ACTION OF LYSOSOMAL ENZYMES ON THE EXTRACELLULAR MATRIX

本项目旨在验证质膜受体 特异性甘露糖-6-磷酸（M6 P）用于调节 某些膜成分，通过锚定溶酶体酶， 接近其细胞表面基质或通过调节 这些酶在细胞外液中的浓度。 我们有 获得的证据表明将M6 P添加到人成纤维细胞中 培养改变了35 SO 4标记的细胞的周转率和模式， 将细胞周糖胺聚糖（GAG）引入细胞外区室。 我们还发现M6 P的添加会导致 放射性蛋白释放到细胞中的多肽谱 细胞外介质 我们建议继续进行这项相关研究， M6 P对细胞中GAG和蛋白质周转的影响 在无M6 P表面受体的情况下，以及在有和无溶酶体的细胞中， 携带M6 P标记的酶。 此外，我们希望确定和 表征衍生自以下的GAG和蛋白质的分子差异： 用和不用M6 P处理的培养物的相应区室。 我们 我还提议测试放射性标记的蛋白质 并且底物附着材料的GAG可以被消化并释放到 培养基被外源添加的细胞降解， 对M6 P敏感。 这些目标的成功实现可能会让我们 为了建立用于纯化负责以下的酶的测定： 在细胞表面的蛋白质和GAG中观察到的分子差异 和细胞外基质作为M6 P添加的结果。 这些研究可能 提供关于溶酶体酶在细胞内代谢物周转中的作用的见解。 表面膜成分，对细胞粘附的一般过程， 基质，细胞运动，以及某些疾病状态， 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.