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

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.

Insight into the Evolving Role of PCSK9

“…The above-mentioned mechanisms lead to the impaired transport of cholesterol from the blood into cells and result in hypercholesterolemia. Furthermore, gain of function (GOF) mutations in PCSK9 genes are the cause of familial hypercholesterolemia with significantly higher levels of cholesterol in the blood [ 7 ], whereas loss-of-function mutations (LOF) are associated with hypocholesterolemia [ 8 ]. Table 2 presents the most frequent mutations of PCSK9 that naturally occur [ 8 , 9 ].…”

Updated Understanding of the Crosstalk Between Glucose/Insulin and Cholesterol Metabolism

“…PCSK9 genetic variants can be divided into gain of function (GOF) mutations and loss of function (LOF) mutations according to their effects on circulating LDL clearance (126,127). Considering the interaction between LDLR and PCSK9, flow cytometry analyses detect the expression level of LDLR in HEK293 cells transfected with PCSK9 variants, which may be an effective and reliable way to distinguish these two distinct types of PCSK9 variants (128). PCSK9-GOF variants tend to increase LDLR degradation in multiple cells, followed by the high level of plasma LDL-C (129).…”

Leu22_Leu23 Duplication at the Signal Peptide of PCSK9 Promotes Intracellular Degradation of LDLr and Autosomal Dominant Hypercholesterolemia

“…PCSK9 was purified from stably transfected HEK293 cells by harvesting the medium followed by one-step nickel affinity chromatography as described before. 27 Purified PCSK9 variants were stored at −80 °C in 50 mmol/L Tris-HCl buffer supplemented with 150 mmol/L NaCl and 10% glycerol, pH 8.0.…”