Fukuta Daisuke, Miyagawa Shuji, Kubo Tomoko, Kusama Tamiko, Shirasu Akio, Hattori Hiroyuki, Shirakura Ryota
Research and Development Laboratory, Nipro Corporation, 3023 Noji, Shiga 525-0055, Japan.
Transpl Immunol. 2004 Jan;12(2):109-14. doi: 10.1016/j.trim.2003.11.001.
The cell membrane-bound forms of whole factor I (fI-PI), the light chain of the serine protease (SP) domain (SP-PI), and the light chain plus the COOH-terminal 45 amino acid (AA) of the heavy chain (SP+45-PI) were constructed. Chinese hamster ovary (CHO) cells, expressing these molecules were established by transfection of cDNA and confirmed by flow cytometry. Amelioration of complement-mediated cell lysis and complement fragment deposition on the cell surface by the transfectant molecules was tested in each CHO cell by means of a lactate dehydrogenase (LDH) assay and flow cytometry, respectively. A highly expressed fI-PI blocked human complement-mediated cell lysis by approximately 84% of the cells. CHO cell transfectants with SP-PI also showed a clear inhibition in cell lysis by human serum, whereas CHO cell transfectants with SP+45-PI showed no inhibition. In addition, fI-PI and SP-PI, but not SP+45-PI, suppressed C5b-9 deposition on CHO cell surface. These data indicate that the last 45 amino acid of the heavy chain, including a disulfide bridge area, did not participate in the serin protease function of factor I. The results suggest that SP-PI has potential for use in clinical xenotransplantation.
构建了全因子I（fI-PI）的细胞膜结合形式、丝氨酸蛋白酶（SP）结构域的轻链（SP-PI）以及轻链加45个氨基酸（AA）的重链COOH末端（SP+45-PI）。通过转染cDNA建立了表达这些分子的中国仓鼠卵巢（CHO）细胞，并通过流式细胞术进行了确认。分别通过乳酸脱氢酶（LDH）测定和流式细胞术在每个CHO细胞中测试了转染分子对补体介导的细胞裂解和补体片段在细胞表面沉积的改善作用。高表达的fI-PI可使约84%的细胞免受人类补体介导的细胞裂解。携带SP-PI的CHO细胞转染体也显示出对人血清介导的细胞裂解有明显抑制作用，而携带SP+45-PI的CHO细胞转染体则无抑制作用。此外，fI-PI和SP-PI可抑制C5b-9在CHO细胞表面的沉积，而SP+45-PI则无此作用。这些数据表明，重链的最后45个氨基酸，包括一个二硫键区域，不参与因子I的丝氨酸蛋白酶功能。结果表明，SP-PI在临床异种移植中有应用潜力。
