Mutating a conserved cysteine in GPIHBP1 reduces amounts of GPIHBP1 in capillaries and abolishes LPL binding

Abstract

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease-chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.

Keywords: chylomicrons; endothelial cells; glycosylphosphatidylinositol-anchored HDL binding protein 1; lipids/chemistry; lipolysis and fatty acid metabolism; lipoprotein lipase; triglycerides.

Fig. 1.

Severe hypertriglyceridemia in Gpihbp1^−/− and Gpihbp1^C63Y/C63Y mice. Plasma triglyceride levels were measured in plasma samples from 3.5- to 4-month-old Gpihbp1^−/− and littermate WT mice (Gpihbp1^+/+) (n = 5/group) along with Gpihbp1^C63Y/C63Y mice and littermate Gpihbp1^C63Y/+ and WT mice (n = 6/group).

Fig. 2.

Gpihbp1 and Lpl transcript levels in heart and BAT from 2-month-old Gpihbp1^C63Y/C63Y, Gpihbp1^C63Y/+, and WT mice along with Gpihbp1^−/− mice. Gpihbp1 (A) and Lpl (B) transcript levels were measured by qRT-PCR (n = 3 for WT, Gpihbp1^C63Y/C63Y, and Gpihbp1^−/− mice; n = 4 for Gpihbp1^C63Y/+ mice). Transcript levels were normalized to expression of cyclophilin A.

Fig. 3.

GPIHBP1-C63Y cannot bind LPL. A: Testing binding of V5-tagged mouse LPL to WT GPIHBP1, GPIHBP1-W108S, and GPIHBP1-C63Y in a co-plating assay (17). In earlier studies, we showed that human GPIHBP1-W109S and human GPIHBP1-C65Y were unable to bind human LPL (9, 15). Here, CHO pgsA-745 cells were electroporated with either an S-protein–tagged version of GPIHBP1 or an expression vector for V5-tagged LPL. The independently transfected cells were then mixed together and plated on coverslips in 24-well plates. Twenty-four hours later, the cells were processed for immunocytochemistry with a goat antibody against the S-protein tag and a mAb against the V5 tag. DNA was stained with DAPI. In this system, cells that expressed WT GPIHBP1 captured LPL produced by LPL-transfected cells; hence, GPIHBP1 and LPL signals colocalized on the merged image. GPIHBP1-W108S and GPIHBP1-C63Y did not bind LPL; hence, there was no colocalization of LPL and GPIHBP1 on the merged image. B: Absent binding of V5-tagged LPL to mouse GPIHBP1-W108S and GPIHBP1-C63Y. CHO pgsA-745 cells were electroporated with S-protein–tagged versions of GPIHBP1 expression vectors. Twenty-four hours after the electroporation, cells were incubated with V5-tagged human LPL. After 1 h, cell extracts were prepared, and the amount of LPL bound to the cells was assessed by Western blotting with an antibody against the V5-tag. The amount of GPIHBP1 in cell extracts was assessed with an antibody against the S-protein tag. The first lane of the Western blot (“LPL”) shows the LPL preparation that was added to the GPIHBP1-transfected cells; the lane labeled (“–DNA”) indicates extracts of nontransfected cells; the lanes labeled WT, W108S, and C63Y show extracts of cells that had been transfected with expression vectors for WT mouse GPIHBP1, GPIHBP1-W108S, or GPIHBP1-C63Y, respectively. Actin was used as a loading control.

Fig. 4.

LPL is mislocalized within the interstitial spaces in Gpihbp1^C63Y/C63Y and Gpihbp1^−/− mice. Immunohistochemistry studies were performed on sections of BAT (A); heart (B); and quadriceps (C). Sections were stained with antibodies for CD31 (cyan), LPL (green), and GPIHBP1 (red). DNA was stained with DAPI (blue). Scale bar, 50 μm.

Fig. 5.

GPIHBP1 protein levels are reduced in tissues of Gpihbp1^C63Y/C63Y mice. Proteins (40 μg) from homogenates of heart (A), BAT (B), lung (C), and gonadal WAT (D) were size-fractionated by SDS-PAGE, and Western blots were performed with the GPIHBP1-specific mAb 11A12 (red) and a polyclonal antibody against β-actin (green).

Fig. 6.

Levels of GPIHBP1 in the capillary lumen are substantially reduced in tissues of Gpihbp1^C63Y/C63Y mice. Gpihbp1^C63Y/C63Y (n = 4), Gpihbp1^C63Y/+ (n = 5), and WT (n = 4) mice, along with littermate Gpihbp1^−/− (n = 3) and Gpihbp1^+/− mice (n = 4) (3.5–4 months old), were injected intravenously with IRDye680-labeled 11A12 (30 μg). After 2.5 min, the mice were perfused, and tissues were harvested. Tissue sections from heart (A), BAT (B), lung (C), quadriceps (D), and liver (E) were prepared, and the intensity of the IRDye680 signal was quantified with an Odyssey infrared scanner. The IRDye680 signal (i.e., from labeled mAb 11A12 in the lumen of capillaries) was normalized to tissue area, and the signal in WT mice was set at 100%. GPIHBP1 levels in Gpihbp1^C63Y/C63Y mice were substantially lower than in WT mice (*P < 0.0001 for heart, BAT, lung, and quadriceps; *P = 0.0055 for liver).

Fig. 7.

Plasma levels of GPIHBP1 in WT (Gpihbp1^+/+), Gpihbp1^C63Y/C63Y, and Gpihbp1^−/− mice. GPIHBP1 levels in the plasma from 6- to 9-month-old Gpihbp1^C63Y/C63Y mice and WT littermate control mice (n = 10/group) were measured by ELISA. Plasma samples from Gpihbp1^−/− mice (n = 3) were included as controls. GPIHBP1 levels in the plasma were somewhat lower in Gpihbp1^C63Y/C63Y mice than in WT mice, but this difference did not reach statistical significance (P = 0.112).

Fig. 8.

GPIHBP1-C63Y forms large amounts of GPIHBP1 dimers/multimers in CHO cells, but minimal amounts of multimers in heart tissue from Gpihbp1^C63Y/C63Y mice. A: Western blot analysis of S-protein–tagged GPIHBP1 proteins released from the surface of GPIHBP1-transfected CHO pgsA-745 cells with PIPLC. Twenty-four hours after the transfection, the cells were washed and incubated for 20 min at 37°C with PIPLC (10 U/ml). PIPLC-released proteins were size-fractionated by SDS-PAGE under nonreducing (NR) and reducing (R) conditions; cell lysates were examined under reducing conditions. GPIHBP1 was detected with an antibody against the S-protein tag; actin was used as a loading control. GPIHBP1 monomers migrate at ∼28 kDa. The GPIHBP1 dimer/multimer-to-monomer ratio was 3.44-fold greater with GPIHBP1-C63Y than with GPIHBP1-W108S, and 1.20-fold higher than with WT GPIHBP1. B: Mouse hearts were isolated and perfused with a mAb against GPIHBP1 (11A12), followed by perfusion with 0.2% Triton X-100. Perfusates were immunoprecipitated with Protein G agarose beads, and eluates from the beads were size-fractionated by SDS-PAGE under nonreducing and reducing conditions. Western blots were performed with an IRDye680-labeled mAb against mouse GPIHBP1 (mAb 11A12). *Position of GPIHBP1 dimer.