Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels.
Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels.
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DOI：
10.1016/0003-9861(90)90666-m
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发表时间：
1990-12
影响因子：
3.9
通讯作者：
N. A. Stearns;J. M. Dong;J. Pan;D. Brenner;G. Sahagian
中科院分区：
文献类型：
--
作者：
N. A. Stearns;J. M. Dong;J. Pan;D. Brenner;G. Sahagian
转化小鼠成纤维细胞（MEP）的主要分泌蛋白是溶酶体半胱氨酸蛋白酶，即组织蛋白酶L。该蛋白在小鼠成纤维细胞中的合成和细胞内运输受生长因子和恶性转化的调节。为了进一步确定这种调节的基础，编码MEP/组织蛋白酶L的cDNA从小鼠肝脏cDNA文库中分离，并用于比较正常和Kirsten肉瘤病毒转化的NIH 3 T3成纤维细胞的组织蛋白酶L。虽然组织蛋白酶L信息水平升高20倍，在转化的成纤维细胞，正常和转化细胞显示相似的组织蛋白酶L基因组DNA消化模式和基因拷贝数，和组织蛋白酶L mRNA序列出现相同的RNA酶保护分析。这些发现表明（i）组织蛋白酶L在正常细胞和转化细胞中由相同的基因合成，（ii）由这些细胞产生的组织蛋白酶L多肽以相同的一级序列翻译。由静止、生长和转化细胞合成的组织蛋白酶L多肽显示出相似的等电聚焦模式，表明相似的翻译后修饰。利用小鼠肝cDNA的定点突变和在COS猴细胞中的表达来检测小鼠组织蛋白酶L的糖基化。结果表明，两个潜在的N-连接糖基化位点中只有一个（Asn 221处的一个）被糖基化。通过QAE-Sephadex离子交换层析和甘露糖6-磷酸受体-Affi-Gel 10亲和层析分析表明，组织蛋白酶L低聚糖在正常细胞和转化细胞中的磷酸化程度相似。虽然观察到几种磷酸化寡糖物质，但主要物质含有两个磷酸单酯部分，并有效地与受体结合。这些研究结果表明，由正常和转化的小鼠成纤维细胞的组织蛋白酶L是相同的，并证实了这一假设，即在这些细胞中的组织蛋白酶L的贩运是由生长诱导的变化在溶酶体蛋白转运系统的调节。
The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system.