A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants.

Steeve Bourane,Ali K Jaafar,Asier Benito-Vicente,Asier Larrea-Sebal,Gilles Lambert,César Martín,Kepa B Uribe,Kevin Chemello,Unai Galicia-Garcia

doi:10.3390/ijms222413602

Abstract

Background: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted. Objective: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants. Methods: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay. Results: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants. Conclusions: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand.

Highlights

Since the discovery of mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene in French families [1] and its identification as the third genetic cause of autosomal dominant familial hypercholesterolemia (FH) [2], PCSK9 has become an attractive therapeutic target for the prevention of hypercholesterolemia and cardiovascular disease (CVD) [3,4].PCSK9 is highly expressed by the liver and to a much lesser extent by the intestine [5]
Analysis of LDLR activity can be assessed in transiently transfected HEK293 cells with the PCSK9 variants
LDL uptake was increased when cells were transfected with loss of function (LOF) variants and reduced when transfected with the Gain of function (GOF) variants compared with wild-type PCSK9 (Figure 1C)

Summary

Introduction

Since the discovery of mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene in French families [1] and its identification as the third genetic cause of autosomal dominant familial hypercholesterolemia (FH) [2], PCSK9 has become an attractive therapeutic target for the prevention of hypercholesterolemia and cardiovascular disease (CVD) [3,4].PCSK9 is highly expressed by the liver and to a much lesser extent by the intestine [5]. Methods: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Results: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Conclusions: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand

Objectives

Methods

Results

Discussion

Conclusion