Idiris Alimjan, Ohtsubo Ken-ichi, Yoza Koh-ichi, Osada Toshiya, Nakamichi Noboru, Matsumura Toshiharu, Ikai Atsushi
Laboratory of Biodynamics, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
J Protein Chem. 2003 Jan;22(1):89-98. doi: 10.1023/a:1023076029496.
The "most primitive" living vertebrate the hagfish has a dimeric proteinase inhibitor, a protein homologous to human alpha2-macroglobulin, in its plasma at high concentration. Although the hagfish proteinase inhibitor has been isolated and its function and quaternary structure studied, its primary structure, subunit composition and fragmentation process remain unclear. In this study, hagfish proteinase inhibitor cDNA was cloned, sequenced and cDNA-deduced amino acid sequence was analyzed. A large fraction of homosubunits in the dimeric structure of the protein has undergone a cleavage at a specific arginyl residue (Arg833) while the rest retained their chain integrity without being processed. Thus random combinations of processed and nonprocessed subunits in the dimeric structure of this protein result in different molecular conformers and generate a complicated multiband pattern in SDS-PAGE. It was further demonstrated by proteolytic analysis that the hagfish inhibitor has no susceptible arginyl residues within its bait region and thus incapable of trapping arginine specific proteinases. This implies that the specific subunit cleavage at Arg833 was caused by an unknown arginine specific proteinase which escaped from the entrapment by the hagfish inhibitor.
“最原始”的现存脊椎动物——盲鳗，其血浆中有一种二聚体蛋白酶抑制剂，该蛋白与人类α2-巨球蛋白同源，且浓度很高。尽管盲鳗蛋白酶抑制剂已被分离出来，其功能和四级结构也已得到研究，但其一级结构、亚基组成和裂解过程仍不清楚。在本研究中，克隆并测序了盲鳗蛋白酶抑制剂的cDNA，并对cDNA推导的氨基酸序列进行了分析。该蛋白二聚体结构中的大部分同型亚基在特定的精氨酰残基（Arg833）处发生了裂解，而其余部分则保持其链完整性，未被加工。因此，该蛋白二聚体结构中已加工和未加工亚基的随机组合导致了不同的分子构象，并在SDS-PAGE中产生复杂的多带模式。蛋白水解分析进一步表明，盲鳗抑制剂在其诱饵区内没有敏感的精氨酰残基，因此无法捕获精氨酸特异性蛋白酶。这意味着在Arg833处的特定亚基裂解是由一种未知的精氨酸特异性蛋白酶引起的，该蛋白酶逃脱了盲鳗抑制剂的捕获。
