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

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.

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

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

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

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

OLIGOSACCHARIDE HOUSE-KEEPING CENTRAL

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

Lysosomal degradation of N-linked oligosaccharides

Lysosomal degradation of N-linked oligosaccharides

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

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

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

Hyaluronan degradation

HEPARAN SULFATE DEGRADATION

HEPARAN SULFATE DEGRADATION

CHONDROITIN SULFATE DEGRADATION

DISEASES OF GLYCOLIPID DEGRADATION **

GSL Degradation Needs Assistants

LIFE CYCLE OF GM2 ACTIVATOR PROTEIN

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.

What determines differences in patient outcome?

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

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

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

Scorecard for Enzyme Replacement Therapy

QUESTIONS ??

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