Department of Biochemistry and Molecular Biology, Universidad del País Vasco UPV/EHU, 48080 Bilbao, Spain.
Department of Molecular Biophysics, Biofisika Institute, University of Basque Country and Consejo Superior de Investigaciones Científicas (UPV/EHU, CSIC), 48940 Leioa, Spain.
Int J Mol Sci. 2023 Feb 7;24(4):3330. doi: 10.3390/ijms24043330.
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.
家族性高胆固醇血症（FH）是一种常染色体显性脂质代谢异常疾病，其特征是血液中 LDL 胆固醇（LDL-C）水平升高。FH 的诊断主要涉及三个基因：LDL 受体（LDLr）、载脂蛋白 B（APOB）和蛋白水解酶原亚基 9（PCSK9），这些基因的基因突变导致血浆 LDL-C 清除减少。迄今为止，已经描述了几种导致 FH 的 PCSK9 功能获得（GOF）变异体，这些变异体具有增强 LDLr 降解的能力。另一方面，降低 PCSK9 对 LDLr 降解活性的突变被描述为功能丧失（LOF）变异体。因此，为了支持 FH 的遗传诊断，对 PCSK9 变异体进行功能表征非常重要。本工作的目的是对在疑似 FH 的患者中发现的 p.(Arg160Gln) PCSK9 变异体进行功能表征。结合了不同的技术来确定自催化裂解效率、蛋白表达、该变异体对 LDLr 活性的影响以及 PCSK9 变异体与 LDLr 的亲和力。p.(Arg160Gln) 变异体的表达和加工与 WT PCSK9 相似。p.(Arg160Gln) PCSK9 对 LDLr 活性的影响低于 WT PCSK9，LDL 内化率更高（13%），p.(Arg160Gln) PCSK9 与 LDLr 的亲和力低于 WT，EC50 分别为 8.6 ± 0.8 和 25.9 ± 0.7。p.(Arg160Gln) PCSK9 变异体是一种 LOF PCSK9，其活性丧失是由于 PCSK9 P' 螺旋的位移引起的，这降低了 LDLr-PCSK9 复合物的稳定性。
