A disordered acidic domain in GPIHBP1 harboring a sulfated tyrosine regulates lipoprotein lipase

Abstract

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (k_on) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.

Keywords: autoimmune disease; electrostatic steering; hypertriglyceridemia; intravascular lipolysis; intrinsically disordered region.

Fig. 1.

Posttranslational modifications of recombinant human GPIHBP1. (A) Human GPIHBP1^1–131 produced in Drosophila S2 cells was analyzed by SDS/PAGE and Coomassie blue staining (nonreduced, lane 1; reduced and alkylated, lane 2). Mass spectra are shown for intact GPIHBP1 before (Inset) and after N-glycanase treatment under native conditions, which reduces the molecular mass by 1,038.5 Da corresponding to one paucimannosidic N-glycan with a core fucose (Man₃GlcNAc₂Fuc), the archetypical insect cell glycan (21). The asterisks indicate the loss of 17 Da due to the formation of pyroglutamate at the N-terminal glutamine in human GPIHBP1. (B) Sensorgrams showing the interactions between immobilized anti-sulfotyrosine mAb 1C-A2 (1,280 RU) and GPIHBP1¹⁻¹³¹ (blue curve), GPIHBP1^1−131/Y18F (green curve), and buffer (black curve). The SPR sensorgrams were recorded with a BiacoreT200 instrument and are shown for sequential injections of twofold dilutions of GPIHBP1 from 125 nM to 2 µM without intervening regenerations. The affinity between GPIHBP1¹⁻¹³¹ and mAb 1C-A2 was 5.4 µM (determined from several runs up to 16 µM GPIHBP1; see Inset).

Fig. 2.

Binding stoichiometry and affinity of LPL–GPIHBP1 complexes. (A–C) The binding stoichiometry between LPL and GPIHBP1 was determined by native PAGE titrating 3 µM LPL (lane 2) with increasing amounts of GPIHBP1 ligand (range, 0.5–5.0 µM) (lanes 3–11). The relative amounts of LPL–GPIHBP1 complexes (marked by asterisks) were determined by scanning the Coomassie blue-stained bands and are superimposed as black diamonds. Data for GPIHBP1¹⁻¹³¹ and GPIHBP1¹⁻³³ (both with Tyr¹⁸-OSO₃) and GPIHBP1^1−131/Y18F are shown in A–C, respectively. (D and E) Single-cycle kinetics with SPR for the interactions between LPL captured on mAb 5D2 and twofold dilutions of GPIHBP1¹⁻¹³¹ (D) or GPIHBP1^1−131/Y18F (E). Shown are two single-cycle runs of five GPIHBP1 concentrations ranging from 0.125–2 nM (red curves) or 0.25–4 nM (green curves). Data were fit to a simple bimolecular interaction (black curves) with the derived kinetic constants and the residuals shown beneath the sensorgrams. (F) Inhibitory capacity of synthetic GPIHBP1¹⁻³³ peptides on the LPL–GPIHBP1¹⁻¹³¹ interaction as assessed by MST. The following peptides were measured: unmodified (orange, IC₅₀ 0.71 ± 0.20 µM); Tyr¹⁸-OSO₃ (red, IC₅₀ 0.31 ± 0.05 µM); Tyr¹⁸–OPO₃ (green, IC₅₀ 0.30 ± 0.05 µM); Tyr¹⁸Phe (blue, IC₅₀ 0.60 ± 0.16 µM); and Tyr¹⁸Glu (purple, IC₅₀ 0.70 ± 0.08 µM). Each titration shows results from four independent replicates. Titrations with Tyr¹⁸ are significantly different from those with Tyr¹⁸-OSO₃ (P < 0.01, Student’s t test). (G) Impact of ionic strength on K_d (green squares) and k_on (red circles) for the LPL–GPIHBP1¹⁻¹³¹ interaction as assessed by SPR.

Fig. 3.

Mitigation of ANGPTL4-mediated LPL unfolding and inactivation by GPIHBP1. (A) We measured LPL unfolding with HDX-MS by quantifying the bimodal distribution of the isotope envelopes for the LPL peptide 131–165 (containing Ser¹³⁴ and Asp¹⁵⁸ of the catalytic triad). Relative amounts of unfolding of 10 µM LPL incubated for 10 min at 25 °C alone (gray bar) or in the presence of 2 µM ANGPTL4¹⁻¹⁵⁹ (black bar) are shown. Incubations with ANGPTL4 were also performed with 30 µM GPIHBP1¹⁻¹³¹ (wt, red bar); 30 µM GPIHBP1^1−131;Y18F (Y18F, blue bar), or 30 µM GPIHBP1³⁴⁻¹³¹ (Δacid, light gray bar). (B) Impact of 30 µM GPIHBP1¹⁻³³ in a similar setting with Tyr¹⁸ being unmodified (-OH), phosphorylated (-OPO₃), or sulfated (-OSO₃). (C) Catalytic activity of 15 nM LPL alone (100% corresponding to 51.9 U/mL) or in the presence of 15 nM ANGPTL4¹⁻¹⁵⁹. These incubations were performed with 15, 30, 45, 150, or 300 nM GPIHBP1¹⁻¹³¹ (red bars) or GPIHBP1^1−131;Y18F (blue bars). Numbers of replicates for each experiment range from 3 to 12. Comparison of data with an unpaired t test: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant.

Fig. 4.

Localization of LPL in the mouse heart. The distribution of LPL in the hearts of wild-type and Gpihbp1^−/− (KO) mice was assessed by immunohistochemistry with antibodies against LPL (red), CD31 (magenta), or β-dystroglycan (green). Nuclei were stained with DAPI (blue). Shown are confocal fluorescence microscopy images of capillary endothelial cells containing an endothelial cell nucleus (which makes it possible to visualize the basolateral and apical membranes). In the wild-type mouse heart, LPL was associated almost exclusively with capillary endothelial cells (arrow). In the heart of a Gpihbp1-deficient mouse, the LPL is mislocalized within the interstitial spaces surrounding both myocytes and capillary endothelial cells, but LPL appeared to bind in close proximity to capillary endothelial cells (arrows). (Scale bars, 5 µm.)

Fig. 5.

Movement of LPL between HSPGs and GPIHBP1. (A) The distribution of LPL in gastrocnemius muscle of wild-type and Gpihbp1^−/− (KO) mice was assessed by confocal immunofluorescence microscopy with antibodies against LPL (green) and CD31 (red). Nuclei were stained with DAPI (blue). In the wild-type mouse LPL was associated almost exclusively with capillary endothelial cells. In the Gpihbp1-deficient mouse the LPL is mislocalized within the interstitial spaces surrounding both myocytes and capillary endothelial cells, but LPL appeared to bind preferentially to capillary endothelial cells. (Scale bars, 20 µm.) (B) Competition of the GPIHBP1–LPL interaction by defined heparin fragments (dp10) by MST. Heparin (red curve; IC₅₀ <14 nM), desulfated on the C2 oxygen of iduronate (green curve; IC₅₀ 59 ± 5 nM), desulfated on the C6 oxygen of glucosamine (blue curve; IC₅₀ 100 ± 10 nM), and desulfated on the C2 amine of glucosamine (orange curve; IC₅₀ 980 ± 14 nM). (C) Mobilization of LPL from a high-density heparin surface by the injection of 200 nM of various GPIHBP1 derivatives. After 1,000-s exposures of GPIHBP1 at a flowrate of 20 µL/min (gray line), different levels of LPL remained on the heparin surface: 23.1% by GPIHBP1¹⁻¹³¹ (blue curve); 27.3% by GPIHBP1^{1−131; Y18F} (red curve); and 97.0% by GPIHBP1³⁴⁻¹³¹ (black curve). Although the difference between GPIHBP1¹⁻¹³¹ and GPIHBP1^{1−131; Y18F} was modest (2.5 ± 1.6%), it was highly significant when comparing 10 consecutive runs with a paired t test (P < 0.001). Buffer control is shown by the green curve.

Fig. 6.

SAXS analyses of GPIHBP1. (A) Concentration-normalized scattering profiles of GPIHBP1¹⁻³³ with Tyr¹⁸–OH (2.6 mg/mL, black curve); Tyr¹⁸-OSO₃ (3.8 mg/mL, blue curve); or Tyr¹⁸–OPO₃ (3.6 mg/mL, green curve). (B) Corresponding pair–distance distribution functions [p(r)]. (C) Kratky plot illustrating the high flexibility and disorder of these peptides, which is best described by an ideal random-walk chain structure (58). (D) Heat-map representation of the flexibility in GPIHBP1¹⁻¹³¹ determined by HDX-MS (17), high exchange (red) to low exchange (blue). Black lines highlight sequences allowed to be flexible during EOM simulations of the SAXS data. (E) SEC-SAXS scattering data for a truncated GPIHBP1 lacking the acidic domain (GPIHBP1³⁴⁻¹³¹, black circles) along with the scattering profile of a rigid GPIHBP1 homology model (17) (CRYSOL; blue curve, χ² 4.6), a model allowing glycosylation flexibility (Allosmod; red curve, χ² 3.9), and a model allowing defined peptide flexibility (EOM; green curve, χ² 2.4). The Inset shows models selected by 10 separate EOM analyses; a cartoon representation shows the nonvariable part of GPIHBP1³⁴⁻¹³¹, and dots (residues 34–42 and 74–83) or spheres (118–131) show the variable parts. The position of Trp⁸⁹, which is important for LPL binding, is shown in a stick representation. (F) SEC-SAXS scattering data for full-length GPIHBP1¹⁻¹³¹ with fits to a rigid homology model (CRYSOL; blue curve, χ² 10.6) and 10 EOM analyses (green curve, χ² 1.6). (G) Conformational ensembles selected for our current model of GPIHBP1¹⁻¹³¹ by the 10 separate EOMs. The illustration was prepared by PyMOL (Schrödinger) using the same settings as in E except that the spheres show residues 1–42 and dots show residues 74–83 and 118–131.

Fig. 7.

Reactivity of GPIHBP1 autoantibodies purified from a subject with the GPIHBP1 autoantibody syndrome. (A) A comparison of the predicted IDRs and reactivity with MHC class II for GPIHBP1. Disorder prediction was assessed by IUPred (59), and reactivity with a MHC class II receptor (HLA-DRB1101 allele) was predicted by NetMHCIIpan version 3.1 (60) using a 15-mer sequence window. The cyan boxes show the positions of predicted secondary structure elements (β-strands). (B and C) Definition of the domain reactivity of a monoclonal antibody against human GPIHBP1 (mAb RF4) by single-cycle kinetics of twofold dilutions (2–32 nM) of GPIHBP1¹⁻¹³¹ (red curve in B), GPIHBP1¹⁻⁴⁵ (green curves in B and C), GPIHBP1³⁴⁻¹³¹ (blue curve in B), GPIHBP1²⁷⁻⁴⁴ (cyan curve in C), and GPIHBP1^27−44/R33M (blue curve in C). (D) Single-cycle kinetics of 2–32 nM GPIHBP1³⁴⁻¹³¹ binding to Protein G–captured total IgG (2 µg/mL) isolated either from a patient with GPIHBP1 autoantibody syndrome [patient 102 (15), red curve] or from a healthy normolipidemic control subject (blue curve). The Inset shows the level of Protein G–captured immunoglobulins, and the box represents the GPIHBP1 binding segment, which is enlarged in the main figure. Comparisons of capture levels and the calculated binding capacities for GPIHBP1 reveal that 2.2 ± 0.3% (n = 9) of the total IgG fraction binds GPIHBP1³⁴⁻¹³¹. (E) Binding profiles for affinity-purified GPIHBP1 autoantibodies to 2–32 nM GPIHBP1³⁴⁻¹³¹ (red curve) or to GPIHBP1¹⁻⁴⁵ (blue curve). These studies showed that 78 ± 7% (n = 4) of the affinity-purified IgG binds to GPIHBP1³⁴⁻¹³¹ (red curve), whereas none of the autoantibodies binds to GPIHBP1’s acidic IDR. Thin black lines in B–E show the kinetic fit of the data to a 1:1 binding model.