The Proprotein Convertase PCSK9 Induces the Degradation of Low Density Lipoprotein Receptor (LDLR) and Its Closest Family Members VLDLR and ApoER2

Suzanne Benjannet,Annik Prat,Steve Poirier,Johannes Nimpf,Eric Bergeron,Nasha Nassoury,Jadwiga Marcinkiewicz,Nabil G. Seidah,Harald Mayer,Gaetan Mayer

doi:10.1074/jbc.m708098200

Abstract

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.

Highlights

One of the unanswered questions is the target specificity of PCSK9, and it is not known, nor obvious, whether other members of the low density lipoprotein receptor (LDLR) family are affected by PCSK9
very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) Are Novel PCSK9 Targets—Whereas LRP1 exhibits a 40% identity to LDLR [3, 12], it was not degraded by wild type PCSK9 or its gain of function mutant S127R [3]
Because the primary sequences of ApoER2 and VLDLR exhibit the highest identity and similarity to that of LDLR (46 and 49 and 59 and 65%; supplemental Fig. S1), it was of interest to assess whether PCSK9 may enhance the degradation of these receptors

Summary

EXPERIMENTAL PROCEDURES

Sequence Alignment—As shown in Fig. S1, the full-length sequence of ApoER2 (NP_150643), VLDLR (NP_003374), and LDLR (NP_000518) were aligned using the Multalin [23] and Genedoc software (National Resource for Biomedical Supercomputing; www.nrbsc.org). CHO-A7 (ldlA7; lacking the LDLR) and its parent CHO-WT were maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum (Invitrogen) [25] These cells were stably transfected with either cDNAs of empty vector pcDNA3, or recombinants of VLDLR and ApoER2. The cells were incubated for 30 min with 1% bovine serum albumin (Fraction V, Sigma) containing 0.1% Triton X-100, followed by overnight incubation at 4 °C with rabbit polyconal antibodies Ab:PCSK9 (1:1000), Ab: VLDLR (a74, 1:200), Ab:ApoER2 (a23, 1:200), and monoclonal Ab:V5 (1:1000, Invitrogen) in blocking solution with or without the late endosomes marker Ab:CI-MPR (cation-independent mannose 6-phosphate receptor (CI-MPR, 1:500, Abcam). The sections were dipped in photographic emulsion (NTB-2, Kodak), exposed for 6 –12 days, developed in D19 solution (Kodak), and stained with hematoxylin and eosin

RESULTS

Because the degradation of

DISCUSSION