Sulfhydryl oxidase (SOx) from mouse epidermis: molecular cloning, nucleotide sequence, and expression of recombinant protein in the cultured cells
Sulfhydryl oxidase (SOx) from mouse epidermis: molecular cloning, nucleotide sequence, and expression of recombinant protein in the cultured cells
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DOI：
10.1016/s0923-1811(02)00061-0
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发表时间：
2002-10-01
影响因子：
4.6
通讯作者：
Ogawa, H
中科院分区：
文献类型：
--
作者：
Matsuba, S;Suga, Y;Ogawa, H
皮肤巯基氧化酶 (SOx) 是一种通过氧化皮肤中的巯基化合物来催化二硫键 (S-S) 交联的酶。在本研究中，我们使用从大鼠精囊中纯化的酶，通过质谱法获得了 Sox 的肽序列。然后，我们通过组装 GeneBank 数据库中的鼠表达序列标签 (EST) 来搜索与大鼠肽序列高度对应的 Sox 核苷酸。组装的小鼠 Sox cDNA 具有 1704 bp 核苷酸的开放阅读框，翻译成 568 个氨基酸。小鼠 Sox 蛋白的计算分子量为 65 kDa。该小鼠序列可以通过逆转录聚合酶链式反应从各种小鼠组织样本的总RNA中扩增，特别是从精囊和表皮的总RNA中高度扩增。随后将该 cDNA 片段克隆到哺乳动物表达载体 (pTARGET-MSSOx) 中，使我们能够在培养细胞中表达小鼠重组 Sox 蛋白。当 pTARGET-MSSOx 转染时，使用抗 SOx 抗血清的蛋白质印迹分析可以在全细胞提取物和收获 HEK 细胞后的培养基中检测到 65 kDa 重组 Sox 条带。在免疫组织化学分析中，引入 pTARGET-MSSOx 后，Pt-K2 细胞似乎产生了一种与细胞中的抗 SOx 抗血清发生反应的 Sox 蛋白。此外，在添加N-乙基马来酰亚胺和二硫苏糖醇后，使用N-(7二甲基氨基-4-甲基香豆素基)马来酰亚胺(DACM)对S-S键进行间接染色，结果表明S-S桥的形成几乎与转染后Pt-K2细胞中SOx表达的定位相匹配。本质上，我们克隆了皮肤 SOx cDNA，并将其表征为 S-S 交联酶之一。来自小鼠表皮的 SOx 克隆似乎有助于研究表皮中酶的潜在功能，特别是对于了解 SOx 在角质形成细胞分化中的生理作用。 (C) 2002 Elsevier Science Ireland Ltd. 保留所有权利。
Skin sulfbydryl oxidase (SOx) is an enzyme that catalyzes disulfide (S-S) cross-linking through the oxidation of sulfhydryl compounds in the skin. In this study, using the enzyme purified from rat seminal vesicle, we obtained peptide sequences for Sox by mass spectrometry. We then searched for Sox nucleotides corresponding highly to the rat peptide sequences by assembling murine-expressed sequence tags (ESTs) from the GeneBank database. The assembled mouse Sox cDNA has an open reading frame of 1704-bp nucleotides, translating into a size of 568 amino acids. The calculated molecular mass of the mouse Sox protein is 65 kDa. This mouse sequence can be amplified from total RNAs of various mouse tissue samples by reverse transcription polymerase chain reaction, especially highly amplified from those of the seminal vesicles and epidermis. The cDNA fragment was subsequently cloned into the mammalian expression vector (pTARGET-MSSOx), allowing us to express mouse recombinant Sox protein in cultured cells. When pTARGET-MSSOx was transfected, Western blot analysis using anti-SOx antiserum could detect a 65 kDa-band of recombinant Sox in both samples from the whole cell extract and the medium after the harvest of the HEK cells. In immimohistochemical analysis, the Pt-K2 cells, following the introduction of pTARGET-MSSOx, seemed to generate a Sox protein reactive to anti-SOx antiserum in the cells. Moreover, the indirect staining of the S-S bonds using N-(7dimethylamino-4-methyl coumarinyl) maleimide (DACM), following the addition of N-ethylmaleimide and dithiothreitol, showed that the formation of S-S bridges almost matched the localization of SOx expression in the Pt-K2 cells after the transfection. In essence, we cloned skin SOx cDNA and characterized it as one of the S-S cross-linking enzymes. The SOx clone from mouse epidermis seems to be useful for investigating the potential function of the enzyme in the epidermis, especially for understanding the physiological role of SOx in the differentiation of keratinocytes. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.