CONTROL OF SYNTHESIS OF A TRANSFORMATION-DEPENDENT SECRETED GLYCOPROTEIN

RNA病毒转化培养的小鼠成纤维细胞 病毒或化学制剂都能分泌39000 mR-磷酸糖蛋白 (主要排泄的蛋白质，MEP)大量存在。未转化的小鼠 成纤维细胞在TPA或TPA等肿瘤促进剂治疗后分泌MEP PDGF等生长因子。纯化的蛋白质含有甘露糖。 6-磷酸，溶酶体识别标记。它是经过处理的 在转化和未转化的细胞内提供两个 特定的低分子量形式，其中最低的具有 主要是溶酶体定位。MEP的分泌形式是 一种低相对分子质量的新型硫醇蛋白酶(组织蛋白酶)的前体 最适pH为3-5，能降解多种蛋白质 包括细胞外基质蛋白胶原、纤维连接蛋白和 层粘连蛋白。已确定了多肽键断裂的特异性 使用氧化的胰岛素B链。分泌型MEP可与甘露糖结合 多种细胞的6-磷酸受体及其内吞和加工 在细胞内。转化、TPA和PDGF通过促进MEP合成 使用MEP特异性基因测得的MEP特异性mRNA水平增加 Cdna探针。MEP mRNA水平升高的机制是增强的 在核径流实验中测得的转录。我们已经克隆了一个 小鼠的功能性MEP基因，并已鉴定出5‘端的侧翼 推测含有MEP启动子的区域。我们正在研究这个系统 作为调节溶酶体蛋白水解酶合成的模型，加工和 分泌物，因为它受到转化和模拟 转化状态，如肿瘤促进剂和生长因子。

Cultured mouse fibroblasts which are transformed by RNA viruses, a DNA virus or a chemical agent, all secrete a 39,000 Mr-phosphoglycoprotein (major excreted protein, MEP) in large amounts. Nontransformed murine fibroblasts secrete MEP after treatment with tumor promoters such as TPA or growth factors such as PDGF. The purified protein contains mannose 6-phosphate, the lysosomal recognition marker. It is processed intracellularly in both transformed and nontransformed cells to give two specific lower molecular weight forms, the lowest of which has a predominantly lysosomal localization. The secreted form of MEP is the precursor to a lower molecular weight novel thiol protease (cathepsin) with a pH optimum of 3-5 capable of hydrolyzing a wide variety of proteins including the extracellular matrix proteins collagen, fibronectin and laminin. The specificity of peptide bond cleavage has been determined using oxidized insulin B chain. Secreted MEP can bind to the mannose 6-phosphate receptor of many cells and be endocytosed and processed intracelluarly. Transformation, TPA and PDGF stimulate MEP synthesis by increasing levels of MEP specific mRNA as measured using an MEP specific cDNA probe. The mechanism of the increase in MEP mRNA levels is increased transcription as measured in nuclear run-off experiments. We have cloned a functional MEP gene from the mouse and have identified the 5' flanking region presumed to contain the MEP promoter. We are studying this system as a model of regulation of lysosomal protease synthesis, processing and secretion as it is affected by transformation and agents which mimic the transformed state, such as tumor promoters and growth factors.