Selective deglycosylation of the heparan sulfate proteoglycan of bovine glomerular basement membrane and identification of the core protein.
The heparan sulfate proteoglycan of the bovine glomerular basement membrane (Mr = 200,000, 30% carbohydrate by weight) has been deglycosylated by various chemical and enzymatic procedures to identify the core protein and provide information about the N- and O-linked saccharide units. Heparitinase digestion of the proteoglycan reduced its Mr to 143,000, consistent with the removal of its four glycosaminoglycan chains with the exception of short segments adjacent to the carbohydrate-protein linkage region, whereas nitrous acid treatment brought about a smaller reduction in size (to Mr = 168,000) which was shown to be due to the resistance of the internal portion of the heparan sulfate polymer to this reagent. Incubation of the heparitinase-digested proteoglycan with peptide N-glycosidase F decreased its Mr by about 8,000 and liberated oligosaccharides which were primarily acidic in nature; since endo-beta-N-acetylglucosaminidase H did not bring about any saccharide release, it appears that the N-linked carbohydrate units (three per molecule) occur exclusively as the complex type. Treatment of the proteoglycan with trifluoromethanesulfonic acid, a reagent which cleaves all saccharide units, yielded the core protein which migrated as a single discrete band (Mr = 128,000) on polyacrylamide gel electrophoresis. Although the native and heparitinase-treated proteoglycan reacted with concanavalin A and Bandeiraea simplicifolia I, the core protein had no affinity for these lectins, and this loss of reactivity can be attributed to the removal of the N- and small O-linked saccharides. However, the immunoreactivity of the deglycosylated protein with antiserum directed against the intact proteoglycan was to a large measure (80%) preserved, suggesting that the polyclonal response to this glomerular basement membrane glycoconjugate is primarily directed against determinants on the polypeptide portion.
D000818:Animals; D001485:Basement Membrane / Q000032:analysis*; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification; D006025:Glycosaminoglycans / Q000302:isolation & purification; D006026:Glycoside Hydrolases; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000302:isolation & purification; D007678:Kidney Glomerulus / Q000032:analysis*; D008970:Molecular Weight; D011509:Proteoglycans / Q000302:isolation & purification
D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D006026:Glycoside Hydrolases; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans