Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase

Abstract

GPIHBP1, a glycosylphosphatidylinositol-anchored endothelial cell protein of the lymphocyte antigen 6 (Ly6) family, binds lipoprotein lipase (LPL) avidly and is required for the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 contains two key structural motifs, an acidic domain and an Ly6 motif (a three-fingered domain specified by 10 cysteines). The acidic domain is required for LPL binding, but the importance of the Ly6 domain is less clear. To explore that issue, we transfected cells with a wild-type GPIHBP1 expression vector or mutant GPIHBP1 vectors in which specific cysteines in the Ly6 domain were changed to alanine. The mutant GPIHBP1 proteins reached the cell surface, as judged by antibody binding studies and by the ability of a phosphatidylinositol-specific phospholipase C to release these proteins from the cell surface. However, cells expressing the cysteine mutants could not bind LPL. The acidic domain of the cysteine mutants appeared to remain accessible, as judged by binding studies with an antibody against the acidic domain. We also developed a cell-free assay of LPL binding. We created a rat monoclonal antibody against the carboxyl terminus of mouse GPIHBP1 and used that antibody to coat agarose beads. We then tested the ability of soluble forms of GPIHBP1 that had been immobilized on monoclonal antibodycoated beads to bind LPL. In this assay, wild-type soluble GPI-HBP1 bound LPL avidly, but the cysteine mutants did not. Thus, our studies suggest that a structurally intact Ly6 domain (in addition to the acidic domain) is essential for LPL binding. Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1)2 is an endothelial cell protein that is required for the lipolytic processing of triglyceride-rich lipoproteins in the plasma (1). In the absence of GPI-HBP1, lipolysis of plasma lipoproteins is virtually abolished, leading to severe hypertriglyceridemia (1). Expression of GPI-HBP1 in cultured cells confers the ability to bind lipoprotein lipase (LPL) (1). That finding, along with the fact that GPIHBP1 is located in endothelial cells, led Beigneux et al.(1) to hypothesize that GPIHBP1 serves as an endothelial cell platform for lipolysis.The discovery of the role of GPIHBP1 in lipolysis prompted interest in defining which portions of GPIHBP1 are important for its function (e.g. for its ability to bind LPL). Mature GPI-HBP1 is a relatively short protein with only two noteworthy structural domains (Fig. 1). First, the amino terminus of the protein contains a strongly acidic domain (amino acids 24 -48 in the mouse sequence) with a large number of aspartates and glutamates (2). This negatively charged domain is absolutely critical for binding LPL, a protein that contains two well characterized positively charged "heparin-binding domains" (3-5). Second, GPIHBP1 contains a cysteine-rich Ly6 domain (amino acids 63-135 in the mouse sequence). The function of the Ly6 domain in LPL binding is less clearly defined.The Ly6 domain is an ancient motif containing ...

Mutations in lipoprotein lipase that block binding to the endothelial cell transporter GPIHBP1

“…Beigneux and coworkers (17,25) showed that defects in either of GPIHBP1's two principal domains (the acidic domain or the cys- Fig. 4.…”

“…We also tested the ability of WT LPL, LPL-C418Y, and LPL-E421K to bind to GPIHBP1 in a cell-free system (16,17). We mixed the LPL, soluble GPIHBP1 (i.e., GPIHBP1 lacking its GPI anchor), and agarose beads coated with the GPIHBP1-specific monoclonal antibody 11A12 and incubated them overnight.…”

“…Soluble mouse GPIHBP1 (no GPI anchor) and 20 μL of concentrated medium containing V5-tagged human LPL preparations were incubated overnight with 40 μL of antibody 11A12-coated agarose beads in 200 μL of PBS solution containing 0.2% Nonidet P-40 (17,25). The beads were washed three times in PBS solution containing 0.2% Nonidet P-40.…”

“…We also tested the ability of WT LPL, C418Y-LPL, and E421K-LPL in a cell free binding assay (16,17). Soluble mouse GPIHBP1 (no GPI anchor) and 20 μL of concentrated medium containing V5-tagged human LPL preparations were incubated overnight with 40 μL of antibody 11A12-coated agarose beads in 200 μL of PBS solution containing 0.2% Nonidet P-40 (17,25).…”

Angiopoietin-like 4 Modifies the Interactions between Lipoprotein Lipase and Its Endothelial Cell Transporter GPIHBP1

“…FLAG-tagged human LPL was concentrated from the medium of a Chinese hamster ovary cell line (CHO-K1) stably expressing FLAG-tagged human LPL as described previously (28). The presence of LPL in the conditioned media was assessed by Western blotting using a mouse antibody against the FLAG tag (1:5000; Sigma).…”