SYNTHESIS AND FUNCTION OF A TRANSFORMATION-DEPENDENT SECRETED LYSOSOMAL PROTEASE

恶性转化的细胞分泌多种蛋白水解酶和蛋白酶 被假设为参与了 侵袭性、转移性、免疫抑制和副肿瘤综合征 与癌症有关。我们的工作集中在一个 分泌型溶酶体半胱氨酸蛋白酶L原，简称MEP 转化细胞的主要排泄蛋白)。组织蛋白L是一个宽泛的人 在溶酶体中发现的光谱酸性蛋白水解酶 酶原进入细胞外空间。我们已经克隆了小鼠的基因 和L的人酶原，并比较了它们的结构和模式 监管。每个外显子有8个外显子和7个内含子，位于相同的位置 在编码区内，但L原蛋白的结构基因 小鼠全长7400bp，而人L原基因全长5100bp， 大小的差异几乎完全由内含子大小来解释。 与小鼠基因不同的是，小鼠基因有一个独特的转录起点和 一个主要的信使核糖核酸物种，至少有两个主要信使核糖核酸转录自 人L原蛋白基因。这两个mRNAs的5‘端不同 未翻译的序列，并代表可选剪接或可选 推动者。确定L原为什么会被大量分泌 恶性转化细胞和其他细胞的活性 过度繁殖，我们已经启动了人类的缺失分析 L蛋白原基因。人原蛋白原缺失形式在L中的表达 在小鼠NIH3T3细胞中导致要么不能形成稳定的 结构(“亲”片段的删除)或不能被分泌 (羧基-末端删除)。评价分泌细胞的功能 原血管紧张素在肿瘤转移中的合成、分泌和 组织蛋白酶L和B在小鼠B16黑色素瘤细胞系中的活性 不同的转移潜能，并且发现与能力没有明显的相关性 转移到转移。

Malignantly transformed cells secrete a variety of proteases and protease precursors which have been hypothesized to be involved in the invasiveness, metastasis, immune suppression, and paraneoplastic syndromes associated with cancer. Our work has concentrated on the function of a secreted lysosomal cysteine proprotease called procathepsin L or MEP (for major excreted protein of transformed cells). Cathepsin L is a broad spectrum acid protease found both in lysosomes and secreted as the proenzyme into the extracellular space. We have cloned the genes for mouse and human procathepsin L and compared their structures and modes of regulation. Each has 8 exons and 7 introns located at the same positions within the coding region, but the structural gene for procathepsin L in the mouse spans 7400 bp, while the human procathepsin L gene is 5100 bp, with the size difference accounted for almost entirely by intron size. Unlike the mouse gene which has a unique start site of transcription and a single major mRNA species, at least two major mRNAs are transcribed from the human procathepsin L gene. These two mRNAs differ in their 5' untranslated sequences, and represent alternative splicing or alternative promoters. To determine why procathepsin L is secreted in large amounts by the activity of malignantly transformed cells and other cells which overproduce it, we have initiated a deletion analysis of the human procathepsin L cDNA. Expression of deleted forms of human procathepsin L in mouse NIH 3T3 cells results in either failure to form a stable structure (deletions of the "pro" piece) or failure to be secreted (carboxy-terminal deletions). To evaluate the function of secreted procathepsins in metastasis we have compared the synthesis, secretion, and activity of cathepsins L and B in a series of mouse B 16 melanoma cells of varying metastatic potential, and find no clear correlation with ability to metastasize.