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

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 antibody-coated beads to bind LPL. In this assay, wild-type soluble GPIHBP1 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 GPIHBP1, lipolysis of plasma lipoproteins is virtually abolished, leading to severe hypertriglyceridemia (1). Expression of GPIHBP1 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 GPIHBP1 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.Open in a separate windowFIGURE 1.Schematic of human GPIHBP1, showing the location of the acidic domain and the 10 highly conserved cysteines of the Ly6 domain. The location of Gln¹¹⁵ is also shown; a Q115P mutation was identified in association with chylomicronemia in a young man (15). This figure is modified, with permission, from a figure published in Ref. 22.The Ly6 domain is an ancient motif containing either 8 or 10 cysteines that are organized in a highly characteristic spacing pattern (6, 7). Mammalian genomes contain ∼25–30 Ly6 proteins, of which the best studied are urokinase-type plasminogen activator receptor and CD59. In those proteins, as well as in other Ly6 proteins, crystallographic studies have shown that each of the cysteines is involved in a disulfide bond, producing a three-fingered structural motif (8–10).In the case of urokinase-type plasminogen activator receptor and CD59, the Ly6 motif is crucial for ligand binding (8–13). But in the case of GPIHBP1, one could argue that this domain might be dispensable, simply because it is plausible that the acidic domain would be sufficient for binding the positively charged domains within LPL. On the other hand, other considerations suggest that the GPIHBP1 Ly6 domain might actually be important for GPIHBP1 function. First, the cysteines that define the Ly6 domain are perfectly conserved in the GPIHBP1 of every mammalian species from platypus to human, suggesting that the three-fingered structure of this domain is important (14). Second, Beigneux et al. (15) found that a missense mutation adjacent to a conserved cysteine (Q115P) impaired the ability of GPIHBP1 to bind LPL. The latter observation led us to entertain the possibility that the Ly6 domain could be functionally important in LPL binding.To explore the functional relevance of the GPIHBP1 Ly6 domain, we decided to mutate the highly conserved cysteines in GPIHBP1 (Fig. 1), because those mutations would be predicted to disrupt the structure of the three-fingered motif. For other Ly6 proteins, mutation of conserved cysteines appears to impair protein function. Mutation of several (but not all) of the Ly6 cysteines in CD59 reduces the ability of that molecule to inhibit complement-mediated membrane attack (13). Also, missense mutations involving cysteines were found in a secreted Ly6 protein, SLURP1, in association with a recessive palmoplantar keratoderma (16). Unfortunately, the function and the binding partner for SLURP1 are unknown, so no one has been able to use biochemical assays to rigorously assess the impact of cysteine mutations on protein function. In contrast, GPIHBP1 has a well established function—binding LPL, and a cell-based system for assessing LPL binding has been developed (1, 15, 17).In the current study, we sought to assess the impact of Ly6 cysteine mutations on the LPL binding capacity of GPIHBP1. But as we embarked on these studies, we worried that the cell-based assay system might be inadequate, for the simple reason that cysteine mutations sometimes interfere with the ability of proteins to reach the cell surface (13, 18). Accordingly, we also developed a cell-free, monoclonal antibody-based assay system for assessing the ability of GPIHBP1 to bind LPL. Both the cell-based binding system and the cell-free system were used to test the possibility that the Ly6 domain of GPIHBP1, like the acidic domain, is critical for binding LPL.