Yamashiro K, Tsuruoka N, Kodama S, Tsujimoto M, Yamamura Y, Tanaka T, Nakazato H, Yamaguchi N
Suntory Institute for Biomedical Research, Osaka, Japan.
Biochim Biophys Acta. 1997 Jan 3;1350(1):11-4. doi: 10.1016/s0167-4781(96)00187-x.
A cDNA encoding a precursor for a novel serine protease (neurosin) was cloned from a cDNA library prepared from a human colon adenocarcinoma cell line, COLO 201. The sequence consisted of 155 bp 5' non-coding region and a 732 bp open reading frame which was followed by a 551 bp 3' non-coding region. The predicted protein consists of 244 amino acids which is possibly processed to an active enzyme of 223 amino acids that shows some similarity (< 30%) to other members of serine protease family. As found in other trypsin-like proteases, the enzyme contains the catalytic triad which is characterized as the essential amino acid residues for the activity. Northern blot analyses of the mRNA showed the strongest expression in brain followed by a lower but significant one in spleen. A construct of cDNA encoding chimeric protein that carries pro-sequence of trypsin II and putative mature neurosin starting from Leu22 was transfected to COS-1 cells. Successful production of the active neurosin was shown after treating the supernatant of the culture of the transfectants with enterokinase.
从人结肠腺癌细胞系COLO 201构建的cDNA文库中克隆出一个编码新型丝氨酸蛋白酶（神经氨酸酶）前体的cDNA。该序列由155bp的5'非编码区和一个732bp的开放阅读框组成，后面跟着一个551bp的3'非编码区。预测的蛋白质由244个氨基酸组成，可能被加工成一个223个氨基酸的活性酶，该酶与丝氨酸蛋白酶家族的其他成员有一些相似性（<30%）。正如在其他胰蛋白酶样蛋白酶中所发现的那样，该酶含有催化三联体，其被表征为活性所必需的氨基酸残基。对mRNA的Northern印迹分析表明，在脑中表达最强，其次在脾脏中有较低但显著的表达。将一个编码嵌合蛋白的cDNA构建体转染到COS-1细胞中，该嵌合蛋白携带胰蛋白酶II的前序列和从Leu22开始的推定成熟神经氨酸酶。在用肠激酶处理转染细胞培养物的上清液后，显示出成功产生了活性神经氨酸酶。
