Objective: The normal function of proprotein convertase subtilisin/kexin type 9 (PCSK9) is to mediate degradation of the low density lipoprotein (LDL) receptors. However, the exact mechanism for this function remains to be determined. Characterization of how the naturally occurring mutations in the PCSK9 gene affect the function of PCSK9, may provide important insight into the mechanism by which PCSK9 degrades the LDL receptors.
Methods: DNA sequencing of the 12 exons with flanking intron sequences of the PCSK9 gene was performed in 1336 unrelated hypercholesterolemic subjects. In vitro assays and bioinformatics analysis were employed to characterize the functional consequences of a novel mutation. EXPERIMENTAL RESULTS: One subject was heterozygous for the novel mutation S462P in exon 9 of the PCSK9 gene. Based upon Western blot analysis of transiently transfected HepG2 cells, S462P-PCSK9 was almost completely retained in the endoplasmic reticulum (ER) even though it did undergo autocatalytic cleavage. Thus, only trace amounts of S462P-PCSK9 were detected in the culture media of transfected cells. Flow cytometric experiments revealed that the S462P-PCSK9 mutant was unable to degrade the LDL receptors.
Discussion: The markedly reduced secretion of S462P-PCSK9 makes S462P a loss-of-function mutation. Ser462 is one of the few residues in the C-terminal domain which is conserved in all known PCSK9 homologs. A hydrogen bond between the side-chain of Ser462 and the backbone of beta-strand 6 of the C-terminal domain, appears to be essential for the proper folding of the C-terminal domain. The S462P mutation is believed to disrupt the normal folding of the C-terminal domain leading to retention of the mutant protein in the ER.