title: Aberrant hetero-disulfide bond formation by the hypertriglyceridemia-associated p.Gly185Cys APOA5 variant (rs2075291).
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.
  
  
  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.
authors.affiliation: 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.).
authors.forename: Vineeta
authors.initials: V
authors.lastname: Sharma
authors.affiliation: 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.).
authors.forename: Andrzej
authors.initials: A
authors.lastname: Witkowski
authors.affiliation: 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.).
authors.forename: H Ewa
authors.initials: HE
authors.lastname: Witkowska
authors.affiliation: 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.).
authors.forename: Andrew
authors.initials: A
authors.lastname: Dykstra
authors.affiliation: 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.).
authors.forename: Jens B
authors.initials: JB
authors.lastname: Simonsen
authors.affiliation: 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.).
authors.forename: Lisa
authors.initials: L
authors.lastname: Nelbach
authors.affiliation: 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.).
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Beckstead
authors.affiliation: 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.).
authors.forename: Clive R
authors.initials: CR
authors.lastname: Pullinger
authors.affiliation: 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.).
authors.forename: John P
authors.initials: JP
authors.lastname: Kane
authors.affiliation: 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.).
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Malloy
authors.affiliation: 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.).
authors.forename: Gordon
authors.initials: G
authors.lastname: Watson
authors.affiliation: 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.).
authors.forename: Trudy M
authors.initials: TM
authors.lastname: Forte
authors.affiliation: 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.
authors.forename: Robert O
authors.initials: RO
authors.lastname: Ryan
mesh_terms: D000818:Animals; D000072040:Apolipoprotein A-V; D001053:Apolipoproteins / Q000172:deficiency / Q000235:genetics; D001054:Apolipoproteins A / Q000235:genetics / Q000378:metabolism*; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D000229:Dependovirus; D004195:Disease Models, Animal; D004220:Disulfides / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D015228:Hypertriglyceridemia / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D014161:Transduction, Genetic; D014162:Transfection; D014280:Triglycerides / Q000097:blood
chemical_list: C437704:APOA5 protein, human; C499058:Apoa5 protein, mouse; D000072040:Apolipoprotein A-V; D001053:Apolipoproteins; D001054:Apolipoproteins A; D015415:Biomarkers; D004220:Disulfides; D014280:Triglycerides
keywords: genetic therapy; lipoproteins; polymorphism, single nucleotide; triglycerides