Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L
Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L
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DOI：
10.1021/bi972251z
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发表时间：
1998-06-09
期刊：
影响因子：
2.9
通讯作者：
Gottesman, MM
中科院分区：
文献类型：
--
作者：
Chauhan, SS;Ray, D;Gottesman, MM
组织蛋白酶L是一种溶酶体半胱氨酸蛋白酶，由恶性转化细胞过度表达和分泌。然而，这种含man 6-磷酸的溶酶体蛋白酶分泌到细胞外介质中的原因尚不清楚。我们希望确定在组织蛋白酶L的酶原形式的一级序列内是否存在影响其亚细胞和细胞外定位的区域。人组织蛋白原L在小鼠NIH 3 T3细胞中的高水平瞬时表达导致该蛋白质的大部分分泌到细胞外介质中。同时，在这些非转化细胞中的内源性小鼠组织蛋白原L被发现在其通常的位置在溶酶体中。具有涉及最后11个氨基酸的羧基末端缺失的人组织蛋白原L的突变体不分泌到培养基中。从羧基末端缺失少至两个氨基酸，Thr和瓦尔，阻断了蛋白质的分泌，但不影响其酶活性、翻译后加工或亚细胞分布。由两个庞大的氨基酸Tyr-Asn的Thr-Val的替换允许的proatepsin L的分泌，但这两个氨基酸的替换由nonbulky丙氨酸阻止其分泌。最后6个氨基酸的单丙氨酸取代（ASYPTV）表明，Y或T的丙氨酸取代不影响hproCAT E的分泌，但S、P或V的丙氨酸取代完全阻断其分泌到培养基中。因此，我们得出结论，前组织蛋白酶L的羧基末端含有一个序列，其分泌所必需的。
Cathepsin L, a lysosomal cysteine protease, is overexpressed and secreted by malignantly transformed cells. However, the reason for secretion of this man 6-phosphate-containing lysosomal protease into the extracellular medium is not clear. We wished to determine whether there is a region within the primary sequence of the proenzyme form of cathepsin L which affects its subcellular and extracellular localization. High-level transient expression of human procathepsin L in mouse NIH 3T3 cells results in the secretion of most of this protein into the extracellular medium. At the same time, the endogenous mouse procathepsin L in these nontransformed cells is found in its usual location in lysosomes. Mutants of human procathepsin L with carboxy-terminus deletions involving the last 11 amino acids are not secreted into the medium. Deletion of as little as two amino acids, Thr and Val, from the carboxy terminus, blocked the secretion of the protein but did not affect its enzyme activity, posttranslational processing, or subcellular distribution. Replacement of Thr-Val by two bulky amino acids Tyr-Asn allowed secretion of the procathepsin L, but the replacement of these two amino acids by nonbulky alanines prevented its secretion. Single alanine substitutions of the last six amino acids (ASYPTV) indicated that substitution by alanine of Y or T does not affect the secretion of hproCAT E, but alanine substitutions of S, P, or V completely blocked its secretion into the culture medium. We therefore conclude that the carboxy terminus of procathepsin L contains a sequence essential for its secretion.