We have focused our recent efforts on studying the biosynthesis and targeting of newly synthesized lysosomal enzymes to lysosomes. The asparagine-linked oligosaccharide units of lysosomal enzymes undergo an extensive series of processing reactions as the newly synthesized enzymes move from the rough endoplasmic reticulum, where they are initially glycosylated, to their final destination in lysosomes. The most important modification is the generation of the phosphomannosyl recognition marker, which occurs in two steps. First, N-acetylglucosamine 1-phosphate is transferred to an acceptor mannose by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase, resulting in a phosphate group in diester linkage between outer N-acetylglucosamine and an inner mannose. Next, a phosphodiester glycosidase removes the N-acetylglucosamine, leaving the phosphate in monoester linkage to the underlying mannose residue. This exposed phosphomannosyl residue serves as an essential component of a recognition marker which leads to binding to high-affinity receptors and subsequent translocation to lysosomes. The transferase acts specifically on lysosomal enzymes, thereby catalyzing the initial, determining step by which newly synthesized lysosomal enzymes are distinguished from other newly synthesized glycoproteins and marked for eventual targeting to lysosomes. We have found that deglycosylated lysosomal enzymes are potent inhibitors of the transferase, indicating that these enzymes contain a common protein sequence or conformation that is recognized by the transferase. Our current experiments are directed toward the identification of this recognition signal. Our preliminary results indicate that the conformation of the lysosomal enzymes is important for the expression of the recognition signal. We have also identified a number of murine cell lines that lack the 215 Kda Man-6-P receptor and yet possess high levels of intracellular acid hydrolases which are contained in dense granules characteristic of lysosomes. We have recently identified and isolated a second Man-6-P receptor in these cells. This receptor, a 46 Kda glycoprotein, is widely distributed among cell types. We are trying to determine the physiologic function of each receptor. (A)
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