Abstract
Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an approximately 150-fold higher affinity at an acidic endosomal pH (K(D) = 4.19 nm) compared with a neutral pH (K(D) = 628 nm). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.
Highlights
High sequence similarity with the proteinase K family of subtilases and contains a catalytic triad (Asp186, His226, and Ser386) responsible for autoprocessing [1, 4]
We have presented biochemical and cell-based data that deepen our understanding of the mechanism by which secreted PCSK9 promotes LDL receptor (LDLR) degradation
All three cell lines had comparable basal low density lipoprotein (LDL) uptake levels that was lowered in a dose-dependent manner by exogenously added wild-type PCSK9, with the maximum potency achieved in HEK293 cells (EC50 ϭ 61 nM; 4.6 g/ml), followed by HepG2 cells (EC50 ϭ 64 nM; 4.8 g/ml) and Chinese hamster ovary (CHO) cells (EC50 ϭ 129 nM; 9.7 g/ml) (Fig. 2B)
Summary
The PCSK9 gene was amplified from human fetal liver QUICK-Clone cDNA (BD Biosciences). The following primers were used for the pre-amplification of the PCSK9 cDNA: 5Ј-GCAACCTCTCCCCTGGCCCTCATG-3Ј (forward) and 5Ј-GCTTCCTGGCACCTCCACCTGGGG-3Ј (reverse). This PCSK9 gene product was used as a template for TOPO TA primers to generate the final PCSK9 sequence. The final PCSK9 insert was ligated into a TOPO TA vector using a pcDNA3.1/V5-His-TOPO-TA expression kit (Invitrogen), followed by transformation into chemically competent TOP10 Escherichia coli cells. The plasmid containing wild-type PCSK9 with C-terminal V5 and His tags is referred to as pcDNA3.1-F1-WT. The following day, cells were transfected with 6 g of wild-type or mutant plasmid DNA per container using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer’s instructions. After 2 days, the medium was changed to 1ϫ DMEM containing 1 mg/ml G418 and supplemented with 10% FBS, and cells were maintained in this medium
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