A protein of capillary endothelial cells, GPIHBP1, is crucial for plasma triglyceride metabolism

Abstract

GPIHBP1, a protein of capillary endothelial cells (ECs), is a crucial partner for lipoprotein lipase (LPL) in the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1, which contains a three-fingered cysteine-rich LU (Ly6/uPAR) domain and an intrinsically disordered acidic domain (AD), captures LPL from within the interstitial spaces (where it is secreted by parenchymal cells) and shuttles it across ECs to the capillary lumen. Without GPIHBP1, LPL remains stranded within the interstitial spaces, causing severe hypertriglyceridemia (chylomicronemia). Biophysical studies revealed that GPIHBP1 stabilizes LPL structure and preserves LPL activity. That discovery was the key to crystallizing the GPIHBP1-LPL complex. The crystal structure revealed that GPIHBP1's LU domain binds, largely by hydrophobic contacts, to LPL's C-terminal lipid-binding domain and that the AD is positioned to project across and interact, by electrostatic forces, with a large basic patch spanning LPL's lipid-binding and catalytic domains. We uncovered three functions for GPIHBP1's AD. First, it accelerates the kinetics of LPL binding. Second, it preserves LPL activity by inhibiting unfolding of LPL's catalytic domain. Third, by sheathing LPL's basic patch, the AD makes it possible for LPL to move across ECs to the capillary lumen. Without the AD, GPIHBP1-bound LPL is trapped by persistent interactions between LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the abluminal surface of ECs. The AD interrupts the HSPG interactions, freeing LPL-GPIHBP1 complexes to move across ECs to the capillary lumen. GPIHBP1 is medically important; GPIHBP1 mutations cause lifelong chylomicronemia, and GPIHBP1 autoantibodies cause some acquired cases of chylomicronemia.

Keywords: endothelial cells; lipoprotein lipase; triglycerides.

Fig. 1.

Confocal immunofluorescence studies of LPL localization in BAT of Gpihbp1^+/+ and Gpihbp1^−/− mice. In Gpihbp1^+/+ mice (+/+), most of the LPL is located on capillary ECs, bound to GPIHBP1 and colocalizing with CD31 (an EC marker). In Gpihbp1^−/− mice (−/−), LPL is mislocalized within the interstitial spaces, bound to HSPGs on the surface of cells (adipocytes and ECs). The LPL in Gpihbp1^−/− mice colocalizes with antibodies against collagen IV, a basal lamina protein of adipocytes (17). (Scale bar, 10 μm.)

Fig. 2.

Immunofluorescence confocal microscopy images of capillary cross-sections in BAT of Gpihbp1^+/+ and Gpihbp1^−/− mice. Sections were stained with antibodies against GPIHBP1, LPL, and CD31. An EC nucleus (n) made it possible to examine distributions of GPIHBP1, CD31, and LPL along the abluminal surface of ECs (blue arrowhead) and the luminal plasma membrane (pink arrowhead). In Gpihbp1^−/− mice, LPL was not transported to the capillary lumen. Different z slices of the capillary cross-sections were shown in a recent paper by Song et al. (30). (Scale bar, 2 µm.)

Fig. 3.

HDX-MS studies revealing temperature-dependent unfolding of the α/β-hydrolase (catalytic) domain of bovine LPL, either alone or in the presence of GPIHBP1 and/or ANGPTL4. Shown here are isotope envelopes of a peptic peptide from the hydrolase domain (residues 131 to 165 in the mature protein sequence of bovine LPL) containing LPL’s active-site serine after pulse labeling in deuterated solvent. LPL was preincubated in protiated solvent for 3 min at the indicated temperatures prior to pulse labeling for 10 s in the deuterated solvent at 25 °C. Emergence of bimodal isotope envelopes is a signature for irreversible protein unfolding. Adapted from Leth-Espensen et al. (23).

Fig. 4.

ANGPTL4-mediated inactivation of LPL triggers irreversible unfolding and collapse of the catalytic pocket in LPL’s N-terminal α/β-hydrolase (catalytic) domain. This figure illustrates the step-wise LPL unfolding elicited by ANGPTL4 binding. Upon binding to ANGPTL4 (N¹), there is greater flexibility in the secondary structure elements of α5 and β6 (blue). The increased flexibility leads to an intermediate state (I) with more global but reversible unfolding of β5 (purple) and α3 (red). Ultimately, those changes trigger irreversible unfolding of structural elements (orange) forming LPL’s catalytic pocket (U). Adapted from Kristensen et al. (64), which is licensed under CC BY 4.0.

Fig. 5.

Crystal structure of the human GPIHBP1–LPL complex. LPL assumed a head-to-tail homodimer conformation, with the lipid-binding Trp-rich loop in the C-terminal PLAT (polycystin-1, lipoxygenase, α-toxin) domain of one monomer buried in the catalytic pocket of the N-terminal α/β hydrolase domain of the partner monomer (34). (Left) A cartoon representation of the structure of a single LPL/GPIHBP1 complex. Both the α/β hydrolase and PLAT domains contain a single N-linked glycan (orange sticks) (34); the hydrolase domain contains a single calcium ion (yellow sphere). Portions of the Trp-rich loop and the lid covering the catalytic pocket were not defined in the electron density map. (Middle and Right) An electrostatic surface potential map of LPL with a ribbon diagram of human GPIHBP1 (bound to LPL’s PLAT domain). N-terminal sequences (residues 21 to 61) containing the disordered acidic domain were not defined in the electron density map. One surface of LPL, spanning both N-terminal domains and CTDs, contained a large basic patch (blue). Residues 21 to 61 of GPIHBP1 are predicted to extend from Leu-62 (the first residue defined in the crystal structure) and project over and form a fuzzy complex with LPL’s basic patch (34). The C terminus of GPIHBP1 (residues 145 to 151) was not defined; a GPI moiety attached to residue G151 anchors GPIHBP1 to the plasma membrane of ECs.

Fig. 6.

Immunofluorescence confocal microscopy images of capillary cross-sections in BAT from a WT mouse (Gpihbp1^+/+) and a mutant mouse (Gpihbp1^S/S) in which the acidic domain was replaced with an S-protein tag. Sections were stained with antibodies against GPIHBP1 (GPI), LPL, and CD31. An EC nucleus (n) made it possible to examine distributions of GPIHBP1, CD31, and LPL along the abluminal surface of ECs (blue arrowhead) and the luminal plasma membrane (pink arrowhead). In Gpihbp1^+/+ mice (+/+), LPL was distributed roughly equally between the abluminal and luminal surfaces of ECs. In Gpihbp1^S/S mice (−/−), LPL was distributed asymmetrically, with only trace amounts in the capillary lumen. (Scale bar, 2 µm.)

Fig. 7.

SPR study demonstrating that WT mouse GPIHBP1 (WT-mGPIHBP1) detaches LPL from a heparin sulfate–coated sensor chip, whereas a mutant mouse GPIHBP1 protein lacking the acidic domain (S-mGPIHBP1) does not. When buffer was injected over the sensor chip, LPL remained stably bound to heparin sulfate chains (green). When WT-mGPIHBP1 was injected over the sensor chip (in five consecutive serial dilutions, 12.5 nM to 200 nM), it dislodged LPL from the heparin sulfate–coated sensor chip (black). When S-mGPIHBP1 was injected over the sensor chip, it simply bound to the LPL and did not dislodge it. Adapted from Song et al. (30), which is licensed under CC BY 4.0.