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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the number of cell surface low-density lipoprotein receptors (LDLRs) and the levels of low-density lipoprotein cholesterol in plasma. Intact cells have not previously been used to determine the characteristics of binding of PCSK9 to LDLR. Using PCSK9 iodinated by the tyramine cellobiose (TC) method ([(125)I]TC-PCSK9), we measured the affinity and kinetics of binding of PCSK9 to LDLR on HepG2 cells at 4 °C. The extent of [(125)I]TC-PCSK9 binding increased as cell surface LDLR density increased. Unlabeled wild-type and two gain-of-function mutants of PCSK9 reduced binding of [(125)I]TC-PCSK9. The Scatchard plot of the binding-inhibition curve was curvilinear, indicative of high-affinity and low-affinity sites for PCSK9 binding on HepG2 cells. Nonlinear regression analysis of the binding data also indicated that a two-site model better fitted the data. The time course of [(125)I]TC-PCSK9 binding showed two phases in the association kinetics. Dissociation of [(125)I]TC-PCSK9 also occurred in two phases. Unlabeled PCSK9 accelerated the dissociation of [(125)I]TC-PCSK9. At low pH, only one phase of dissociation was apparent. Furthermore, the dissociation of [(125)I]TC-PCSK9 under pre-equilibrium conditions was faster than under equilibrium conditions. Overall, the data suggest that PCSK9 binding to cell surface LDLR cannot be described by a simple bimolecular reaction. Possible interpretations that can account for these observations are discussed., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interferes with the recycling of low-density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss-of-function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain-of-function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 vertebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9-mediated degradation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site-directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for investigations on the function of this regulator of cholesterol homeostasis.

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low-density lipoprotein receptor. To date, more than 900 different mutations have been described. Transport-defective mutations (class 2) causing partial or complete retention of the receptor in the endoplasmic reticulum are the predominant class of mutations. In a cell culture system (Chinese hamster ovary cells), we show that chemical chaperones are able to mediate rescue of a transport-defective mutant (G544V), and that the ability to obtain rescue is mutation dependent. In particular, the low molecular mass fatty acid derivative 4-phenylbutyrate mediated a marked increase in the transport of G544V-mutant low-density lipoprotein receptor to the plasma membrane. Thirty per cent of the mutant receptor was able to escape from the endoplasmic reticulum and reach the cell surface. The rescued receptor had reduced stability, but was found to be as efficient as the wild-type low-density lipoprotein receptor in binding and internalizing low-density lipoprotein. In addition to 4-phenylbutyrate, we also studied 3-phenylpropionate and 5-phenylvalerate, and compared their effect on rescue of the G544V-mutant low-density lipoprotein receptor with their ability to increase overall gene expression caused by their histone deacetylase inhibitor activity. No correlation was found. Our results indicate that the effect of these agents was not solely mediated by their ability to induce gene expression of proteins involved in intracellular transport, but rather could be due to a direct chemical chaperone activity. These data suggest that rescue of mutant low-density lipoprotein receptor is possible and that it might be feasible to develop pharmacologic chaperones to treat familial hypercholesterolemia patients with class 2 mutations.