Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase.
Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase.
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角质层糜蛋白酶的克隆、表达和表征。
DOI：
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发表时间：
1994
影响因子：
4.8
通讯作者：
T. Egelrud
中科院分区：
文献类型：
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作者：
L. Hansson;M. Strömqvist;A. Bäckman;P. Wallbrandt;A. Carlstein;T. Egelrud
从角质形成细胞来源的文库中分离到编码人角质层糜蛋白酶(SCCE)的基因。获得的核苷酸序列含有一个开放阅读框，足以编码一个由253个氨基酸残基组成的前原蛋白。检测到两种与SCCE基因杂交的基因在人皮肤中的表达，分别为1.2kb和2.0kb。这两种形式在3‘-非翻译序列的长度方面不同。对来自人体不同组织的mRNA的分析表明，SCCE基因的大量表达仅限于人类皮肤。将克隆的基因导入牛乳头瘤病毒表达系统，纯化并鉴定重组蛋白。结果表明，重组SCCE由一个22个氨基酸残基组成的信号肽和一个由7个氨基酸残基组成的前肽组成。胰酶消化去除了这一前肽，并产生了一种蛋白水解性蛋白，其NH2末端氨基酸序列与天然SCCE相同。推导出的氨基酸序列包含丝氨酸蛋白酶的保守活性部位区域。计算出未糖基化的活性单链氨基酸的相对分子质量为24.4 kDa。该序列表明在C末端附近有一个暂定的N-糖基化位点。重组SCCE被发现在糖基化方面是异质性的，其方式类似于天然酶。
The cDNA encoding human stratum corneum chymotryptic enzyme (SCCE); an epidermal serine-proteinase which was recently purified from human stratum corneum, was isolated from a keratinocyte derived library. The obtained nucleotide sequence contained an open reading frame sufficient to encode a preproprotein consisting of 253 amino acid residues. Expression of two mRNA species hybridizing with SCCE cDNA, 1.2 and 2.0 kilobases, respectively, was detected to human skin. These two forms differ with respect to the length of the 3'-untranslated sequence. Analysis of mRNA derived from various human tissues showed that abundant expression of the SCCE gene was restricted to human skin. The cloned cDNA was introduced to a bovine papilloma virus-based expression system and recombinant protein was purified and characterized. The results show that recombinant SCCE is produced with a 22-amino acid residue signal peptide and a propeptide of 7 amino acid residues. Tryptic digestion removed this propeptide and yielded a proteolytically active protein with the same NH2-terminal amino acid sequence as native SCCE. The deduced amino acid sequence contains the conserved active site regions of serine proteinases. The calculated molecular mass of unglycosylated active SCCE was 24.4 kDa. The sequence indicates one tentative N-glycosylation site located near the C terminus. Recombinant SCCE was found to be heterogenous regarding glycosylation in a manner similar to that of the native enzyme.