ACTION OF LYSOSOMAL ENZYMES ON THE EXTRACELLULAR MATRIX

该项目建议检验质膜受体的假说 甘露糖-6-磷酸(M6P)专一性调节细胞的周转 某些膜成分，通过锚定溶酶体酶 接近其细胞表面底物或通过调节 这些酶在细胞外液中的浓度。我们有 已有证据表明M6P添加到人成纤维细胞中 培养改变35SO4标记菌株的周转速度和模式 细胞周围的糖胺多聚糖(GAG)进入细胞外隔室。 我们还发现，M6P的加入导致了 释放到人体内的放射性蛋白质的多肽图谱 胞外培养液。我们建议继续进行这项相关研究， M6P对慢性粒细胞白血病细胞GAG和蛋白质周转的影响 没有M6P表面受体的细胞，以及有和没有溶酶体的细胞 携带M6P标记的酶。此外，我们希望确定和 描述来源于GAG和蛋白质的分子差异 用M6P和不用M6P处理的相应的培养隔室。我们 也建议测试放射性标记的蛋白质 附着在基材上的物质可以被消化并释放成 通过外源添加的细胞，这种降解过程是 对M6P敏感。这些目标的成功实现可能会让我们 建立酶的纯化方法(S) 观察到细胞表面蛋白质和GAG的分子差异 和细胞外基质作为M6P添加的结果。这些研究可能 提供有关溶酶体酶在周转中的作用的见解 表面膜成分，对细胞黏附的一般过程 底物，细胞运动，以及某些疾病状态，如 骨关节炎和转移。

This project proposes to test the hypothesis that plasma membrane receptors specific for mannose-6-phosphate (M6P) serve to modulate the turnover of certain membrane components, either by anchoring lysosomal enzymes proximate to their cell surface substrates or by regulating the concentration of these enzymes in the extracellular fluid. We have obtained evidence that indicates the addition of M6P to human fibroblast culture alters the rate and pattern of turnover of 35SO4-labeled pericellular glycosaminoglycans (GAGs) into the extracellular compartment. We have also found that the addition of M6P results in changes in the polypeptide profiles of radioactive proteins released into the extracellular medium. We propose to continue this correlative study on the effect of M6P on the turnover of GAGs and proteins in cells with and without surface receptors for M6P, and in cells with and without lysosomal enzymes carrying the M6P marker. In addition, we wish to identify and characterize the molecular differences in GAGs and proteins derived from corresponding compartments of cultures treated with and without M6P. We also propose to test the possibility that radioactively-labeled proteins and GAGs of substrate-attached material can be digested and released into the medium by exogenously added cells and that this degradation process is sensitive to M6P. The successful achievement of these goals may allow us to establish assays for the purification of the enzyme(s) responsible for molecular differences observed in the proteins and GAGs of the cell surface and extracellular matrix as a result of M6P addition. These studies may provide insights on the role of lysosomal enzymes on the turnover of surface membrane components, on the general processes of cell adhesion to the substratum, cell movement, as well as certain diseased states such as osteoarthritis and metastasis.