1. O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc
Notch
Thrombospondin
Factor IX
Yeast mannoproteins
a-dystroglycan
cadherins
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
Glc
Ser/Thr
GlcNAc
Notch
Coagulation Factors
Fibrinolytic Factors
Nuclear Proteins
Cytoplasmic Proteins
SEPARATE LECTURE
Mucins
ALSO: Proteoglycans, Hydroxyproline/Hydroxylysine Glycosylation
After Esko, J

2. O-Glycosidic Linkage
O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)
b-elimination
GalNAc
GalNAc a a
Ser
After Esko, J

3. Glycan synthesis in a cellular context Most O-Glycosylated
proteins are synthesized
in the secretory pathway

4. O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc
GlcNAc

5. Mucin-Type O-GalNAc Glycansb4 b3 a3
Ser/Thr b6
Major extracellular vertebrate O-glycan
Begins in cis-Golgi (ERGIC) by attachment of GalNAc in a-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
After Esko, J

7. Polypeptide GalNAc Transferases
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).
>20 ppGalNAcT family members
Share structural features in active site
Some have lectin (ricin) domain
After Esko, J

8. Core 1 and Core 2 Synthesis
T (TF)
Antigen Tn
Core 2
GlcNAcT
Core 1
GalT
(cis)
Ser/Thr b3 b6 a6 a3
ST6GalNAc1
(trans)
Ser/Thr
Sialyl Tn
Antigen b3
Disialyl T
After Esko, J

9. From: Tongzhong Ju and Richard D. Cummings

10. Core 3 and Core 4 Synthesis
GlcNAcT
Core 4
GlcNAcT b3 b3 b6
Ser/Thr
Ser/Thr
Ser/Thr
After Esko, J

11. Unusual Core O-Glycan Structures
Ser/Thr b3
Core 4 b6
Core 1
Core 2
Core 7
Ser/Thr a6
Core 6? b6
Core 5 a3
Core 8
After Esko, J

12. 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

13. Mucin Production Lung Epithelium Goblet cells in intestinal crypts
After Esko, J

14. 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

15. 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

16. O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc
GlcNAc

21. O-Fuc Two Flavors: Mono and Tetrasaccharide
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?

22. O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc
GlcNAc

23. O-Glc Pathway
Shao, L. et al. Glycobiology : ; doi: /glycob/cwf085

25. Rumi is OGluT
KDEL Retention Signal
Temp. Sensitive Mutation

27. 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?

28. O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc
GlcNAc

31. POMT1
in the ER In
Complex
With
POMT2
Uses
Dol-Man
As Donor

32. Alpha-Dystroglycan

34. Muscular Dystrophies associated with glycosylation of a – 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
POMGnT1
Addition of GlcNac b
2 to O
Man
Underglycosylated a
DG,
uncapped O
Fukuyama
type
MDC
Fukutin
Glycosyltransferase
like protein DG
Limb
Girdle and
MDC 1C
Related
Protein l
ike Golgi protein
Myodystrophy,
myd
Mouse
MDC 1D
LARGE
like Golgi protein
Other Disease causing genes: B4GAT1, POMGnT2, DPM1-3, ISPD, TMEM5 ….

35. Mammalian O-Mannosylation
POMGnT POMGnT1+GnTVb POMGnT2

37. GnTV-B Primarily Responsible
O-Man b-6 Branching in the Brain (primarily)
GnTV-B Primarily Responsible

38. A New Glycan Structure on a-DG that is Large-dependent
Science (2010) 327:88-92

39. The ECM binding O-Man Structure
(also the receptor for certain clades of arenaviruses including Lassa)
Proposed role of B4GAT1 in the O-mannosylation pathway.The molecular link between the phospho-O-mannose trisaccharide synthesized on α-dystroglycan and the LARGE synthesized repeating disaccharide crucial for laminin binding reactivity is still not fully characterized (represented as [X]). B4GAT1 appears to possess a priming activity for LARGE and likely adds to an underlying β-xylose that is added by an as yet undefined glycosyltransferase (represented with a question mark).DOI:
Praissman J L et al. eLife Sciences 2014;3:e03943

40. +1, 1100
a-DG KO Brain
MS/MS Analysis of the O-Man Classical Tetrasaccharide
Glycan Structure Released from Brain Proteins Derived from the
a-DG knock-out animal. Greyed out structures represent their loss.

41. Other O-Mannosylated Proteins:
The Cadherin Family of Proteins
-also involved in adhesion
RPTPb
-Receptor protein tyrosine receptor
3. OTHERS???

42. O-Man O-Man is clearly involved in CMD
What mammalian proteins (especially in the brain) are O-Man modified besides a-DG?
What are the functions of fukutin, ispd, TMEM5 in O-mannosylation?
Why the heterogeneity in O-Man structures, what specific structures at what sites on the protein modulate specific interactions?
What is relationship between O-Man and O-GalNAc?

43. O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc
GlcNAc

44. A few more O-glycans…… GalNAc Man Fuc Glc Gal Xyl GlcNAc Ser/Thrb
Ser/Thr
Ser/Thr
Ser/Thr
Ser/Thr
Hyl
Ser
Hyp
Ser/Thr
O-Xyl….precursor for GAGs…
O-GlcNAc….separate lecture—note: recent report
of O-GlcNAc on Notch extracellular domain (different
enzyme (eOGT) than the intracellular one (OGT))

45. O-Glycosylation of Hyl

46. 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?

47. The Glycosaminoglycans
After Esko, J

48. O-Glycosylation -less studied (until recently?)
-tools to study are underdeveloped
-in many cases, clearest functional data (not including folding/quality control)