title: Degradation of LDLR protein mediated by 'gain of function' PCSK9 mutants in normal and ARH cells.
abstract: Dominant gain-of-function mutations in proprotein convertase subtilisin kexin type 9 (PCSK9) cause familial hypercholesterolaemia (FH) and result in accelerated atherosclerosis and premature coronary heart disease. It is believed that PCSK9 binds to LDL-receptor (LDLR) protein and prevents its recycling to the cell surface; gain-of-function PCSK9 mutants enhance LDLR degradation. Several new variants of PCSK9 have been identified, but their effect on PCSK9 activity has not been determined. We describe a new procedure for assessing the activity of four putative gain-of-function mutations identified in FH patients (D129N, D374H, N425S, R496W). All four mutant proteins were secreted normally from transfected HEK293T cells. Immortalized lymphocytes from normolipaemic controls were incubated with conditioned medium from transfected cells and cell-surface LDLR protein was determined by FACS. D374H was as potent as D374Y in reducing cell-surface LDLR, while the other three mutations were more potent than wild type, but less so than the D374 mutants; this correlated with total serum cholesterol in the patients. Substitution of different amino acids at 374 showed that aspartate in this position was critical; even glutamate at residue 374 increased LDLR degradation. When the assay was carried out with ARH-negative lymphocytes that are unable to internalise the LDLR, D374Y-PCSK9 was able to reduce cell-surface LDLR by 35%, compared with approximately 70% for normal lymphocytes. Thus, PCSK9-mediated LDLR degradation is not entirely dependent on ARH function. We propose a novel ARH-independent pathway for PCSK9 activity on LDLR.
authors.affiliation: MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, DuCane Road, London W12 ONN, UK.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Fasano
authors.forename: Xi-Ming
authors.initials: XM
authors.lastname: Sun
authors.forename: Dilipkumar D
authors.initials: DD
authors.lastname: Patel
authors.forename: Anne K
authors.initials: AK
authors.lastname: Soutar
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism* / Q000502:physiology; D002384:Catalysis; D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D011973:Receptors, LDL / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics* / Q000502:physiology; D013997:Time Factors; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C469745:LDLRAP1 protein, human; D011973:Receptors, LDL; D018698:Glutamic Acid; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases