1. Synthesis and Structure of Major Glycan Classes
1/24/05

2. Large O-linked Glycosaminoglycans and poly-lactosamine structures
Glycoprotein N-linked and O-linked oligosaccharides
Glycolipid oligosaccharides

3. Symbolic Representation Simplified Traditional
The building blocks
Symbolic Representation
Simplified Traditional
After Varki, A

4. Glycan synthesis
in a cellular context

5. Overview
From ER
through
Trans-Golgi and
points inbetween

6. ER processing of N-linked glycans

7. Major Classes of N-Glycans
“Hybrid”
“Complex”
“High-Mannose”
(oligo-mannose)
After Varki, A

8. Biosynthesis of N-Glycans: Production of GlcNAc-P-P-Dolichol
Man
Gal
Sia
Fuc
Glc
Biosynthesis of N-Glycans: Production of GlcNAc-P-P-Dolichol
Tunicamycin
Blocks - not
very specific!
Dolichol
Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

9. GlcNAc
Man
Gal
Sia
Fuc
Glc
Biosynthesis of the N-Glycan Precursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER)
CDG = Congenital Disorder of Glycosylation in Humans
Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

10. Biosynthesis of the N-Glycan Precursor on Lumenal Leaflet of ER
GlcNAc
Man
Gal
Sia
Fuc
Glc
Biosynthesis of the N-Glycan Precursor on Lumenal Leaflet of ER
Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

11. GlcNAc
Man
Gal
Sia
Fuc
Glc
Completion of Biosynthesis of N-Glycan Precursor on Lumenal Leaflet of ER - and Transfer to Protein
Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

12. Yeast OST complex contains nine membrane-bound subunits After Varki, A
Oligosaccharyltransferase complex (OST) in the ER membrane transfers the dolichol N-glycan precursor to asparagine residues on nascently translated proteins
Target “sequon” for N-glycosylation
Necessary but not sufficient
X = any amino acid except proline
Rarely can be Asn-X-Cys
Transfer co-translational/immediate
post-translational before folding
~2/3 of proteins have sequons
~ 2/3 sequons actually occupied
(some variably)
Yeast OST complex contains nine membrane-bound subunits
After Varki, A

13. Initial Processing of N-Glycans in the ER and Golgi
GlcNAc
Man
Gal
Sia
Fuc
Glc
Initial Processing of N-Glycans in the ER and Golgi ER
Golgi
Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

14. Calnexin (and Calcireticulin) function during glycoprotein folding in the endoplasmic reticulum
Improperly folded proteins are re-glucosylated by glucosyltransferase which acts as “sensor” for improper folding
3 Glucose
Residues
After Varki, A

15. ER glycolipid synthesis

16. Biosynthesis of Ceramide and Glucosylceramide

17. ER glycolipid synthesis

18. Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
After Hart, G

19. Examples of GPI-Anchored Proteins
Cell surface hydrolases
alkaline phosphatase
acetylcholinesterase
5’ nucleotidase
Protozoal antigens
trypanosome VSG
leishmanial protease
plasmodium antigens
Adhesion molecules
neural cell adhesion molecule
heparan sulfate proteoglycan
Mammalian antigens
Thy-1
carcinoembryonic antigen
Others
decay accelerating factor
scrapie prion protein
folate receptor
After Hart, G

20. Structure of the Basic GPI Anchor
After Hart, G

21. Structural Analysis of the GPI Anchor Enzymatic and chemical cleavage sites are useful in identifying GPI anchored membrane proteins
After Hart, G

22. Examples of C-Terminal Sequences Signaling the Addition of GPI-Anchors
Bold AA is site of GPI attachment Sequence to right is cleaved by the transpeptidase upon Anchor addition
After Hart, G

23. Golgi processing of N-linked glycans

24. Completion of Processing of N-Glycans in ER and Golgi
GlcNAc
Man
Gal
Sia
Fuc
Glc
Completion of Processing of N-Glycans in ER and Golgi
Final products often show
“microheterogeneity” at each
N-Glycosylation site
Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

25. GlcNAc-Transferases Determine Number of “Antennae” of N-glycans
After Varki, A

26. Some representative examples of mammalian complex-type N-glycans
After Varki, A

27. Evolutionary Variations of the N-glycan Processing Pathway
Asn
Yeast
Slime
Mold N
Asn a3 6a b 2
Plants a3
“Pauci-
mannose” a3
Insects 6a a6
Vertebrates a 3 4 N
Asn
After Varki, A

28. Golgi processing of O-linked glycans

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

30. Examples of O-Glycans GalNAc Ser/Thr Man Fuc GlcNAc Ser/Thr Ser/Thr
Yeast mannoproteins
a-dystroglycan
Nuclear Proteins
Cytoplasmic Proteins
GalNAc
Ser/Thr
Man
Fuc
GlcNAc
Ser/Thr
Ser/Thr
Ser/Thr
Mucins
Notch
Coagulation Factors
Fibrinolytic Factors
After Esko, J

31. Mucin-Type O-GalNAc Glycansb4 b3 a3
Ser/Thr b6
Major 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 a
After Esko, J

32. 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).
12 members of mammalian ppGalNAcT family
Estimated size of family = 24
Share structural features in active site
Some have lectin (ricin) domain
After Esko, J

33. Core 1 and Core 2 Synthesis
GalT
Core 2
GlcNAcT
Ser/Thr b3
Ser/Thr b3 b6
Ser/Thr
After Esko, J

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

35. 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

36. 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 tend clustered - bottle brush
Expressed by epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts
Gelation fun cation mediated by interchain disulfide bonding
Note that O-GalNAc addition can occur readily at adjacent Ser/Thr residues,allowing clustering to occur. Can't happen with N-linked chains since large precursor added en bloc.
After Esko, J

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

38. 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
Individuals lacking salivary glands have abnormal mucosal surfaces and chronic low level inflammation
Mucins can agglutinate certain pathogenic bacteria (oral streptococci)
After Esko, J

39. Questions
What is the function of multiple polypeptide GalNAc transferases?
Do various transferases within a family act on the same or different substrates?
How is tissue specific expression of transferases regulated?
How does competition of transferases for substrates determine the glycoforms expressed by cells and tissues?
After Esko, J

40. Golgi processing of Glycosphingolipids

41. Major Classes of Glycosphingolipids
Series Designation Core Structure
Lacto (LcOSe4) Gal3GlcNAc3Gal4Glc1Ceramide
Lactoneo (LcnOSe4) Gal4GlcNAc3Gal4Glc1Ceramide
Globo (GbOSe4) GalNAc3Gala4Gal4Glc1Ceramide
Isoglobo (GbiOSe4) GalNAc3Gal3Gal4Glc1Ceramide
Ganglio (GgOSe4) Gal3GalNAc4Gal4Glc1Ceramide
Muco (MucOSe4) GalGal3Gal4Glc1Ceramide
Gala (GalOSe2) Gal4Gal1Ceramide
Sulfatides Sulfo-Gal1Ceramide
Different Core structures generate unique shapes and are expressed in a cell-type specific manner
After Varki, A

43. Turnover and Degradation of Glycosphingolipids
Internalized from plasma membrane via endocytosis
Pass through endosomes (some remodelling possible?)
Terminal degradation in lysosomes - stepwise reactions by specific enzymes.
Some final steps involve cleavages close to the cell membrane, and require facilitation by specific sphingolipid activator proteins (SAPs).
Individual components, available for
re-utilization in various pathways.
At least some of glucosylceramide may
remain intact and be recycled
Human diseases in which specific enzymes or SAPs
are genetically deficient (“storage disorders”

44. Biological Roles of Glycosphingolipids
Thought to be critical components of the epidermal (skin) permeability barrier
Organizing role in cell membrane. Thought to associate with GPI anchors in the trans-Golgi, forming “rafts” which target to apical domains of polarized epithelial cells
May also be in glycosphingolipid enriched domains (“GEMs”) which are associated with cytosolic oncogenes and signalling molecules
Physical protection against hostile environnments
Binding sites for the adhesion of symbiont bacteria.
Highly specific receptor targets for a variety
of bacteria, toxins and viruses.

45. Biological Roles of Glycosphingolipids
Specific association of certain glycosphingolipids with certain membrane receptors.
Can mediate low-affinity but high specificity carbohydrate-carbohydrate interactions between different cell types.
Targets for autoimmune antibodies in Guillian-Barre and Miller-Fisher syndromes following Campylobacter infections and in some patients with human myeloma
Shed in large amounts by certain cancers - these are found to have a strong immunosuppressive effects, via as yet unknown mechanisms

46. Ganglioside synthetic enzyme KOs
GlcCer synthase (GlcT): Early embryonic lethal, differentiation and germ layer formation okay
LacCer synthase (GalT): ??
GM3 synthase (SiaTI): Increased sensitivity to insulin (skeletal muscle receptor phosphorylation)
GM2 synthase (GalNAcT): Male sterility, demyelinating disorder
GD2 synthase (SiaTII): Ok
GM2 synthase/GD2 synthase double KO: sudden death, auditory seizures, demylination

47. Sheikh, KA, et al. (1999) PNAS 96, 7532

48. Golgi processing of Glycosaminoglycans

49. After Esko, J

50. But first, an exception:
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)
After Esko, J

51. Physical Properties After Esko, J
HA distribution
HA physical properties
Discuss intramoleuclar hydrogen bonds stabilize structure.
System will recover after breaking these interactions.
Gels of high viscosity, but 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.
After Esko, J

52. HA synthase(s) located in plasma membrane, spans membrane 12 times
Copolymerization of UDP-GlcNAc and UDP-GlcA occurs independently of a core protein
HA can contain ,000 disaccharides ( Da, ~2 µm in length)
After Esko, J

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

54. 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

55. 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

56. 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
After Esko, J

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

58. Heparan Sulfate After Esko, J 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
After Esko, J

59. 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
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

60. 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

61. 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

62. Overview

63. General Principles Glycan synthesis is compartmentalized within cells
Precursors begin as lipid-linked species on the cytoplasmic face of the ER, requiring that substrates be flipped for further processsing
Addition of N-linked glycans generally occurs co-translationally
ER glycosylation may have evolved as a method for quality control, Golgi modifications are layered on top of this well-conserved function
Within the Golgi and distal secretory pathway, specific glycan structures can impart targeting information
The assembly-line model holds well for N-linked proteins but may not be as applicable to glycolipid and GAG synthesis