R306S gain-of-function mutation enhances PCSK9-mediated lowering of LDL-R expression

To investigate the effect of missense mutation in PCSK9 gene R306S on the metabolism of LDL and to analyze its relationship between structure and function. METHODS The wild type PCSK9 gene and the second or the third grade structures of the mutant R306S coding protein were all forecasted with the aid of Expert Protein Analysis System. The PCSK9 gene eukaryocyte expression vector was constructed and the nucleotide sequence was determined; The pathotype recombinant eukaryotic expression plasmid which carrying PCSK9 gene was constructed by directed-mutation method; Liposorne was used as intermediate to transfect the recombinant plasmid into BEL-7402 cell, the cells receiving no plasmid transfection served as control during this process; The LDL-R level was detected by Western Blot. RESULTS Through simulating the second and the third grade structures of protein, conformations changes were found in the two major coding protein parts of mutated PCSK9 gene: carboxy-terminal domain and catalyzing subunit site domain, the space between these two domains increased as well. And that was not found in wild type of PCSK9 gene. Tansfecting wild type of PCSK9 plasmid into BEL-7402 cell could lead to the decrease of LDL-R mature protein expression compared with controlled group(P<;0.05) and there was a significant decrease when cells transfected with positive control plasmids (P<;0.01). CONCLUSIONS The R306S mutation, gene mutant of PCSK9, could significantly lead to the decrease of LDL-R mature protein expression. Analysis results obtained from computer analogy confirmed that there were changes on the second and the third grade structures of R306S coding protein.