ON THE MECHANISM OF LYSOSOMAL ENZYME RESISTANCE TO AUTOPROTEOLYSIS (GLYCOPROTEINS, PROTEIN DEGRADATION)

Lysosomes are actively involved in the degradation of a wide variety of biological molecules including proteins. The lysosomal enzymes which are responsible for this activity are themselves preferentially resistant to proteolytic degradation This acid stability is not dependent on lysosomal organelle structure and must therefore be an intrinsic property of the enzyme structures. Since all lysosomal enzymes are glycosylated and since oligosaccharide moieties have been implicated in the protection of a few proteins from proteolytic attack, the role of oligosaccharides in lysosomal enzyme resistance to autolysis was investigated. The glycosylation of cellular proteins can be inhibited via treatment of fibroblast with the antibiotic tunicamycin. Under optimal conditions, tunicamycin treatment results in a reproducible decrease in fibroblast lysosomal enzyme levels. The observed decrease in cellular enzyme levels cannot be explained by inhibition of protein synthesis nor by increased secretion of enzyme into the extracellular medium. Furthermore, there is no significant appearance of lysosomal enzymes having low affinity for Sepharose Con A. These data suggest that addition of carbohydrate at the time of enzyme synthesis is important for the formation of enzymes which are both active and stable. When crude cell homogenates are incubated with endo-(beta)-N-acetylglucosaminidase H to remove high mannose oligosaccharides, there is a decreased affinity of the endogenous lysosomal enzyme DAP-I for Sepharose Con A, indicating a deglycosylation of the enzyme. Separation of DAP-I populations with differing Con A affinities reveals no correlation between the extent of glycosylation and enzyme stability at acid pH. These data suggest that while the oligosaccharide components of lysosomal enzymes may be critical for the proper synthesis of active enzyme, once the native structures are formed, surface carbohydrate moieties are not required to maintain lysosomal enzyme resistance to acid inactivation. Lysosomal enzymes are less stable when fibroblast sonicates are incubated at neutral pH. By contrast, non-lysosomal enzymes and the majority of cell proteins are resistant to degradation at neutral pH. These data indicate that the low pH of the lysosomes serves two purposes: (1) to provide an environment where lysosomal enzymes may retain their native conformation, and (2) to disrupt the native conformations of extralysosomal proteins thus sensitizing them to degradation.