title: Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa.
abstract: We analysed the Proprotein Convertase Subtilisin Kexin type 9 (PCSK9) exons and intronic junctions of 71 patients with familial hypercholesterolemia (FH) in whom LDL receptor (LDLR) or apolipoprotein B100 mutations were excluded. The previously reported S127R and R237W mutations were found in South African families, whereas new missense mutations D129G and A168E were found in families from New Zealand. Only, the S127R and D129G mutations modify a highly conserved residue and segregate with the FH phenotype. We overexpressed those mutants in hepatoma cells and found that both S127R and D129G have reduced autocatalytic activity compared with wild-type PCSK9, whereas the A168E mutant is processed normally. The S127R and D129G mutants were not secreted from cells, unlike the A168E mutant and wild-type PCSK9. By immunoblot, we showed that the expression of the LDLR was reduced by 40% in cells overexpressing wild-type or A168E PCSK9 and further reduced by 30% when the S127R or D129G mutants were used. Paralleling the LDLR levels, LDL cellular binding decreased by 25% upon wild-type PCSK9 or A168E overexpression, and by 45% with both S127R and D129G mutants. Our study therefore indicates that PCSK9 mediated inhibition of the LDLR does not require PCSK9 autocatalytic cleavage or secretion, suggesting that PCSK9 may also function intracellularly.
authors.affiliation: Canterbury Health Laboratories, 8001 Christchurch, New Zealand.
authors.forename: Vivienne M
authors.initials: VM
authors.lastname: Homer
authors.forename: A David
authors.initials: AD
authors.lastname: Marais
authors.forename: Francesca
authors.initials: F
authors.lastname: Charlton
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Laurie
authors.forename: Nicola
authors.initials: N
authors.lastname: Hurndell
authors.forename: Russel
authors.initials: R
authors.lastname: Scott
authors.forename: Fabien
authors.initials: F
authors.lastname: Mangili
authors.forename: David R
authors.initials: DR
authors.lastname: Sullivan
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Barter
authors.forename: Kerry-Anne
authors.initials: KA
authors.lastname: Rye
authors.forename: Peter M
authors.initials: PM
authors.lastname: George
authors.forename: Gilles
authors.initials: G
authors.lastname: Lambert
mesh_terms: D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D053299:Apolipoprotein B-100 / Q000235:genetics; D015331:Cohort Studies; D005260:Female; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics*; D008875:Middle Aged; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009520:New Zealand; D010375:Pedigree; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D011973:Receptors, LDL / Q000235:genetics; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D013019:South Africa
chemical_list: D053299:Apolipoprotein B-100; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases