Thr

GlcNAc*

Nuclear ProteinsCytoplasmic Proteins

After Esko, J

Ser/Thr

Glc*

NotchThrombospondin

Factor IX

ALSO: Proteoglycans, Hydroxyproline/Hydroxylysine Glycosylation

* SEPARATE LECTURES

O-Glycosidic Linkage

α

Ser

GalNAc

After Esko, J

O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)

β-elimination

O

OH

H

H

HO

H

O

NAcHH

OH

NH2CHC

H2C

O

GalNAc

α

Glycan synthesisin a cellular context

Most O-Glycosylatedproteins are synthesizedin the secretory pathway

O-Glycosylation

Ser/Thr

GalNAc

Ser/Thr

Man

Ser/Thr

Fuc

Ser/Thr

GlcNAc

Ser/Thr

Glc

Mucin-Type O-GalNAc Glycans•  Major extracellular vertebrate

O-glycan•  Begins in cis-Golgi (ERGIC) by

attachment of GalNAc in α-linkage to specific Ser/Thr residues

•  Assembly is simpler than N-linked chains - no lipid intermediate is used

•  Always involves nucleotide sugars

•  Always occurs by addition to non-reducing terminus or by branching

β4

β3

β4

β3

β4

α3

α3

α3

Ser/Thr

β3 β6

After Esko, J

Polypeptide GalNAc Transferases

•  >20 ppGalNAcT family members•  Share structural features in active site•  Some have lectin (ricin) domain

Regions in white, pink, red, and black represent, respectively, 0–29%, 30–69%, 70–99%, and 100% sequence identity (Hagen et al. (2003) Glycobiology 13:1R-16R).

After Esko, J

Core 1 and Core 2 Synthesis

After Esko, J

T (TF)Antigen

TnAntigen

Core 2GlcNAcT

Core 1GalT(cis)

Ser/Thr

β3 β6

Ser/Thr

β3

Ser/Thr

α6

α3

ST6GalNAc1(trans)

Ser/Thr

SialylTn

AntigenSer/Thr

β3

Ser/Thr

β3 α6Disialyl

TAntigen

From: Tongzhong Ju and Richard D. Cummings

Core 4GlcNAcT

Core 3GlcNAcT

Ser/Thr

β3 β6

Ser/Thr

β3

Ser/Thr

Core 3 and Core 4 Synthesis

After Esko, J

Core 3

Ser/Thr

β3

Core 4

Ser/Thr

β3 β6

Ser/Thr

β3

Core 1

Ser/Thr

β3 β6

Core 2

Core 7

Ser/Thr

α6

Core 6?

Ser/Thr

β6

Core 5

Ser/Thr

α3

Core 8

Ser/Thr

α3

Unusual Core O-Glycan Structures

After Esko, J

Mucins are Heavily O-glycosylated

•  Apomucin contain tandem repeats (8-169 amino acids) rich in proline, threonine, and serine (PTS domains)

•  Glycosylation constitutes as much as 80% of mass and tends to be clustered - bottle brush

•  Expressed by epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts

After Esko, J

Lung Epithelium

Goblet cells in intestinal crypts

Mucin Production

After Esko, J

Mucins: Protective Barriers for Epithelial Cells

•  Lubrication for epithelial surfaces•  Modulate infection:

– Receptors for bacterial adhesins– Secreted mucins can act as decoys

•  Barrier against freezing:– Antifreeze glycoproteins–  [Ala-Ala-Thr]n≤40 with Core 1

disaccharides

After Esko, J

Antigenic epitopes found in mucins

β4

β3

β4

β3

β4

α3

α3

α3

Ser/Thr

β3 β6

Type-3 H, A, and B antigens that form the O (H), A, and B blood group determinants on N- and O-glycans

Chapter 13, Figure 6Essentials of Glycobiology

Second Edition

A1 and A2 blood group antigens

Chapter 13, Figure 9Essentials of Glycobiology

Second Edition

Type-1 and -2 Lewis determinants

Chapter 13, Figure 10Essentials of Glycobiology

Second Edition

Lewis blood groups

Chapter 13, Figure 11Essentials of Glycobiology

Second Edition

Terminal GlcNAc residues are usually galactosylated

Chapter 13, Figure 2Essentials of Glycobiology

Second Edition

Poly-N-acetyllactosamine chains

Chapter 13, Figure 3Essentials of Glycobiology

Second Edition

i and I antigen synthesis

Chapter 13, Figure 4Essentials of Glycobiology

Second Edition

Synthesis and structure of the HNK-1 epitope

Chapter 13, Figure 21Essentials of Glycobiology

Second Edition

Structure and synthesis of the Galα1-3Gal antigen

Chapter 13, Figure 13Essentials of Glycobiology

Second Edition

NOT IN HUMANS!!!

Synthesis of glycoproteins bearing terminal α2-3 sialic acid transferred by the ST3Gal family of sialyltransferases

Chapter 13, Figure 17Essentials of Glycobiology

Second Edition

Synthesis of the human Sda or mouse CT antigen

Chapter 13, Figure 16Essentials of Glycobiology

Second Edition

Synthesis of α2-6 sialic acid on O-glycans and glycolipids by the ST6GalNAc family of sialyltransferases

Chapter 13, Figure 18Essentials of Glycobiology

Second Edition

Complex O-GalNAc glycans with different core structures

Chapter 9, Figure 2Essentials of Glycobiology

Second Edition

Questions

•  What is the function of multiple polypeptide GalNAc transferases?

•  How is tissue specific expression of transferases regulated?

•  How does competition of transferases for substrates determine the glycoforms expressed by cells and tissues?

•  What roles do chaperones play?

After Esko, J

O-Glycosylation

Ser/Thr

GalNAc

Ser/Thr

Man

Ser/Thr

Fuc

Ser/Thr

GlcNAc

Ser/Thr

Glc

O-Fuc

•  One of the clearest examples of glycosylation (Fringe) modulating signal transduction

•  What other proteins carry O-Fuc and how does glycosylation modulate activity?

•  How is glycosylation regulated?

•  Separate Lecture (Haltiwanger)

O-Glycosylation

Ser/Thr

GalNAc

Ser/Thr

Man

Ser/Thr

Fuc

Ser/Thr

GlcNAc

Ser/Thr

Glc

Shao, L. et al. Glycobiology 2002 12:763-770; doi:10.1093/glycob/cwf085

O-Glc Pathway

Rumi is OGluT

KDEL Retention Signal

Temp. Sensitive Mutation

O-Glc•  Always a trisaccharide?•  Glc & Xyl (except for proteoglycans) rarely

used on mammalian glycoproteins--why both here? Many of the same proteins as O-Fuc modifed, why?

•  Role in Modulating Signaling? Regulated by enzymes or sugar nucleotide availability?

•  Separate Lecture (Haltiwanger)

O-Glycosylation

Ser/Thr

GalNAc

Ser/Thr

Man

Ser/Thr

Fuc

Ser/Thr

GlcNAc

Ser/Thr

Glc

POMT1in the ER

In Complex

WithPOMT2

Uses Dol-ManAs Donor

Alpha-Dystroglycan

Muscular Dystrophies associated with glycosylation of α – DG (oversimplified)

Disease Species Affected

Gene Biochemical

Lesion Biochemical Phenotype Walker - Warburg Syndrome Human POMT1 O - Man addition to

Ser/Thr Decreased protein O - mannosylation

Muscle - Eye - Brain D isease Human POMGnT1 Addition of GlcNac

β 2 to O - Man Underglycosylated α - DG, uncapped O - Man

Fukuyama - type MDC Human Fukutin Glycosyltransferase -

like protein Underglycosylated α - DG

Limb - Girdle and MDC 1C Human

Fukutin - Related Protein

Glycosyltransferase - l ike Golgi protein Underglycosylated α - DG

Myodystrophy, myd Mouse

MDC 1D Human LARGE Glycosyltransferase -

like Golgi protein Underglycosylated α - DG

Other Disease causing genes: B4GAT1, POMGnT2, DPM1-3, ISPD, TMEM5 ….

Mammalian O-Mannosylation

POMGnT1 POMGnT1+GnTVb POMGnT2

The ECM binding O-Man Structure (also the receptor for certain clades of arenaviruses including Lassa)

Praissman J L et al. eLife Sciences 2014;3:e03943

Other O-Mannosylated Proteins:

1.  The Cadherin Family of Proteins -also involved in adhesion

2.  RPTPβ-Receptor protein tyrosine receptor

3. OTHERS???

O-Man

•  O-Man is clearly involved in CMD•  What mammalian proteins (especially in the

brain) are O-Man modified besides α-DG?•  What are the roles for M1, M2, and M3

glycans?•  What is relationship between O-Man and O-

GalNAc?

•  Separate Lecture (Wells)

O-Glycosylation

Ser/Thr

GalNAc

Ser/Thr

Man

Ser/Thr

Fuc

Ser/Thr

GlcNAc

Ser/Thr

Glc

-6-P

-6-P

-6-P

-6-P

-1-P

UDP-

GlycogenPPP Glycolysis Hexosamine Biosynthetic Pathway

(HBP)

UDP-

- -O

UDP-

CMP-

Complex Glycosylation

A.

B.

Key:

= Glc

= Fru

= GlcN

= GlcNAc= GalNAc= Neu5Ac

Features of O-GlcNAc•  NOT elongated•  Nucleocytoplasmic Proteins•  Dynamic & Inducible•  Enzymes for its Addition (OGT) and

Removal (O-GlcNAcase) are Nucleocytoplasmic

•  OGT Knock-Out is Lethal at the ES cell stage–  X-linked OGT, Females Heterozygotes are NOT mosaic

•  Reciprocity with Phosphorylation (Yin-Yang) on Some Proteins

Classes of Proteins Modified By O-GlcNAc ���>1,000 Proteins Identified to Date

❖ Cytoskeletal ComponentsTau, Vinculin

❖ Hormone ReceptorsERα&β

❖ Kinases & Other Signaling Molecules CKII, eNOS, IRS-2

❖ Nuclear Pore ProteinsNUP62, NUP155

❖ Oncogenes & Tumor Suppresors

p53, Rb

❖  Transcription Factorsc-myc, Sp1, Pax6

❖ Metabolic EnzymesGAPDH, Pyruvate Kinase

❖  Transcriptional and Translational Machinery

RNA Pol II, EIF4A

❖ Viral ProteinsSV40 T Antigen, v-Erb-a

❖ Heat-Shock ProteinsHSP90, α-crystallin

O-GlcNAc Challenges•  Assigning function to specific site on particular protein

(mapping sites)–  Recent data has demonstrated this–  Great example CAMKIV (O-GlcNAc in active site)–  Another Example: Fox01 repression of gluconeogenic

enzymes in liver–  Another Example: O-GlcNAc of Histone required for

mono-Ub modification•  Regulation of OGT/OGA (PTM, protein:protein

associations)

•  Separate Lecture (Wells)

Ser/Thr

GalNAc

Ser/Thr

Man

Ser/Thr

Fuc

Ser/Thr

GlcNAc

Ser/Thr

Glc

Hyl HypSer

Gal Xyl

β

A few more O-glycans……

O-Xyl….precursor for GAGs…Separate Lecture (Bergmann)

O-GlcNAc (nucleocytoplasmic….separate lecturenote: recent report of O-GlcNAc on Notch extracellular domain (different enzyme (eOGT) than the intracellular one (OGT))

O-Glycosylation of Hyl

O-Glycosylation of Hyl

•  Found on Collagen and Adiponectin (which has a “collagen-like” domain)

•  Glycosylation Essential for Basement Membrane Formation in Tissues

•  Modulates Collagen Cross-linking?•  Other proteins with modification?

After Esko, J

The Glycosaminoglycans

���O-Glycosylation���

-less studied (until recently?)-tools to study are underdevelopment-in many cases, clear functional effect