Assessing the Role of the Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1) Three-finger Domain in Binding Lipoprotein Lipase

Anne P Beigneux,Brandon S.J Davies,Shelly Tat,Jenny Chen,Peter Gin,Constance V Voss,Michael M Weinstein,André Bensadoun,Clive R Pullinger,Loren G Fong,Stephen G Young

doi:10.1074/jbc.m111.242024

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.

Highlights

Microvascular endothelial cells and is responsible for shuttling lipoprotein lipase (LPL) from the interstitial spaces to the capillary lumen
To assess the impact of each amino acid substitution on the cell surface expression of Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), Chinese hamster ovary (CHO) K1 cells were electroporated with wild-type or mutant GPIHBP1 constructs, and the total amount of GPIHBP1 in cells and the amount of GPIHBP1 expressed on the cell surface were measured by Western blotting
We used alanine-scanning mutagenesis to investigate the notion that the GPIHBP1 lymphocyte antigen 6 (Ly6) domain is crucial for LPL binding

Summary

EXPERIMENTAL PROCEDURES

GPIHBP1 LPL-binding Domain described previously [9]. Site-directed mutagenesis with the QuikChange kit (Agilent Technologies) was used to construct a series of mutant GPIHBP1 constructs in which each amino acid in the Ly6 domain (Cys-65 to Cys-136) was replaced with an alanine. The presence of LPL in the conditioned medium was assessed by Western blotting with a mouse monoclonal antibody against the V5 tag (1:200; Invitrogen). GPIHBP1-transfected CHO-K1 cells plated in triplicate wells of a 24-well plate were incubated with a rabbit polyclonal antibody against the S-protein tag (10 ␮g/ml; Abcam) for 2 h at 4 °C in ice-cold PBS/Mg/Ca). LPL Transport Assays—RHMVECs were transfected with either wild-type or mutant human GPIHBP1 constructs or a human CD59 construct (all S-protein-tagged) and grown on fibronectin-coated Millicell filters (Millipore) [1]. LPL was detected with CF568-conjugated monoclonal antibody 5D2 (1:50), and GPIHBP1 was detected with a goat antibody against the S-protein tag (1:100) followed by an Alexa647-conjugated donkey anti-goat IgG (1:800). Cross-sections of endothelial cells through nuclei were obtained by generating three-dimensional images with Volocity Visualization software version 5.3 (PerkinElmer Life Sciences)

RESULTS

Decrease in LPL binding

DISCUSSION