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

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)

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
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

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

15. ER glycolipid synthesis

16. Biosynthesis of Ceramide and Glucosylceramide

17. ER glycolipid synthesis

18. Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid

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

20. Structure of the Basic GPI Anchor

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

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

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

26. Some representative examples of mammalian complex-type N-glycans

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

28. Golgi processing of O-linked glycans