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Published on: January 5, 2017
Only the light chain is sufficient for the serine protease function of the membrane bound form of factor I on a
Daisuke Fukuta1, Shuji Miyagawa, Tomoko Kubo
1Research and Development Laboratory, Nipro Corporation, 3023 Noji, Shiga 525-0055, Japan.
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.
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.
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