title: Assessing the role of the glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) three-finger domain in binding lipoprotein lipase.
abstract: Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is an endothelial cell protein that transports lipoprotein lipase (LPL) from the subendothelial spaces to the capillary lumen. GPIHBP1 contains two main structural motifs, an amino-terminal acidic domain enriched in aspartates and glutamates and a lymphocyte antigen 6 (Ly6) motif containing 10 cysteines. All of the cysteines in the Ly6 domain are disulfide-bonded, causing the protein to assume a three-fingered structure. The acidic domain of GPIHBP1 is known to be important for LPL binding, but the involvement of the Ly6 domain in LPL binding requires further study. To assess the importance of the Ly6 domain, we created a series of GPIHBP1 mutants in which each residue of the Ly6 domain was changed to alanine. The mutant proteins were expressed in Chinese hamster ovary (CHO) cells, and their expression level on the cell surface and their ability to bind LPL were assessed with an immunofluorescence microscopy assay and a Western blot assay. We identified 12 amino acids within GPIHBP1, aside from the conserved cysteines, that are important for LPL binding; nine of those were clustered in finger 2 of the GPIHBP1 three-fingered motif. The defective GPIHBP1 proteins also lacked the ability to transport LPL from the basolateral to the apical surface of endothelial cells. Our studies demonstrate that the Ly6 domain of GPIHBP1 is important for the ability of GPIHBP1 to bind and transport LPL.
authors.affiliation: Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, USA. abeigneux@mednet.ucla.edu
authors.forename: Anne P
authors.initials: AP
authors.lastname: Beigneux
authors.forename: Brandon S J
authors.initials: BS
authors.lastname: Davies
authors.forename: Shelly
authors.initials: S
authors.lastname: Tat
authors.forename: Jenny
authors.initials: J
authors.lastname: Chen
authors.forename: Peter
authors.initials: P
authors.lastname: Gin
authors.forename: Constance V
authors.initials: CV
authors.lastname: Voss
authors.forename: Michael M
authors.initials: MM
authors.lastname: Weinstein
authors.forename: André
authors.initials: A
authors.lastname: Bensadoun
authors.forename: Clive R
authors.initials: CR
authors.lastname: Pullinger
authors.forename: Loren G
authors.initials: LG
authors.lastname: Fong
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Young
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D008071:Lipoprotein Lipase / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010449:Peptide Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D018110:Receptors, Lipoprotein
chemical_list: D002352:Carrier Proteins; C523701:GPIHBP1 protein, human; D018110:Receptors, Lipoprotein; C548261:LPL protein, human; D008071:Lipoprotein Lipase