STRUCTURE, BIOSYNTHESIS AND FUNCTION OF GLYCOPROTEINS

我们最近的努力集中在研究生物合成和 新合成的溶酶体酶对溶酶体的靶向性。这个 天冬酰胺连接的溶酶体酶低聚糖单位经历 作为新合成的酶，广泛的一系列加工反应 从粗面内质网移出，它们最初在那里 糖基化，最终到达溶酶体的目的地。最重要的 修饰是磷脂酰基识别标记的产生， 这发生在两个步骤中。首先，N-乙酰氨基葡萄糖1-磷酸是 UDP-N-乙酰氨基葡萄糖：溶酶体转移到受体甘露糖 N-乙酰氨基葡萄糖1-磷酸转移酶，产生磷酸 外N-乙酰氨基葡萄糖与内N-乙酰氨基葡萄糖之间的二酯键基团 甘露糖。接下来，一种磷酸二酯糖苷酶去除 N-乙酰氨基葡萄糖，使磷酸盐以单酯连接到 潜在的甘露糖残留物。这种暴露的磷酸甘露醇残留物用作 识别标记的基本组件，它导致绑定到 高亲和力受体和随后的转位到溶酶体。这个 转移酶特异性地作用于溶酶体酶，从而催化 最初，决定新合成的溶酶体酶的步骤 区别于其他新合成的糖蛋白，并标记为 最终以溶酶体为靶标。我们已经发现脱糖基 溶酶体酶是转移酶的有效抑制物，这表明 这些酶包含一个共同的蛋白质序列或构象， 被转移酶识别。我们目前的实验针对的是 该识别信号的识别。我们的初步结果 提示溶酶体酶的构象对 识别信号的表达。我们还确认了一个号码 缺乏215 KDA Man-6-P受体的小鼠细胞株 高水平的细胞内酸性水解酶，包含在密集的 溶酶体特有的颗粒。我们最近确认了 在这些细胞中分离出第二个Man-6-P受体。这个受体，一个46kDa的 糖蛋白，广泛分布于细胞类型中。我们正在努力 确定每个受体的生理功能。(A)

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)