GPIHBP1 Missense Mutations Often Cause Multimerization of GPIHBP1 and Thereby Prevent Lipoprotein Lipase Binding

Abstract

Rationale GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1 missense mutations that interfere with LPL binding cause familial chylomicronemia. Objective We sought to understand mechanisms by which GPIHBP1 mutations prevent LPL binding and lead to chylomicronemia. Methods and Results We expressed mutant forms of GPIHBP1 in Chinese hamster ovary cells, rat and human endothelial cells, and Drosophila S2 cells. In each expression system, mutation of cysteines in GPIHBP1’s Ly6 domain (including mutants identified in chylomicronemia patients) led to the formation of disulfide-linked dimers and multimers. GPIHBP1 dimerization/multimerization was not unique to cysteine mutations; mutations in other amino acid residues, including several associated with chylomicronemia, also led to protein dimerization/multimerization. The loss of GPIHBP1 monomers is quite relevant to the pathogenesis of chylomicronemia because only GPIHBP1 monomers—and not dimers or multimers—are capable of binding LPL. One GPIHBP1 mutant, GPIHBP1-W109S, had distinctive properties. GPIHBP1-W109S lacked the ability to bind LPL but had a reduced propensity for forming dimers or multimers, suggesting that W109 might play a more direct role in binding LPL. In support of that idea, replacing W109 with any of 8 other amino acids abolished LPL binding—and often did so without promoting the formation of dimers and multimers. Conclusions Many amino acid substitutions in GPIHBP1’s Ly6 domain that abolish LPL binding lead to protein dimerization/multimerization. Dimerization/multimerization is relevant to disease pathogenesis, given that only GPIHBP1 monomers are capable of binding LPL.

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

“…The amounts of LPL that moved to beads coated with GPIHBP1(25-50) and GPIHBP1-W109S (normalized to the amount of GPIHBP1 on the beads) averaged 87.9% and 23.3%, respectively, in three separate experiments. so without causing inappropriate intermolecular disulfide bond formation and protein multimerization (27). The discovery that GPIHBP1 lacking the acidic domain is capable of capturing LPL from HSPGs is consistent with recent findings by Mysling et al (24).…”

“…We produced secreted versions of human wild-type GPIHBP1, GPIHBP1-W109S, and GPIHBP1 in Drosophila S2 cells (27). These GPIHBP1 proteins contained a uPAR tag (27) and the epitope for the GPIHBP1-specific monoclonal antibody 11A12 (28).…”

“…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).…”

“…CHO pgsA-745 cells (2 × 10 6 ) were electroporated with 2 g of a plasmid for S-protein-tagged versions of wild-type human GPIHBP1, GPIHBP1-W109S (27), or GPIHBP1-D,E(25-50)N,Q [a mutant GPIHBP1 in which all acidic amino acids within residues 25-50 were replaced with uncharged amino acids (Asn or Gln)]; the transfected cells were plated on coverslips in 24-well plates. Twenty-four hours later, the transfected cells were incubated with V5-tagged LPL (30) for 1 h at 4°C, washed, and then processed for Western blotting.…”

An LPL–specific monoclonal antibody, 88B8, that abolishes the binding of LPL to GPIHBP1

“…A variety of missense mutations in GPIHBP1's LU domain have been identified in patients with chylomicronemia (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), and all of those abolish the ability of GPIHBP1 to bind LPL (6). Most of these mutations interfere with the formation of disulfide bonds in the LU domain, leading to disulfide-linked dimers and multimers (23). Alanine-scanning mutagenesis studies showed that the highly conserved second finger of the threefingered LU domain is particularly important for binding LPL (24).…”