Functional Characterization of p.(Arg160Gln) PCSK9 Variant Accidentally Found in a Hypercholesterolemic Subject

Abstract

Familial hypercholesterolaemia (FH) is an autosomal dominant dyslipidaemia, characterised by elevated LDL cholesterol (LDL-C) levels in the blood. Three main genes are involved in FH diagnosis: LDL receptor (LDLr), Apolipoprotein B (APOB) and Protein convertase subtilisin/kexin type 9 (PCSK9) with genetic mutations that led to reduced plasma LDL-C clearance. To date, several PCSK9 gain-of-function (GOF) variants causing FH have been described based on their increased ability to degrade LDLr. On the other hand, mutations that reduce the activity of PCSK9 on LDLr degradation have been described as loss-of-function (LOF) variants. It is therefore important to functionally characterise PCSK9 variants in order to support the genetic diagnosis of FH. The aim of this work is to functionally characterise the p.(Arg160Gln) PCSK9 variant found in a subject suspected to have FH. Different techniques have been combined to determine efficiency of the autocatalytic cleavage, protein expression, effect of the variant on LDLr activity and affinity of the PCSK9 variant for the LDLr. Expression and processing of the p.(Arg160Gln) variant had a result similar to that of WT PCSK9. The effect of p.(Arg160Gln) PCSK9 on LDLr activity is lower than WT PCSK9, with higher values of LDL internalisation (13%) and p.(Arg160Gln) PCSK9 affinity for the LDLr is lower than WT, EC50 8.6 ± 0.8 and 25.9 ± 0.7, respectively. The p.(Arg160Gln) PCSK9 variant is a LOF PCSK9 whose loss of activity is caused by a displacement of the PCSK9 P' helix, which reduces the stability of the LDLr-PCSK9 complex.

Keywords: GOF; LOF; PCSK9; activity; characterisation; familial hypercholesterolemia.

Figure 1

Family pedigree showing the carriers of the p.(Arg160Gln) PCSK9 variant. Half-blackened indicates heterozygous carriers of the p.(Arg160Gln) variant. The arrow represents the proband. Age (in years) and LDL-C (mg/dL) are given. “?” unknown.

Figure 4

The p.(Arg160Gln) PCSK9 variant shows lower affinity to the LDLr compared to WT. Affinity curves representing the binding affinity of PCSK9 variants for the LDLr determined by solid-phase immunoassay at pH 7.4. Data represents the means of three independent experiments. EC₅₀ values are shown in Table 3.

Figure 2

Expression, maturation and secretion of the p.(Arg160Gln) PCSK9 variant is similar to WT PCSK9. (A) Representative immunoblots of expression and secretion of PCSK9 on HEK293 cells transiently transfected with mock (empty plasmid), WT, D374Y and p.(Arg160Gln). (B) Ratio between processed/non-processed PCSK9 quantified by densitometry. (C) Amount of expressed PCSK9 determined as the ratio between intracellular PCSK9/GAPDH quantified by densitometry. (D) Amount of secreted PCSK9 determined as the ratio between media/GAPDH quantified by densitometry. Histograms represent the mean ± SD of three independent experiments. * p < 0.05 compared to WT PCSK9.

Figure 3

Expression of the p.(Arg160Gln) PCSK9 variant increases LDL uptake compared to WT. Transiently transfected HEK293 cells with the different PCSK9 variants were incubated with FITC-labelled LDL and lipoprotein uptake was measured by flow cytometry. Histograms represent the mean ± SD of three independent experiments. * p < 0.01 compared to WT. Mock corresponds to an empty plasmid. LDL uptake was normalised to the total amount of PKSC9 secreted to the culture medium 48 h post-transfection.

Figure 5

AlphaFold-2 structure prediction of the p.(Arg160Gln) PCSK9 variant compared to WT. Structures of the area of interest (amino acids 153–160) in both WT and p.(Arg160Gln) were compared to understand the nature of the variant. (A) Front view, (B) top view and (C) side view. In green, prodomain (31–152); in purple, catalytic domain (154–451); in pink, C-terminal domain (453–692). Inside the catalytic domain, Arg160 residue is coloured in blue, the catalytic triad in yellow (186, 226 and 386) and the LDLr binding site in grey (367–381).

Figure 6

Suggested mechanisms leading to loss of affinity of the p.(Ard160Gln) PCSK9 variant for the LDLr. (A) Upon prodomain cleavage, P′ helix is located next to the LDLr-binding site and strengthens the electrostatic forces for binding and maintaining the PCSK9-LDLr complex. Additionally, the Arg160 residue forms a salt bridge with the LDLr Asp343 residue. (B) Replacement of Arg160 by a Gln modifies the change the direction of rotation of the P’ helix, which no longer remains close to the EGF-A residues implicated in PCSK9 binding.