Binding Preferences for GPIHBP1, a Glycosylphosphatidylinositol-Anchored Protein of Capillary Endothelial Cells

Abstract

Objective-To define the ability of GPIHBP1 to bind other lipase family members and other apolipoproteins (apos) and lipoproteins. Methods and Results-GPIHBP1, a GPI-anchored lymphocyte antigen (Ly)6 protein of capillary endothelial cells, binds lipoprotein lipase (LPL) avidly, but its ability to bind related lipase family members has never been evaluated. As judged by cell-based and cell-free binding assays, LPL binds to GPIHBP1, but other members of the lipase family do not. We also examined the binding of apoAV-phospholipid disks to GPIHBP1. ApoAV binds avidly to GPIHBP1-transfected cells; this binding requires GPIHBP1's amino-terminal acidic domain and is independent of its cysteine-rich Ly6 domain (the latter domain is essential for LPL binding). GPIHBP1-transfected cells did not bind high-density lipoprotein. Chylomicrons bind avidly to GPIHBP1-transfected Chinese hamster ovary cells, but this binding is dependent on GPIHBP1's ability to bind LPL within the cell culture medium. Conclusion-GPIHBP1 binds LPL but does not bind other lipase family members. GPIHBP1 binds apoAV but does not bind apoAI or high-density lipoprotein. mice display severe hypertriglyceridemia, even on a low-fat diet, with plasma triglyceride levels of 3000 to 6000 mg/ dL. 1,3,4 Subsequent studies proved that GPIHBP1 is responsible for transporting lipoprotein lipase (LPL) into the lumen of capillaries. 5 In the absence of GPIHBP1, LPL is mislocalized to the interstitial spaces 5 and, therefore, cannot hydrolyze triglycerides in plasma lipoproteins.GPIHBP1 contains a single lymphocyte antigen 6 (Ly6) domain containing 10 cysteines, which are arranged in a characteristic spacing pattern. 6 Mutation of any of these cysteines abolishes GPIHBP1's capacity to bind LPL. 7 A second noteworthy feature of GPIHBP1-and the one that distinguishes it from other Ly6 protein family members-is a striking acidic domain at its amino terminus. This domain is highly enriched in negatively charged amino acids (21 of 26 consecutive residues in human GPIHBP1 are aspartate or glutamate). Mutating the acidic domain abolishes GPIHBP1's ability to bind LPL. 8 The binding of LPL to GPIHBP1 appears to depend on a positively charged heparin-binding domain in LPL. 9,10 Mutating the positively charged amino acids in LPL's principal heparin-binding domain eliminates its ability to bind to GPIHBP1. 8 Two other lipase family members, hepatic lipase (HL) and endothelial lipase (EL), also contain heparinbinding domains. [11][12][13][14] Whether these other heparin-binding lipases also bind to GPIHBP1 has never been assessed.Transfection of Chinese hamster ovary (CHO) pgsA-745 cells with GPIHBP1 confers the ability to bind apolipoprotein (apo) AV-phospholipid disks. 1 ApoAV contains a strong heparinbinding domain, 15 In the current study, we sought to define binding preferences for GPIHBP1. We tested the ability of other lipase family members to bind to GPIHBP1 and assessed the structural features of GPIHBP1 required for apoAV binding. In addition, we investigated th...

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

“…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.…”

“…Antibody binding was detected with IR dye-conjugated secondary antibodies (Li-Cor) and an Odyssey infrared scanner (Li-Cor). For the immunofluorescence microscopy assay (16), cells transfected with WT GPIHBP1 were mixed with cells transfected with WT or mutant LPL and then plated on coverslips (25,000 cells/well) in 24-well plates. After incubating the cells overnight at 37°C, cells were fixed in 3% paraformaldehyde, blocked in 10% donkey serum, and incubated with the goat antibody against the S-protein tag (to detect GPIHBP1) and antibody 5D2 (to detect LPL).…”

“…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).…”

“…These cysteines are conserved in the LPL of nearly all vertebrates, and also in hepatic lipase, which does not bind to GPIHBP1 (16). The C418-C438 linkage is not required for catalytic activity (19), but the inability of LPL-C418Y to bind to GPIHBP1 prompted us to consider whether the disulfide bond is required for GPIHBP1 binding.…”

Mobility of “HSPG-bound” LPL explains how LPL is able to reach GPIHBP1 on capillaries

“…These GPIHBP1 proteins contained a uPAR tag (27) and the epitope for the GPIHBP1-specific monoclonal antibody 11A12 (28). The GPIHBP1 proteins were purified over agarose beads coated with the uPAR-specific monoclonal antibody R24 (29).…”