The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation

Abstract

Edited by George M. CarmanFamilial hypercholesterolemia (FH) is characterized by severely elevated low density lipoprotein (LDL) cholesterol. Herein, we identified an FH patient presenting novel compound heterozygote mutations R410S and G592E of the LDL receptor (LDLR). The patient responded modestly to maximum rosuvastatin plus ezetimibe therapy, even in combination with a PCSK9 monoclonal antibody injection. Using cell biology and molecular dynamics simulations, we aimed to define the underlying mechanism(s) by which these LDLR mutations affect LDL metabolism and lead to hypercholesterolemia. Our data showed that the LDLR-G592E is a class 2b mutant, because it mostly failed to exit the endoplasmic reticulum and was degraded. Even though LDLR-R410S and LDLR-WT were similar in levels of cell surface and total receptor and bound equally well to LDL or extracellular PCSK9, the LDLR-R410S was resistant to exogenous PCSK9-mediated degradation in endosomes/lysosomes and showed reduced LDL internalization and degradation relative to LDLR-WT. Evidence is provided for a tighter association of LDL with LDLR-R410S at acidic pH, a reduced LDL delivery to late endosomes/ lysosomes, and an increased release in the medium of the bound/ internalized LDL, as compared with LDLR-WT. These data suggested that LDLR-R410S recycles loaded with its LDL-cargo. Our findings demonstrate that LDLR-R410S represents an LDLR loss-of-function through a novel class 8 FH-causing mechanism, thereby rationalizing the observed phenotype.

Functional analysis of natural PCSK9 mutants in modern and archaic humans

“…Accordingly, in IHH cells only the H449R mutation led to a significant GOF on the LDLR, but not H449L or H449A (Fig. C), reinforcing the notion that the parameters regulating the activity of PCSK9 are different in both pathways , and that a positive charge at residue 449 (e.g., Arg) enhances the intracellular activity of PCSK9, but not its extracellular one.…”

“…C). Immunocytochemical analysis of these cells under nonpermeabilizing conditions allowed us to assess the levels of the LDLR at the cell surface (green) and DiI‐LDL uptake (red) probed the LDLR functional activity . The data revealed that all natural mutants of PCSK9, including H449L, represent significant LOF mutations as clearly evident by increases in both LDLR levels and DiI‐LDL internalization compared to WT (Fig.…”

“…). Accordingly, we collected all C‐terminally V5‐tagged recombinant proteins from the media of HEK293 cells overexpressing an empty bicistronic pIRES vector (CTL) or the same vector also expressing wild‐type (WT) or mutant PCSK9 . As a positive control, we used the GOF PCSK9‐D374Y and the negative control consisted of the LOF PCSK9‐RRRR 218 ↓EL (4REL) that is completely furin‐inactivated by cleavage at Arg 218 ↓ .…”

“…These included PCSK9‐4REL, ‐S127R, ‐D374Y, ‐R96L, ‐R105W, ‐P174S, ‐H449L, H449A, and H449R. Since both S127R and D374Y have been shown to be GOF mutations in the intracellular and extracellular pathways, we used these mutants as GOF controls in our studies of the intracellular pathway. A significant LOF was observed for the R96L, R105W, and P174S, especially in IHH cells (Fig.…”

“…A second, relatively minor intracellular pathway sorts the complex from the TGN to lysosomes directly , seemingly occurring in some nonhepatic tissues such as small intestine and possibly pancreas . We have developed robust assays to estimate the implication of PCSK9 and its mutants in each pathway, including the use of cell transfections, cell incubation using media swaps, western blot analyses, and LDLR immunocytochemistry coupled with fluorescent DiI‐LDL uptake .…”