Ohkubo K, Ogata S, Misumi Y, Takami N, Ikehara Y
Department of Biochemistry, Fukuoka University School of Medicine.
J Biochem. 1991 Feb;109(2):243-50.
A glycoprotein with Mr 63,000 purified from rat serum was found to inhibit trypsin activity but not chymotrypsin or elastase activity, resembling contrapsin purified from mouse serum. To obtain further information on the molecular structure, a cDNA clone (lambda CPi-21) for this contrapsin-like protease inhibitor was isolated from a rat liver cDNA library. The 1.6-kb cDNA insert contained an open reading frame that encodes a 416-residue polypeptide (CPi-21), in which the first 29 residues were suggested to comprise a signal peptide by comparison with the NH2-terminal sequence of the purified protein. The predicted structure also contained other peptide sequences determined by Edman degradation. Four potential N-linked glycosylation sites were found in the molecule, presumably accounting for the larger molecular mass of the mature form. Further screening of the cDNA library with a Pst-XbaI fragment (302 bp) of lambda CPi-21 as a probe yielded two other cDNA clones (lambda CPi-23 and lambda CPi-26), which encode 413-residue and 418-residue polypeptides, respectively. A comparison of their amino acid sequences revealed that CPi-21 has 89 and 71% homology with CPi-23 and CPi-26, respectively. The primary structure of each of the three proteins has about 70% homology with that of mouse contrapsin, in contrast to 43-46% homology with that of rat alpha 1-protease inhibitor. These results indicate that all the CPi proteins presented here belong to a subfamily of "serpins" of which mouse contrapsin was the first member to be identified.
从大鼠血清中纯化出一种分子量为63,000的糖蛋白，它能抑制胰蛋白酶活性，但不抑制糜蛋白酶或弹性蛋白酶活性，这与从小鼠血清中纯化出的抗胰蛋白酶相似。为了进一步了解其分子结构，从大鼠肝脏cDNA文库中分离出了一个编码这种抗胰蛋白酶样蛋白酶抑制剂的cDNA克隆（λCPi-21）。这个1.6kb的cDNA插入片段包含一个开放阅读框，编码一个416个氨基酸残基的多肽（CPi-21），通过与纯化蛋白的NH2末端序列比较，推测前29个残基构成信号肽。预测结构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，这可能是成熟形式分子量较大之原因。用λCPi-21的Pst-XbaI片段（302bp）作为探针进一步筛选cDNA文库，得到另外两个cDNA克隆（λCPi-23和λCPi-26），它们分别编码413个氨基酸残基和418个氨基酸残基的多肽。比较它们的氨基酸序列发现，CPi-21与CPi-23和CPi-26的同源性分别为89%和71%。这三种蛋白质的一级结构与小鼠抗胰蛋白酶的一级结构有大约70%的同源性，相比之下，与大鼠α1-蛋白酶抑制剂的同源性为43-46%。这些结果表明，本文所述的所有CPi蛋白都属于“丝氨酸蛋白酶抑制剂”亚家族，其中小鼠抗胰蛋白酶是被鉴定出的首个成员。
