| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Mechanism in the secretion of congenital FXI variant with a novel mutation near the C-terminal region
To elucidate the mechanism in the secretion of FXI W599R, we studied the expression experiments.
And, the structural model of FXIW599R was visualized using the co-ordinates of FXIa monomer extracted from the rhFXI37O-607-ecotin complex (1XXd.PBD). Structural modeling showed that the W599 residue is positioned on an alpha helix in the C-terminal region of the FXI molecule. This residue belongs to a structurally conserved region among human FXI, rabbit FXI, bovine FXI, rat FXI and mouse FXI sequences. It was suggested that W599 in the alpha helix at the C-terminal region FXI may be structurally important for secretion from cells.
FXI cDNA with the unique restriction enzyme sites were amplified by PCR using single strand cDNA from normal human liver. To investigate the influence of the FXIW599R on FXI biosynthesis, the expression experiments were performed in COS-1 cells and CHO-K1 cells usi
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ng the pcDNA3.0 Zeo vector containing either wild type FXIcDNA or mutant FXIcDNA. FXIc and FXIag levels of FXIW599R secreted into the conditioned media were <1%, and the intracellular FXI was present at 60 to 70% of the wild type recombinant protein level in cells. And FXIW599G, FXIW599E and FXIW599F were same results as FXIW599R. The real-time quantitative transcript analysis were performed to confirm that gene transcription and mRNA processing were not impaired in the cultured cells transfected the wild type pcDNA XI and pcDNA XI599R. It was demonstrated that comparative amounts of FXI mRNA were same in cultured cells transfected the pcDNA XI wild type and pcDNA XI599R, respectively. Both wild type FXI and mutant types were present in cells as disulfide bond-linked dimer corresponding to 160KDa with a small amount of monomer corresponding to 80KDa under non-reducing condition on a 7.5% SDS-PAGE using the intracellular FXI labeled with ^<35>S-methionin. The mutant FXI in the conditioned media was much lower than that of wild-type FXI at all time points in pulse-chase studies with [^<35>S] methionine. We analyzed the effects of tunicamycin, castanosperin and nojirimycin, potent specific inhibitor of glycosylation in ER, and the effects of lactacystin, potent specific inhibitor of 20S-proteasome degradation and leupeptin, inhibitor of lysosomal degradation to investigate the mechanism of accumulation and degradation of FXI in stably transfected CHO cells. The intracellular levels of FXI W599R did not change in the presence of tunicamycin, castanosperin and nojirimycin. The intracellular levels of FXI W599R did not change in the presence of leupeptin and lactacystain, whereas those of the mutant FXI decreased in the presence of brefeldin A. Less
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