A Proprotein Convertase Subtilisin-like/Kexin Type 9 (PCSK9) C-terminal Domain Antibody Antigen-binding Fragment Inhibits PCSK9 Internalization and Restores Low Density Lipoprotein Uptake

Abstract

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of ␤-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9⌬C, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) 4 is a key regulator of plasma low density lipoprotein (LDL) cholesterol and has emerged as a promising target for prevention and treatment of coronary heart disease. A strong link between PCSK9, LDL, cholesterol, and coronary heart disease has been established by multiple laboratories. Human genetic studies demonstrated remarkable correlations between several nonsense or missense PCSK9 mutations with plasma LDL cholesterol levels and the risk of coronary heart disease. Thus, putative gain-or loss-of-function mutants were found to correlate with increased or reduced plasma LDL levels and cardiovascular risk, respectively (1-7). A recent genome-wide association study further bolstered the importance of PCSK9 by establishing a linkage between a single nucleotide polymorphism at a locus near PCSK9 with early-onset myocardial infarction (8).There is extensive evidence that plasma PCSK9 raises LDL cholesterol levels by binding to cell surface LDLR and targeting the receptor to lysosomes for degradation (9 -13). Accordingly, inhibition of PCSK9 by recombinant LDLR fragments (14 -16) or by mono-or polyclonal antibodies (17, 18) restored LDL cholesterol uptake in cells. Moreover, either RNAi targeting liver PCSK9 (19) or intravenous injection of a monoclonal antibody disrupting the PCSK9-LDLR inter...

Novel Loss-of-Function PCSK9 Variant Is Associated with Low Plasma LDL Cholesterol in a French-Canadian Family and with Impaired Processing and Secretion in Cell Culture

“…2 and 3). Consequently, this PCSK9 variant no longer affects the downregulation of cell surface LDLR through the endosomal/lysosomal pathway, a major route for PCSK9-mediated LDLR degradation (40 ). Our cell biology findings are consistent with the findings of our human studies, in which individuals carrying the PCSK9-Q152H mutation showed very low plasma PCSK9 concentrations (Ͼ79% reduction compared with the general Canadian population) and consequently low concentrations of circulating LDLC owing to the upregulation of liver LDLR.…”

Plasma levels of PCSK9 and phenotypic variability in familial hypercholesterolemia

“…These fi ndings, combined with the fact that decreased PCSK9 activity is associated with lower LDL-C levels and a reduced risk of coronary heart disease ( 18 ), support the inhibition of PCSK9 as a target of great signifi cance. In fact, several agents are already being investigated in humans (19)(20)(21)(22)(23)(24).…”

PCSK9 inhibition fails to alter hepatic LDLR, circulating cholesterol, and atherosclerosis in the absence of ApoE

“…1A ) ( 57,58 ). Pcsk9 Ϫ / Ϫ mice exhibit increased hepatic LDLR levels leading to lower circulating cholesterol levels ( 26 ). However, it is unclear if PCSK9 also infl uences the levels of circulating cholesterol independent of the LDLR ( 47 ).…”

“…Whether this disproportionate reduction in risk is due to PCSK9 having a direct negative effect at the atherosclerotic lesion or if the additional benefi t is driven by a modest lifelong reduction in serum cholesterol is unclear. These observations have led to the development of PCSK9 inhibitors as a means to therapeutically reduce LDL-C and the associated CVD risk (25)(26)(27)(28)(29). Inhibition of PCSK9 by monoclonal antibodies, adnectins, or siRNAs reduces LDL-C levels in patients, and clinical trials designed to assess the effect of anti-PCSK9 therapies on cardiovascular outcomes are underway (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42).…”