1. The Essentials of Nucleotide Sugars Wells

2. Sugar Nucleotides (high energy donors) in Mammals
What mammalian sugar is missing?

3. General Principles for Synthesis
1: initiated by kinase reaction, consumes 2 NTP
example: Glc  Glc-6-PGlc-1-PUDP-Glc
2: multiple ways (epimerization, decarboxylation, etc.)
example: UDP-GlcNAcUDP-GalNAc ; UDP-GlcA UDP-Xyl
3: nucleotide-exchange reaction
example Gal-1-P +UDP-Glc  Glc-1-P + UDP-Gal

4. Glucose is only Sugar Needed for Synthesis of All Sugar Nucleotides
Note: No salvage
Pathway for GlcA!
Key Control Points:
Early step enzymes that are
inhibited by end-product
Sugar nucleotide

5. Dolichol phosphate: The “other” high energy sugar donor
Chapter 8, Figure 2
Essentials of Glycobiology
Second Edition
Dol-P-Man N-link, O-Man, C-Man, GPI anchors
Dol-P-Glc N-linked biosynthesis
Dol-P-GlcNAc N-linked biosynthesis

6. Really complex O-glycans: Sulfated glycosaminoglycans and hyaluronic acid Tiemeyer
with significant input from Carl Bergmann (CCRC)
and Jeffrey Esko (UCSD)

7. The glycospace of animal glycans

8. The Glycosaminoglycans

9. Keratan sulfate I (corneal) and II (skeletal)
Keratan sulfates consist of a sulfated polylactosamine extended on either Asn or Ser/Thr linked glycan. 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.

10. Proteoglycans and Human Disease
Simpson-Golabi-Behmel Overgrowth Syndrome (SGBS)
Skeletal Achondroplasias
Hereditary Multiple Exostoses (HME)

11. Vulval Morphogenesis in C. elegans
The vulva is a passageway that accepts sperm from males and expels eggs from the gonad

12. sqv Mutants of C. elegans
Herman and Horvitz (1999) described a set of C. elegans mutants defective in vulval development (sqv, squashed vulva)
wild-type
sqv
sqv mutations alter epithelial invagination

13. SQV Proteins Encode the Enzymes Required for Chondroitin Biosynthesis
Cytosol
Golgi
SQV-4
UDP-Glc
Dehydrogenase
UDP-
SQV-1
UDP-GlcA
Decarboxylase
UDP-
SQV-6
Xylosyltransferase
UDP-
SQV-7
Nucleotide sugar
multi-transporter
SQV-2
Galactosyltransferase II
Ser
UDP- n
SQV-5
Chondroitin Synthase
SQV-8
Glucuronosyltransferase I
SQV-3
Galactosyltransferase I
Xyl
Gal
GlcA
GalNAc
Glc

14. Chondroitin Proteoglycan (non-sulfated)
Xyl
Gal
GlcA
Chondroitin is one type of glycosaminoglycan (GAG) chain
GalNAc

15. wild-type
sqv
One theory of cellular invagination is that the adjacent epithelial cells may secrete a chondroitin proteoglycan in a polarized fashion
Hydration of the matrix might cause expansion and an inward curvature of the cell layer.

16. How does this apply to vertebrates?4 3 b4 b3
GalNAc
GlcA
Non-sulfated chondroitin not found in vertebrates
Instead, they make hyaluronic acid which is similar in structure
GlcNAc
GlcA b 4 3 b4 b3

17. Hyaluronan (HA) b 4 3 b4 b3
n≥1000
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
Abundant in skeletal tissues, synovial fluid, and skin
Synthesis is elevated in expanding tissues (morphogenesis, invasion)

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

19. After Hascall and Laurent
Physical Properties
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

20. HA Synthases Three HAS genes (HAS1-3) known in vertebrates
Has2 as the primary source of HA during embryonic development
Has2-/- embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10) Nat Med Aug;8(8):850-5
A single HAS in Streptococcus (capsules)
Assembly process occurs differently
Virulence factor
Molecular mimicry
None detected in insects or nematodes
Homologs of genes suggest a relationship to chitin synthases (GlcNAcb1,4)n
Light micrograph of Strep. mutans. From Dr. Timothy Paustian, University of Wisconsin-Madison

21. 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

22. HA turnover Eukaryotic hyaluronidase (HYAL) gene family 3p21.3
HYAL1, 2 and 3
Turnover of hyaluronan in most tissues is rapid
t1/2 of ~1 day in epidermal tissues
Large hyaluronan molecules in the extracellular space interact with cell surface receptors
Fragments produced by an associated GPI anchored hyaluronidase, most likely Hyal2.
Fragments transported to lysosomes for complete degradation, most likely involving Hyal1
HYAL2 null mice are embryonic lethal
Lysosomal storage disorder in a person with a mutation in HYAL1

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

24. Hyaluronan Binding Proteins
Aggrecan forms link-protein stabilized complexes with HA, load bearing function

25. Lectican Family
This family of proteoglycans consist of a group of homologous core proteins
Have in common an hyaluronic acid binding motif
Chondroitin sulfate

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

27. Chondroitin Sulfate 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

28. CS types, not mutually exclusive
Taken from Essentials of Glycobiology, 1st edition

29. Chondroitin Sulfate Proteoglycan

30. Cartilage - Proteoglycan Aggregates
Forms aggregates with hyaluronic acid (HA)
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
Hyaluronic
Acid

31. Diseases - Achondroplasias
Gene
Mice
Human
Aggrecan
Cartilage Matrix Deficiency (CMD) ?
Sulfate Transporter (DTDST) -
Atelosteogenesis Type II
Achondrogeneis Type Ib
Diastrophic dysplasia (DTD)
PAPS synthetase
Brachymorphism
Spondylo-
epimetaphyseal dysplasia
PAPS
ATP
SO42-
DTDST
out in
GAG Sulfation

32. Extracellular Matrices
Cells are surrounded by an extracellular matrix
Fibroblasts and other connective tissue cells produce a fibrillar matrix (ground substance) composed of small interstitial proteoglycans, fibrillar collagens, and glycoproteins

33. Interstitial Proteoglycans SLRPs - Small Leucine Rich Proteoglycans
Gene
Knockouts
Decorin
(single CS chain)
Fragile skin
Thin dermis
Increased resistance to Borrelia burgdorfei
Biglycan
(two CS chains)
Decrease in bone mass and dentin mineralization
Fewer osteoblasts
Mild muscular dystrophy
Fibromodulin
(keratan sulfate
Ectopic tendon ossification
Lumican (keratan sulfate
Corneal opacification

34. Extracellular Matrices
Epithelial cells produce basement membranes composed of heparan sulfate 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

35. Perlecan - Basement Membrane Proteoglycan
467 KDa
muscle development and differentiation appear to be normal and the nerve terminals are normally formed
Neuromuscular junction of the mutant mouse muscles appear normal.
Acetylcholine esterase is completely absent at the neuromuscular junctions, although it is synthesized normally.
LDL
receptor-like
repeats
Laminin
repeats
Ig-like repeats
Laminin &
EGF motifs
Perlecan knockouts: Perinatal lethal chondrodysplasia
Human mutations: Schwartz-Jampel syndrome, Silverman-Handmaker type Dyssegmental dysplasia
Glycoconj J 19, 263–267, 2003)

36. Heparan Sulfate Proteoglycans
Chondroitin sulfate 4S
GlcA
GlcNAc
IdoA

37. Heparan Sulfate 6 S 6 S 6 S N S N S N S 2 S N S 3 S
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

38. Ulf Lindahl, Uppsala University

39. Biosynthesis of a 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
GlcA
Gal
Gal
Xyl 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)

40. The Heparin Anti-thrombin III binding motif
Heparin vs. Heparan, tomato vs. tomahto?
Heparin is the most highly sulfated form of heparan found in nature. Still, signficant microheterogeneity imparts important function. Anti-thrombin III binding to Heparin is periodate sensitive, but other heparin and heparan activities are not.
The Heparin Anti-thrombin III binding motif
Lindahl, U

41. Despite heparin’s ability to disturb blood coagulation, it’s normal function in the mast cells that make it is not clear. Heparin is discharged from the mast cell only in special emergency situations, such as anaphylactic shock.
Marine mussels have no blood in the conventional sense to anticoagulate (no vasculature), yet the polysaccharide in their "mast cells" contains the specific antithrombin-binding pentasaccharide sequence and is very high in anticoagulant activity.
It seems reasonable to assume that mussels express a protease inhibitor related to antithrombin (ie: belonging to the serpin family), but for a functional reason quite unrelated to blood coagulation.

42. Heparan Sulfate Proteoglycans: Co-receptors and Signaling Molecules
Wnts
TGF-/BMPs
HGF
HB-EGF
Hedgehog
FGF
VEGF
Angiopoietin
Heparan sulfate
FGF
FGF
Signaling Event
Mitogenesis

43. Membrane Heparan Sulfate Proteoglycans Type I Membrane Proteins
Syndecans
Type I Membrane Proteins
4 members
Glypicans
GPI anchored proteins
6 members

44. Syndecans Cell-Binding Domain TM C1 C2 V
Syndecan cytoplasmic domains composed of two regions that are conserved among the syndecans (C1 and C2) and a variable region (V)
C2 domain is a binding site for PDZ domains in cytoskeletal proteins (e.g., syntenin) and signaling molecules (e.g., CASK)
These domains are also phosphorylated on tyrosine by PKCa, which may regulate binding
Syndecan core protein can initiate downstream signaling when it participates as an adhesion receptor
Having said all this, syndecan-1 and syndecan-4 knockouts have mild phenotypes. Not clear about syndecan-2 and -3
Transgenic expression of syndecan-1 uncovered a physiological control of feeding behavior by syndecan-3 (Cell 2001, 106: )
Bacterial infection seems to depend on syndecan-1 (Nature 2001, 411:98-102)

45. Glypicans
Glypicans have a large globular domain with the 2-3 heparan sulfate chains lying between this and the membrane
Glypicans can initiate downstream signaling and participate as an adhesion receptors in vitro.
Knockout of glypican-1 is unremarkable - redundancy?
Knockout of glypican-3 has remarkable phenotype, identical to Simpson-Golabi-Behmel Syndrome
Underlying mechanism unclear
Modulation of growth factor(s)?

46. Biosynthetic Knockouts
Gene
Mice
Human
EXT1/EXT2
Embryonic lethal (null)
Heterozygotes develop rib exostoses
Hereditary Multiple Exostoses
NDST1
Perinatal lethal (null)
Various developmental defects (forebrain, lung)
Tissue specific knockouts have various physiological alterations ?
NDST2
Viable, mucosal mast cell deficiency
GlcA C5 Epimerase
Perinatal lethal (null) Renal agenesis, lung defects, and skeletal malformations
Uronosyl 2-O-sulfotransferase
Perinatal lethal (null) Renal agenesis,eye and skeletal defects

47. 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

48. 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) b-galactosidase: mild 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 b-glucuronidase: hepatosplenomegaly, dystosis multiplex
Michael W. KING, IU School of Medicine

49. Summary
Proteoglycans contain glycosaminoglycans: chondroitin sulfate, dermatan sulfate, or heparan sulfate
Chondroitin and dermatan sulfate proteoglycans are found in the matrix and play structural roles in cartilage, bone and soft tissues
Tissue architecture
Heparan sulfate, chondroitin sulfate, and dermatan sulfate proteoglycans are found at the cell surface and play roles in cell adhesion and signaling during development
Growth control, positive and negative
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
Regulated degradation of these compounds is also important (MPS)