Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa
Introduction
Familial hypercholesterolaemia (FH) is predominantly an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) or in the ligand-binding domain of apolipoprotein (APO) B100, the protein component of LDL that interacts with the LDLR [1]. Since 2003, a handful of missense mutations in a new gene, PCSK9, have also been associated with FH [2], [3], [4], [5], [6]. In 2005, the establishment of a causative association between nonsense mutations in PCSK9 and low plasma LDL-cholesterol (LDL-C) levels as well as reduced incidence of cardiovascular disease was a major breakthrough in the investigation of PCSK9 [7], [8]. In addition, several missense mutations and one inframe deletion in PCSK9 have also been associated with hypocholesterolemia [9], [10], [11], [12]. Experimental evidence for a direct role for PCSK9 in lipoprotein metabolism has been provided by studies showing that overexpression of PCSK9 promotes the accumulation of LDL in the plasma of control mice but not in LDLR-deficient animals [13], [14], [15], [16], [17]. This effect in control mice was associated with decreased hepatic LDLR levels [13], [14], [15], [17]. Conversely, PCSK9 knockout mice present with increased hepatic LDLR expression and LDL plasma clearance rates compared with controls [18].
The molecular mechanism(s) by which PCSK9 modulate(s) LDLR expression is not transcriptional [17], [19]. PCSK9 appears to accelerate the intracellular degradation of mature LDLR. Because LDLR expression is normally decreased by PCSK9 overexpression in mice lacking the ARH adaptor protein, required for LDLR clustering into clathrin coated pits and subsequent endocytosis, it was first envisaged that PCSK9 acted on the LDLR before it reaches the basolateral surface of the hepatocyte (i.e. intracellularly) [17]. But PCSK9 is also secreted and when exogenously added to cells it requires LDLR mediated internalization to inhibit LDLR expression [20], [21], [22], [23]. The PCSK9 D347Y mutant displayed a greater affinity toward the LDLR than wild-type PCSK9, explaining the severe hypercholesterolemic phenotype of patients carrying this particular mutation [21], [23]. It has also been shown that secreted PCSK9 is cleaved by furin, leading to a shorter and inactive truncated protein. In vitro, the PCSK9 F216L mutant is not processed by furin, which is thought to be the mechanism leading to hypercholesterolemia in patients carrying this particular mutation [24].
PCSK9 is synthesized as a 72 kDa pro-protein that undergoes autocatalytic intramolecular processing at the FAQ152⇓SIP site to form a 63 kDa mature enzyme [25], [26]. PCSK9 3D structure indicates that (i) mature PCSK9 binds the extracellular domain of the LDLR and (ii) that the catalytic site of PCSK9 remains inaccessible after auto-cleavage, suggesting that PCSK9 catalytic activity is not required for LDLR degradation [23]. Among the naturally occurring mutants of PCSK9 associated with FH, only one (S127R) does not undergo proper autocatalytic cleavage and is not secreted from cells [27], [28]. Among PCSK9 mutants associated with hypocholesterolemia, three are not secreted or are poorly secreted, whereas two are normally cleaved and secreted from cells [27], [29]. Together the PCSK9 mutations studies have not yet provided an answer as to whether (i) PCSK9 acts primarily intracellularly or as a secreted factor and whether (ii) PCSK9 catalytic activity is required for LDLR degradation. Here, we report four PCSK9 variants identified in patients from New Zealand and South Africa, among which two missense mutations within the prodomain of PCSK9 segregate with FH. We characterized those two mutants and found that they poorly undergo autocatalytic cleavage, are not secreted from cells, and dramatically inhibit the expression of the LDLR.
Section snippets
Patients
A total of 234 FH patients diagnosed on clinical criteria adapted from the Simon Broome Study Group [i.e. TC > 7.5 mMol with either tendon xanthomas in the proband or a first degree relative (definite FH) or coronary heart disease or hypercholesterolemia in a first degree relative (possible FH)] were screened for mutations in the LDLR or APOB100 genes. Of these 234 patients, 47 were found to have LDLR mutations (44 different mutations – prevalence 20%), 4 had FDB (apoB3500 mutation – prevalence
Identification of PCSK9 mutations in FH patients
All coding regions of the PCSK9 gene, including the exon/intron boundaries and the promoter region were sequenced in 71 definite FH patients not carrying LDLR or APOB genes defects. DNA sequencing of exon 2 in a South African patient revealed the presence of a T/A mutation translating into the previously reported S127R substitution associated with FH [2], while a novel A/G mutation translating to a D129G substitution was detected in a patient from New Zealand. Sequencing of exon 3 revealed a
Discussion
In this study, we identified four PCSK9 variants in individuals with clinical features of familial hypercholesterolemia: the previously reported S127R and R237W mutants in South Africa and two new missense mutations in New Zealand, D129G and A168E. Among pedigrees, we found that the D129G missense mutation segregated with hypercholesterolemia whereas the A168E substitution did not. In addition, both S127 and D129 residues of PCSK9 are highly conserved among species whereas A168 is not. To
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