Vineeta Sharma1, Andrzej Witkowski1, H Ewa Witkowska1, Andrew Dykstra1, Jens B Simonsen1, Lisa Nelbach1, Jennifer A Beckstead1, Clive R Pullinger1, John P Kane1, Mary J Malloy1, Gordon Watson1, Trudy M Forte1, Robert O Ryan2. 1. From the Children's Hospital Oakland Research Institute, CA (V.S., A.W., J.B.S., L.N., J.A.B., G.W., T.M.F., R.O.R.); Department of Obstetrics, Gynecology and Reproductive Sciences, UCSF Sandler-Moore Mass Spectrometry Core Facility, San Francisco, CA (H.E.W., A.D.); and Cardiovascular Research Institute, University of California, San Francisco (C.R.P., J.P.K., M.J.M.). 2. From the Children's Hospital Oakland Research Institute, CA (V.S., A.W., J.B.S., L.N., J.A.B., G.W., T.M.F., R.O.R.); Department of Obstetrics, Gynecology and Reproductive Sciences, UCSF Sandler-Moore Mass Spectrometry Core Facility, San Francisco, CA (H.E.W., A.D.); and Cardiovascular Research Institute, University of California, San Francisco (C.R.P., J.P.K., M.J.M.). rryan@chori.org.
Abstract
OBJECTIVE: Apolipoprotein A-V (apoA-V) is a low-abundance plasma protein that modulates triacylglycerol homeostasis. Gene transfer studies were undertaken in apoa5 (-/-) mice to define the mechanism underlying the correlation between the single-nucleotide polymorphism c.553G>T in APOA5 and hypertriglyceridemia. APPROACH AND RESULTS: Adeno-associated virus (AAV) 2/8-mediated gene transfer of wild-type apoA-V induced a dramatic lowering of plasma triacylglycerol in apoa5 (-/-) mice, whereas AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T single-nucleotide polymorphism: rs2075291; p.Gly185Cys when numbering includes signal sequence) had a modest effect. Characterization studies revealed that plasma levels of wild-type and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike wild-type apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Nonreducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked heterodimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for wild-type APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1, and others. CONCLUSIONS: Substitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein-binding and triacylglycerol-modulation functions are compromised.
OBJECTIVE:Apolipoprotein A-V (apoA-V) is a low-abundance plasma protein that modulates triacylglycerol homeostasis. Gene transfer studies were undertaken in apoa5 (-/-) mice to define the mechanism underlying the correlation between the single-nucleotide polymorphism c.553G>T in APOA5 and hypertriglyceridemia. APPROACH AND RESULTS:Adeno-associated virus (AAV) 2/8-mediated gene transfer of wild-type apoA-V induced a dramatic lowering of plasma triacylglycerol in apoa5 (-/-) mice, whereas AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T single-nucleotide polymorphism: rs2075291; p.Gly185Cys when numbering includes signal sequence) had a modest effect. Characterization studies revealed that plasma levels of wild-type and G162CapoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162CapoA-V indicated that, unlike wild-type apoA-V, >50% of G162CapoA-V was recovered in the lipoprotein-free fraction. Nonreducing SDS-PAGE immunoblot analysis provided evidence that G162CapoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked heterodimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for wild-type APOA5 and c.553G>T APOA5 revealed that G162CapoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1, and others. CONCLUSIONS: Substitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein-binding and triacylglycerol-modulation functions are compromised.
Authors: H N van der Vliet; M G Sammels; A C Leegwater; J H Levels; P H Reitsma; W Boers; R A Chamuleau Journal: J Biol Chem Date: 2001-09-27 Impact factor: 5.157
Authors: Peter J O'Brien; William E Alborn; John H Sloan; Maverick Ulmer; Amechand Boodhoo; Michael D Knierman; Albert E Schultze; Robert J Konrad Journal: Clin Chem Date: 2004-11-04 Impact factor: 8.327
Authors: Aivar Lookene; Jennifer A Beckstead; Solveig Nilsson; Gunilla Olivecrona; Robert O Ryan Journal: J Biol Chem Date: 2005-05-06 Impact factor: 5.157
Authors: Neda M Bogari; Ashwag Aljohani; Amr A Amin; Faisal A Al-Allaf; Anas Dannoun; Mohiuddin M Taher; Atalla Elsayed; Dareen Ibrahim Rednah; Osama Elkhatee; Massimo Porqueddu; Francesco Alamanni; Soud Abdulraof A Khogeer; Ahmed Fawzy Journal: BMC Cardiovasc Disord Date: 2019-01-03 Impact factor: 2.298