Ohkubo K, Ogata S, Misumi Y, Takami N, Sinohara H, Ikehara Y
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Biochem J. 1991 Jun 1;276 ( Pt 2)(Pt 2):337-42. doi: 10.1042/bj2760337.
A cDNA clone (lambda MC-2) for contrapsin, a serine-proteinase inhibitor, was isolated from a lambda ZAP mouse liver cDNA library. The 1.6 kb cDNA insert of lambda MC-2 contained an open reading frame that encodes a 418-residue polypeptide (46,970 Da), in which a signal peptide of 21 residues was identified by comparison with the N-terminal sequence of the purified protein. The predicted structure (MC-2) also contained other peptide sequences determined by Edman degradation. Four potential sites for N-linked glycosylation were found in the molecule, accounting for the difference in molecular mass between the predicted form and the purified protein (63 kDa). Further screening of the cDNA library with an EcoRI-EcoRI fragment (510 bp) of lambda MC-2 as a probe yielded another cDNA clone (lambda MC-7), which encodes a 418-residue polypeptide (MC-7) with a calculated mass of 47,010 Da. MC-2 showed 83% similarity at the amino acid level to MC-7, in contrast with 44% similarity to alpha 1-proteinase inhibitor. The possible reactive site (P1-P'1) for serine proteinase is suggested to be Lys-Ala for MC-2 and Ser-Arg for MC-7. Northern-blot analysis revealed that both MC-2 and MC-7 mRNAs have the same size of 1.8 kb and are markedly induced in response to acute inflammation. Construction of the expression plasmids pSVMC-2 and pSVMC-7 and their transfection into COS-1 cells demonstrated that pSVMC-2 directs the synthesis of a 63 kDa form whereas pSVMC-7 expresses a 56 kDa form. The difference in molecular mass between the two may be explained by the fact that the MC-7 sequence contains three potential sites for N-glycosylation, one site less than that of MC-2.
从λZAP小鼠肝脏cDNA文库中分离出丝氨酸蛋白酶抑制剂抗胰蛋白酶的一个cDNA克隆（λMC - 2）。λMC - 2的1.6kb cDNA插入片段包含一个开放阅读框，编码一个418个氨基酸残基的多肽（46,970Da），通过与纯化蛋白的N端序列比较，确定其中有一个21个氨基酸残基的信号肽。预测结构（MC - 2）还包含通过埃德曼降解确定的其他肽序列。在该分子中发现了四个潜在的N - 糖基化位点，这解释了预测形式与纯化蛋白（63kDa）之间分子量的差异。用λMC - 2的EcoRI - EcoRI片段（510bp）作为探针进一步筛选cDNA文库，得到另一个cDNA克隆（λMC - 7），它编码一个418个氨基酸残基的多肽（MC - 7），计算分子量为47,010Da。MC - 2在氨基酸水平上与MC - 7的相似性为83%，而与α1 - 蛋白酶抑制剂的相似性为44%。推测丝氨酸蛋白酶的可能反应位点（P1 - P'1）对于MC - 2是Lys - Ala，对于MC - 7是Ser - Arg。Northern杂交分析显示，MC - 2和MC - 7的mRNA大小均为1.8kb，并且在急性炎症反应中明显上调。表达质粒pSVMC - 2和pSVMC - 7的构建及其转染到COS - 1细胞中表明，pSVMC - 2指导合成一种63kDa的形式，而pSVMC - 7表达一种56kDa的形式。两者分子量的差异可能是由于MC - 7序列包含三个潜在的N - 糖基化位点，比MC - 2少一个位点。
