1. ESSENTIALS OF GLYCOBIOLOGY
LECTURE 14
GLYCOCONJUGATE DEGRADATION
and
LYSOSOMAL STORAGE DISEASES
Hud Freeze

2. USUAL TURNOVER
Most glycans are extracellular or on cell surface
Membrane recycling (50%/hr)
Receptor and non-receptor
mediated endocytosis To
Endosome
Lysosome
Lysosomal exoglycosidases degrade glycans at low pH
Specific lysosomal transporters carry neutral
hexose, acetylatedaminohexose (GlcNAc, GalNAc), and
Anionic sugars (GlcA, Sia) to the cytoplasm.

3. SUGAR CHAIN DEGRADATION ENZYMES
Most Are:
Lysosome/Endosome
Low pH optimum,
Sugar/anomeric specificity
Exo-glycosidases
Targeted to lysosome through P-lectins and Man-6-P
But Some Are:
Non-lysosomal
Active near neutral pH
Endoglycosidases
Targeted as membrane bound molecules
Not in the lysosome

4. Special Features for Degradation of Different Glycoconjugates
TYPE FEATURE
Glycoproteins
N-linked ER/Golgi/Cytoplasm/ Lysosome
O-linked Unexpected Products
Proteoglycans Endoglycosidases
Unique ORDER
Non-glycohydrolase enzymes
Glycosphingolipids Assisting proteins

5. Special Problems for N-Linked Sugar Chains
N-glycosylation occurs in ER-Topology for lysosomal degradation is wrong
~50% of ER proteins misfold and are degraded - what happens to the sugar chain? To glycopeptides?
Protein synthetic rate and glycosylation rate must be coordinated
Competition for lectin-based chaperones

7. lots of Man( )
Released
What happens to the released mannose?

8. OLIGOSACCHARIDE HOUSE-KEEPING CENTRAL

9. MANNOSE METABOLISM IN CELLS AND MORE
Mannose in plasma comes from
Oligosaccharide turnover in cells
Cells also produce mannose
From glucose
Glc Glc-6-P Fru-6-P Man-6-P
Golgi
cytosol
Lysosome
Glycans ER

10. Lysosomal degradation of N-linked oligosaccharides

11. Lysosomal degradation of N-linked oligosaccharides

12. Enzymatic defects are usually found by accumulation of
Partially degraded oligosaccharides in urine

13. O-LINKED OLIGOSACCHARIDE DEGRADATION
Same enzymes used for N-linked oligosaccharide degradation
a-GalNAc’ase deficiency--produce GalNAc terminated
Oligosaccharides?
Excretion of GalNAc-a-Ser/Thr?
No!!
Why Not?
The oligosaccharides are larger size!
How to explain this? a
Ser/Thr a
Ser/Thr

14. ** Partially degraded polysaccharides accumulate in tissues and urine.
Structural analysis of glycans used to work out pathway

15. Hyaluronan degradation

16. HEPARAN SULFATE DEGRADATION

17. HEPARAN SULFATE DEGRADATION

18. CHONDROITIN SULFATE DEGRADATION

19. DISEASES OF GLYCOLIPID DEGRADATION**

20. GSL Degradation
Needs Assistants

21. LIFE CYCLE OF GM2 ACTIVATOR PROTEIN

22. Model for the degradation of membrane-bound GlcCer by glucocerebrosidase and SAP-C and Cer by acid ceramidase and SAP-D, respectively. Besides the interaction of lysosomal enzyme and activator protein, the model emphasizes binding of activator protein and lysosomal enzymes to the vesicular surface containing BMP.

23. What determines differences in patient outcome?

25. S I = SE TREATMENT FOR LYSOSOMAL STAORAGE DISEASES Tissue Destruction
SUBSTRATE DEPLETION THERAPY
ENZYME REPLACMENT THERAPY
Tissue Destruction
Phagocytosis
Synthesis
Cell proliferation
Uptake
Cell loss
Exosystosis
catabolism
Cell turnover
S I = SE
SUBSTRATE ACCUMULATION

26. ENZYME REPLACEMENT THERAPY
WHAT PROBLEMS WOULD YOU ENCOUNTER ?
Get it to the right cells?
Is it stable in the cells?
Does it need co-factors?
Can it degrade already accumulated materials?
Does it cross the blood brain barrier?
Does it generate antibodies?
SUBSTRATE DEPLETION THERAPY
Potential advantages
Small molecules cross the blood-brain barrier
Targeting initial step reduces all intermediates

27. ENZYME REPLACEMENT THERAPY
FIRST ATTEMPTED IN ~1980 Gaucher Disease Glucosyl Ceramide deficiency. Modified the N-linked glycans targets enzyme to the liver macrophage through a Man/GlcNAc receptor
Used in >3000 patients as product Cerezyme (Genzyme)
Other enzymes used to treat Fabry (a-Galactosidase), MPSI
a-L-iduronidase
SUBSTRATE DEPLETION THERAPY
First Clinical trials using N-Butyldeoxynojirimycin, (miglustat 'Zavesca') on Gaucher patients were successful
Results for animal models for other disorders appear promising

28. Scorecard for Enzyme Replacement Therapy

29. QUESTIONS ??

30. Different glycans have unique degradation pathways
REMEMBER THAT
Different glycans have unique degradation pathways
Mutations in degradative enzymes lead to rare diseases that are cell/organ specific in their pahology
Limiting glycan synthesis or replacing deficient enzymes in genetic disorders may reduce some pathology