The long range goal of this research is to characterize how lysosomes function in cells by examining degradation of a specific rat serum glycoprotein, alpha1-acid glycoprotein, in a perfused rat liver. This substrate has been radiolabeled in either its peptide, galactosyl, N-acetylglucosaminyl, or fucosyl components and can be rapidly targeted into lysosomes by a beta-D- galactosyl-receptor system present in that tissue. Experiments done in the presence of active-site inhibitors of lysosomal proteinases or glycosidases produce hydrolase deficiencies analogous to human disorders of lysosomal metabolism. Based on chemical structures of fragments that accumulate in inhibitor- treated liver, we have discovered that disassembly of asialo- alpha1-acid glycoprotein is a highly ordered bidirectional pathway. Two sequences are initiated by 1) removal of galactose from non-reducing ends of the oligosaccharide chains and 2) digestion of the polypeptide. The latter half of catabolism also encompasses removal of Asn's that link the carbohydrate to the peptide and hydrolysis of nearby sugar units (the reducing end GlcNAc and any attached Fuc). Breaking of the N- acetylglucosaminyl bond that forms the core di-N- acetylchitobiose unit of each Asn-linked oligosaccharide separates the two oppositely directed pathways. Our major aim is to substantiate this degradative scheme and to characterize biochemical details of the individual steps, especially cleavage of the dividing-point GlcNAc-GlcNAc unit. We will purify the di-N- acetylchitobiase responsible for this reaction from both rat and human livers, determine its presence in other species and tissues, characterize its substrate specificity and mechanism of action, and study biosynthesis of this glycosidase in cultured hepatocytes. We will also examine interrelationships between the two halves of the degradative pathway. By using proteinase inhibitors to diminish peptide breakdown, we can examine what parts of the polysaccharide are thereby sterically protected from hydrolysis. Similarly, fucose hydrolysis will be inhibited and any effects of this on proteolysis and removal of other sugars will be noted. Finally, we will study steps that involve outer-chain residues of Ga1, G1cNAc and Man. In each case we will test whether these sugars are removed in a branch-specific order by different isoenzymes.
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