Cellular and secreted pro-protein convertase subtilisin/kexin type 9 catalytic activity in hepatocytes

Abstract

ObjectivesPro-protein convertase subtilisin/kexin type 9 (PCSK9) impairs the low density lipoprotein receptor (LDLr) recyling. To reach the LDLr, the pro-protein must cleave itself in the endoplasmic reticulum. Using a fluorogenic peptide corresponding to the cleavage site, we directly monitored for the first time the cleavage activity of purified human PCSK9 and that of endogenous human wild-type PCSK9 and naturally occurring variants in hepatocytes. MethodsValidation of the assay was performed with wild type or PCSK9 deficient primary mouse hepatocytes and immortalized human hepatocytes transfected with antiPCSK9 siRNA. An analysis of the cleaved peptide was performed using mass spectrometry. Pharmacological regulation of the enzyme was studied in human hepatocytes. Expression vectors coding for the variants S127R, D374Y, F216L, S386A were transfected in primary hepatocytes from PCSK9 deficient mice. ResultsPCSK9 activity was measured in cell lysates and media, at levels 100 times higher than with the human purified recombinant protein. The assay is highly specific for PCSK9 in cell lysate and cell culture media but not in plasma. Pharmacological up- or down-regulation of PCSK9 expression produced paralleled effects on the activity. The catalytic activity of gain-of-function variants S127R, D374Y recapitulated roughly the maturation efficiency estimated by western blots, in contrast with the F216L variant that presented with a 54% lower catalytic activity than the wild-type protein, despite similar proPCSK9 to PCSK9 ratios. Thus, other factors might be involved in the maturation of PCSK9. ConclusionAll together, these results shed a new light on PCSK9 enzymatic activity and could help identifying proPCSK9 inhibitors.