1. O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc
Notch
Thrombospondin
Factor IX
Yeast mannoproteins
a-dystroglycan
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 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?
After Esko, J

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. 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?
After Esko, J

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

31. Alpha-Dystroglycan

32. Muscular Dystrophies associated with glycosylation of a - DG
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
MDC, Congenital Muscular Dystrophy; POMT, Protein
Mannosyltransferase;
POMGnT1, Protein
Mannose, N
cetylglucosaminyltransferase 1

33. POMT1
in the ER

35. Figure 3: Glycans linked to Ser/Thr through Man or GalNAc in mammalian brain and muscle. Detected structures on a-DG highlighted by red checks.

37. 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 and large 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?
After Esko, J

38. O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc
GlcNAc
O-GlcNAc and O-Glycosylation of Hydroxyproline
---SEPARATE LECTURES (Wells, West, & Hahn)

39. O-Glycosylation of Hyl

40. 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?

41. REALLY COMPLICATED O-Glycosylation Carl Bergmann

42. The Glycosaminoglycans
After Esko, J

43. Hyaluronan (HA) After Esko, J b 4 3 b4 b3
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
Abundant in skeletal tissues, synovial fluid, skin, and vitreous
Ovulation/ fertilization
Angiogenesis
Cell migration
Macrophage stimulation
Morphogenesis and differentiation
After Esko, J

44. Karl Meyer, Columbia University
Simoni, R. D. et al. J. Biol. Chem. 2002;277:e27

45. Physical Properties After Hascall and Laurent
Gels of high viscosity, and a great lubricant since at high shear its viscosity drops, but remains resilient
Interglycosidic H-bonding restricts rotations across glycosidic bonds
Promotes rapid recovery after mechanical perturbations
Hydrated matrices rich in hyaluronan expand the extracellular space, facilitating cell migration.
There is both a polar and a hydrophobic face for interaction with other macromolecules
HA distribution
HA physical properties
Discuss intramoleuclar hydrogen bonds stabilize structure.
System will recover after breaking these interactions.
After Hascall and Laurent

46. After Weigel, P HA synthase(s) located in plasma membrane
                                                                      
HA synthase(s) located in plasma membrane
Copolymerization of UDP-GlcNAc and UDP-GlcA occurs independently of a core protein
HA can contain ,000 disaccharides (105- 4x 107 Da, ~10 µm, the length of an erythrocyte)
Half-life rate ranges from 2 weeks in synovial fluid to 5 minutes in the bloodstream
After Weigel, P

47. Diagram of a putative metabolic scheme for hyaluronan degradation.
From Stern, R.

48. Hyaluronan Binding Proteins
Aggrecan forms link-protein stabilized complexes with HA, load bearing function
After Esko, J

49. Cartilage - Proteoglycan Aggregates
High charge density creates osmotic pressure that draws water into the tissue (sponge)
Absorbs high compressive loads, yet resilient
Aggrecan: Large chondroitin sulfate proteoglycan present in cartilage and other connective tissues
Core protein ~400 kDa
~100 chondroitin sulfate chains of ~20 kDa
Aggrecan
Forms aggregates with hyaluronic acid (HA)
Hyaluronic
Acid
After Esko, J

50. Albert Dorfman, University of Chicago
Kresge, N. et al. J. Biol. Chem. 2005;280:e28

51. Chondroitin Sulfate After Esko, J b 4 3 b4 b3 6S 6S 6S 4S 4S 4S
IdoA 6S 6S 6S b 4 3 b4 b3 4S 4S 4S
GalNAc
GlcA
Non-sulfated chondroitin is rare in vertebrates, but multiple types of sulfated chondroitins are known (A, B, C, D, etc)
Multiple sulfotransferases decorate the chain
An epimerase can flip the stereochemistry of D-GlcA to L-IdoA (Dermatan Sulfate)
The chains are easily characterized using bacterial chondroitinases which degrade the chain to disaccharides
After Esko, J

52. Extracellular Matrices
Epithelial cells produce basement membranes composed of proteoglycans, reticular collagens and glycoproteins
Laminin
Entactin/Nidogen
Collagen Type IV
Perlecan:
Heparan Sulfate
Proteoglycan
Bamacan:
Chondroitin Sulfate
Acts as a selective barrier to the movement of cells
Tissue regeneration after injury, provides a scaffolding along which regenerating cells can migrate
Kidney glomerulus has an unusually thick basement membrane that acts as a molecular filter dependent on perlecan, a heparan sulfate proteoglycan
Neuromuscular junction basement membrane contains the heparan sulfate proteoglycan agrin, essential for synaptogenesis
After Esko, J

53. Aggrecan CS/KS brain, cartilage Versican CS brain, mesenchyme
Secreted PGs
Aggrecan CS/KS brain, cartilage
Versican CS brain, mesenchyme
Neurocan CS brain
Brevican*CS brain
Decorin CS/DS brain, connective tissue
Biglycan CS/DS brain, connective tissue
Testican CS/HS brain, testis
Perlecan HS brain, basement membrane
Dystroglycan HS brain, muscle
Agrin HS brain, muscle
Claustrin KS brain
Glycoword

54. NG2-PG CS brain, cartilage
Transmembrane PGs
NG2-PG CS brain, cartilage
RPTP zeta/beta (phosphacan) CS/KS brain, cartilage
Neuroglycan C CS brain
Appican CS brain, connective tissue
N-syndecan HS brain
SV2 KS brain
GPI-anchored PGs
Glypican HS brain, muscle
K-glypican HS brain, kidney
Cerebroglycan HS brain
Glycoword

55. Ulf Lindahl, Uppsala University

56. Heparin/Heparan Sulfate
IdoA
GlcNAc 6 S 6 S 6 S N S N S N S 2 S N S
GlcA
Gal
Gal
Xyl 3 S
Characterization of heparan sulfate is based on different criteria
- GlcNAc vs GlcNS
- 3-O-Sulfo and 6-O-sulfo groups
-IdoA vs GlcA
Heparinases degrade chain into disaccharide units
Nitrous acid degrades chains at GlcNS
Disaccharides characterized by HPLC or mass spectrometry
After Esko, J

57. Biosynthesis of a Heparin/Heparan Sulfate Chain
GlcNAc/S
6-O-sulfotransferases
(6OST) (3+ isozymes) 6 S
Copolymerase
Complex
EXT1/EXT2
IdoA
Epimerase
GlcNAc
EXTL3
EXTL2? 6 S 6 S
Xyl
GlcA
Gal
Gal N S N S 2 S
Uronic acid
2-O-sulfotransferase N S N S 3 S
GlcNH2/S
3-O-sulfotransferases
(3OST) (6 isozymes)
GlcNAc N-deacetylase
N-sulfotransferases
(NDST) (4 isozymes)
After Esko, J

58. Heparan Sulfate Proteoglycans
Chondroitin sulfate 4S
GlcA
GlcNAc
IdoA
After Esko, J

59. The Heparin Anti-thrombin III binding motif
Lindahl, U

60. Heparin’s function in the mast cell appears to have nothing to do with disturbed blood coagulation. Heparin is discharged from the mast cell only in special emergency situations, such as anaphylactic shock or other inflammatory reactions.
Marine mussels have no blood in the conventional sense to anticoagulate, yet the polysaccharide in the "mast cells" turns out to contain the specific antithrombin-binding pentasaccharide sequence. It also has very high anticoagulant activity. It seems reasonable to assume that the clams contain a protease inhibitor related to antithrombin (belonging to the serpin family), but for a functional reason quite unrelated to blood coagulation.

61. Keratan sulfate I (corneal) and II (skeletal)
Figure Keratan sulfates consist of a sulfated polylactosamine linked either to Asn or Ser/Thr residues. The actual order of the various sulfated and nonsulfated disaccharides occurs somewhat randomly along the chain. Not shown are sialic acid and fucose residues that may be present at the termini of the chains.

62. Golgi processing of Glycosaminoglycans

63. Proteoglycan Turnover
Shedding by exoproteolytic activity, MMP-7 for one
Endosulfatase recently discovered that removes sulfate groups on proteoglycans at cell surface: remodeling
Heparanase (endohexosaminidase) clips at certain sites in the chain. Outside cells, it plays a role in cell invasion processes
Inside cells it’s the first step towards complete degradation in lysosomes by exoglycosidases and sulfatases P l a s m e b r n E d o c y t i S p 1 g 2 3 x u f G H h ( ~ k D ) 5
Mucopolysaccharidoses
After Esko, J

64. Mucopolysaccharidoses - Lysosomal Storage diseases
I H: Hurler's a-L-iduronidase corneal clouding; dwarfism; mental retardation; early mortality
MPS I S: Scheie's a -L-iduronidase corneal clouding; aortic valve disease; joint stiffening; normal intelligence and life span
MPS I H/S: Huler/Scheie a -L-iduronidase similar to both I-H and I-S
MPS II: Hunter's L-iduronate-2-sulfatase mild and severe forms, X-linked, also a possible autosomal form, facial and physical deformities; mental retardation
MPS III A: Sanfilippo(A) Heparan N-sulfatase skin, brain, lungs, heart and skeletal muscle are affected in all 4 types of MPS-III
MPS III B: Sanfilippo(B) N-acetyl-a -D-glucosaminidase congestive heart failure; progressive mental retardation
MPS III C: Sanfilippo(C) N-acetylCoA: a-glucosamine- N-acetyltransferase coarse facial features; organomegaly
MPS III D: Sanfilippo(D) N-acetyl-a -glucosamine-6-sulfatase moderate physical deformities; progressive mental retardation
MPS IV A: Morquio's(A) N-acetylgalactosamine- 6-sulfatase corneal clouding, thin enamel, aortic valve disease, skeletal abnormalities
MPS IV B: Morquio's(B) ß -galactosidasemild skeletal abnormalities, normal enamel, hypoplastic odontoid, corneal clouding
MPS VI Maroteaux-Lamy N-acetylgalactosamine- 4-sulfatase 3 distinct forms from mild to severe, aortic valve disease, normal intellect, corneal clouding, coarse facial features
MPS VII Sly ß –glucuronidase hepatosplenomegaly, dystosis multiplex
Michael W. KING, IU School of Medicine

65. GAG Summary
Proteoglycans contain glycosaminoglycans: chondroitin sulfate, dermatan sulfate, or heparan sulfate
Heparan sulfate, Chondroitin, and dermatan sulfate proteoglycans are found in the matrix and play structural roles in cartilage, bone and soft tissues and are found at the cell surface and play roles in cell adhesion and signaling during development
Proteoglycans in the extracellular matrix can also act as a reservoir of growth factors, protect growth factors from degradation, and facilitate the formation of gradients
Human diseases in proteoglycan assembly are rare
Degradation of these compounds is also important (MPS)
After Esko, J