Affinity and kinetics of proprotein convertase subtilisin/kexin type 9 binding to low-density lipoprotein receptors on HepG2 cells

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.