We introduced 2 different mutations, i.e CGC to TGA in codon 2159 and CGA to CAA in codon 2209 within the C-domain of factor VIII(FVIII) gene that had previously been reported as genetic defects in hemophilia A. The FVIII gene mutants corresponding to R2159C and R2209Q were made by site-directed mutagenesis. Synthesis and secretion of abnormal FVIII were analyzed by immunoprecipitation of cell extracts (CE) and condi-tioned media (CM) from
35S-methionine pulse-labeled and chased transfected cells. SDS-PAGE analysis demonstrated that both mutants were normally synthesized, but disappear-ed from the CE during 4 hours of chase. Addition of ALLN, an inhibitor of cysteine-protease, increased the intracellular accumulation of the R2159C mutant in the CE, without affecting the amount recovered in the CM. These results indicate that defective secretion with subsequent intracellular degradation is responsible for the loss of the mutant FVIII from the CE, leading to the hemophilia A phenotype for both mutants.
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