Rosa M Sánchez-Hernández1, Maria Donata Di Taranto2, Asier Benito-Vicente3, Kepa B Uribe3, Itziar Lamiquiz-Moneo4, Asier Larrea-Sebal3, Shifa Jebari3, Unai Galicia-Garcia5, F Javier Nóvoa1, Mauro Boronat1, Ana M Wägner1, Fernando Civeira4, César Martín6, Giuliana Fortunato7. 1. Sección de Endocrinología y Nutrición, Complejo Hospitalario Universitario Insular Materno Infantil de Gran Canaria, Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS) de la Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain. 2. Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli and CEINGE S.C.a r.l, Biotecnologie Avanzate, Napoli, Italy. 3. Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080, Bilbao, Spain. 4. Hospital Universitario Miguel Servet. IIS Aragon. CIBERCV. Universidad de Zaragoza, Zaragoza, Spain. 5. Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, 48940, Leioa, Bizkaia, Spain. 6. Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080, Bilbao, Spain. Electronic address: cesar.martin@ehu.eus. 7. Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli and CEINGE S.C.a r.l, Biotecnologie Avanzate, Napoli, Italy. Electronic address: fortunat@unina.it.
Abstract
BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is a monogenic disease characterized by high levels of low-density lipoprotein cholesterol and premature atherosclerotic cardiovascular disease. FH is caused by loss of function mutations in genes encoding LDL receptor (LDLR), and Apolipoprotein B (APOB) or gain of function (GOF) mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, we identified a novel variant in PCSK9, p.(Arg499His), located in the C-terminal domain, in two unrelated FH patients from Spain and Italy. METHODS: We studied familial segregation and determined variant activity in vitro. RESULTS: We determined PCSK9 expression, secretion and activity of the variant in transfected HEK293 cells; extracellular activity of the recombinant p.(Arg499His) PCSK9 variant in HEK 293 and HepG2 cells; PCSK9 affinity to the LDL receptor at neutral and acidic pH; the mechanism of action of the p.(Arg499His) PCSK9 variant by co-transfection with a soluble construct of the LDL receptor and by determining total PCSK9 intracellular accumulation when endosomal acidification is impaired and when an excess of soluble LDLr is present in the culture medium. Our results show high LDL-C concentrations and FH phenotype in p.(Arg499His) carriers. In vitro functional characterization shows that p.(Arg499His) PCSK9 variant causes a reduction in LDLr expression and LDL uptake. An intracellular activity for this variant is also shown when blocking the activity of secreted PCSK9 and by inhibiting endosomal acidification. CONCLUSIONS: We demonstrated that p.(Arg499His) PCSK9 variant causes a direct intracellular degradation of LDLr therefore causing FH by reducing LDLr availability.
BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is a monogenic disease characterized by high levels of low-density lipoprotein cholesterol and premature atherosclerotic cardiovascular disease. FH is caused by loss of function mutations in genes encoding LDL receptor (LDLR), and Apolipoprotein B (APOB) or gain of function (GOF) mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, we identified a novel variant in PCSK9, p.(Arg499His), located in the C-terminal domain, in two unrelated FHpatients from Spain and Italy. METHODS: We studied familial segregation and determined variant activity in vitro. RESULTS: We determined PCSK9 expression, secretion and activity of the variant in transfected HEK293 cells; extracellular activity of the recombinant p.(Arg499His) PCSK9 variant in HEK 293 and HepG2 cells; PCSK9 affinity to the LDL receptor at neutral and acidic pH; the mechanism of action of the p.(Arg499His) PCSK9 variant by co-transfection with a soluble construct of the LDL receptor and by determining total PCSK9 intracellular accumulation when endosomal acidification is impaired and when an excess of soluble LDLr is present in the culture medium. Our results show high LDL-C concentrations and FH phenotype in p.(Arg499His) carriers. In vitro functional characterization shows that p.(Arg499His) PCSK9 variant causes a reduction in LDLr expression and LDL uptake. An intracellular activity for this variant is also shown when blocking the activity of secreted PCSK9 and by inhibiting endosomal acidification. CONCLUSIONS: We demonstrated that p.(Arg499His) PCSK9 variant causes a direct intracellular degradation of LDLr therefore causing FH by reducing LDLr availability.