ANGPTL3/8 is an atypical unfoldase that regulates intravascular lipolysis by catalyzing unfolding of lipoprotein lipase

Figures

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  Fig. 1.. Oligomeric states and inhibitory properties of ANGPTL3 and ANGPTL3/8. (A) SEC of ANGPTL3 and ANGPTL3/8 in 10 mM HEPES, 150 mM NaCl, pH 8.0 using a Superose™ 6 Increase 10/300 CL column. Purity of the ANGPTL3 and ANGPTL3/8 preparations is shown by a Coomassie-stained polyacrylamide gel after SDS-PAGE (Inset). (B and C) Mass photometry of 10 nM ANGPTL3 (B) and 10 nM ANGPTL3/8 (C) revealed that ANGPTL3 is a homotrimer whereas ANGPTL3/8 is a heterotrimer containing two ANGPTL3 protomers and one ANGPTL8 protomer. The calculated mass is 159 kDa for ANGPTL3 trimers and 126 kDa for a 2:1 heterotrimeric complex of ANGPTL3 and ANGPTL8 without N-linked carbohydrates. Loss of 38 kDa (*) or loss of 75 kDa (⁑) corresponds to proteolytic removal of one or two fibrinogen-like domains (FLD). (D) Concentration-dependent inhibition of LPL activity by ANGPTL3, ANGPTL4, E40K ANGPTL4, or ANGPTL3/8. LPL inhibition was assessed by incubating 15 nM human LPL with a twofold dilution series of the various ANGPTLs (0.08 to 50 nM) for 10 min at 25 °C before the residual LPL activity toward long-chain triacylglycerols was determined by a NEFA assay. LPL activity is plotted relative to the activity of LPL…

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  Fig. 2.. Binding sites on ANGPTL3 for LPL and ANGPTL8. (A) Differential deuterium uptake plots displaying the difference in the deuterium uptake of ANGPTL3 sequences between ANGPTL3/8 heterotrimers and ANGPTL3 trimers normalized to maximally labeled control. (B) Differential deuterium uptake plots showing the relative deuterium uptake in ANGPTL3 peptides from ANGPTL3 trimers incubated in deuterated solvent either alone or in the presence of a molar excess of LPL. (C) Same analysis as in (B) but for heterotrimeric ANGPTL3/8 complexes. The shaded gray areas in A–C correspond to the largest SD in the dataset for each peptide (n = 3). Identities of the 65 peptic ANGPTL3 peptides are shown in SI Appendix, Fig. S2. (D) Deuterium uptake plots for selected ANGPTL3 sequences present in ANGPTLT3 trimers (solid blue) or ANGPTL3/8 heterotrimers (solid red). The impact of a molar excess of LPL on the deuterium uptake in these regions is shown by the corresponding dashed lines. Heatmaps of the data displayed in panels (B) and (C) are shown in SI Appendix, Fig. S4.

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  Fig. 3.. LPL binding site on ANGPTL8 in ANGPTL3/8 complexes. (A) Differential deuterium uptake plots showing the imprint from bovine LPL on the relative deuterium uptake in ANGPTL8 sequences by incubating 0.5 µM ANGPTL3/8 heterotrimers in the absence or presence of 1 µM LPL. The shaded gray area corresponds to the largest SD in the dataset for each peptide (n = 3). The corresponding heatmaps are shown in SI Appendix, Fig. S6. (B) Deuterium uptake plots for selected ANGPTL8 sequences in the absence (blue) and presence of a molar excess of LPL (red). Identities of the 31 peptic ANGPTL8 peptides are shown in SI Appendix, Fig. S5. (C) Model for the ANGPTL3/8 complex with a 2:1 stoichiometry of the oligomer. ANGPTL3 protomers are colored wheat and the ANGPTL8 protomer is in light gray. The LPL binding site based on reduced deuterium uptake is highlighted for the ANGPTL3 protomer in purple and for the ANGPTL8 protomer in green. Stabilization of regions in the ANGPTL3 protomers by ANGPTL8 is shown in blue. The epitope on the ANGPTL8 protomer for a neutralizing monoclonal antibody (mAb7) is shown in red (27). (D) Negative stain electron transmission microscopy image of ANGPTL3/8 bound to a…

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  Fig. 4.. Binding sites for ANGPTL3 and ANGPTL3/8 and their allosteric impact on LPL conformation. (A) Differential deuterium uptake plots depicting the changes in relative deuterium uptake for 49 peptic bLPL peptides recovered for LPL•GPIHBP1 incubated alone or in the presence of ANGPTL3/8 (Upper plot) or ANGPTL3 (Lower plot) for 5 s (orange), 25 s (red), and 50 s (blue) at 25 °C. The identity of the peptides is shown in SI Appendix, Fig. S10. Reduced deuterium uptake in β2–α3 and β3–α3 is highlighted by cyan bars while increased uptake in β6 and α5 due to allostery is highlighted by purple bars. The shaded gray area corresponds to the largest SD in the dataset for each peptide (n = 3). (B) Deuterium uptake of LPL peptides representing i) binding sites for ANGPTL3 and ANGPTL8 i.e. β2–α3 [51–62] and β3–α3 [84–101]; ii) regions showing allostery with increased deuterium uptake but without isotope bimodality i.e. β6–β7 [180−195] and α5 [239–249], iii) binding site for GPIHBP1 i.e. β9′–β10′ [396–419], and iv) the catalytic pocket i.e. β4–α4–β5 [131–165]. The arrow indicates correlated exchange as observed by the emergence of bimodal isotope envelopes. Uptake plots for all 49 LPL peptides are…

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  Fig. 5.. Changes in thermostability of LPL in the presence of ANGPTL3, ANGPTL3/8, and GPIHBP1. (A) LPL thermostability was measured by pulse-labeling HDX-MS using bimodality in isotope envelopes for peptide 131–165 as proxy for LPL unfolding. In brief, 7.5 µM LPL was incubated for 3 min in protiated solvent at the stated temperatures either alone or in the presence of 5 µM ANGPTL3 or 5 µM ANGPTL3/8 followed a 10 s pulse labeling at 25 °C after a 10-fold dilution in deuterated solvent. Shown are the binomial fittings to the native LPL conformation (blue); the unfolded conformation (red), and an intermediate conformation (green). (B) same as (A) but in the presence of 12 µM GPIHBP1. (C) Thermostability of LPL measured with nano-DSF. In this experiment, 10 µM LPL alone (red) or in the presence of substoichiometric molar amounts of 0.5 µM ANGPTL3 (blue) or ANGPTL3/8 (red) were subjected to differential scanning fluorimetry with a ramp temperature of 1 °C /min. Shown are the first derivatives for fluorescence emissions at 350 nm/330 nm (Upper panel) and for turbidity (Lower panel). (D) Same as (C) but where ANGPTL3 and ANGPTL3/8 are replaced by 0.5 µM FLD (green). (E) Same as (C) but where 10…

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  Fig. 6.. ANGPTL3 and ANGPTL3/8 catalyze LPL unfolding. (A) Progression in unfolding of the β4–α4–β5 foldon as assessed by bimodal isotope distributions for peptide 131–165. In brief, 7.5 µM LPL was incubated in the absence or presence of 0.75 µM of ANGPTL3 or ANGPTL3/8 for 5 to 100 s in protiated solvent at 37 °C followed by a 10 s pulse labeling in 90 % D2O at 25 °C. Relative LPL unfolding was calculated by binomial fitting of the isotope envelopes and is shown as the mean ± S.D. for the high mass envelope (n = 3). Of note, substoichiometric amounts of ANGPTL3 and ANGPTL3/8 relative to LPL (1:10) lead to a complete unfolding of all LPL molecules after 100 s. (B) same as in (A) except that the incubation mixture also contained 12 µM GPIHBP1.

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  Fig. 7.. Model of LPL in complex with ANGPTL3/8. (A) Molecular model for LPL•ANGPTL3/8 complexes in a cartoon presentation. Human LPL is depicted in gray, ANGPTL3 protomers in wheat, and the single ANGPTL8 protomer in light blue. Regions in LPL protected from deuterium exchange are highlighted in green (β2–α2, β3–α3, and lid), whereas regions with an accelerated deuterium uptake are shown in orange (β6–β7 and α5). (B) Expanded view of the binding interface between LPL and ANGPTL3/8 highlights the central position of ANGPTL8 in the binding interface with LPL where its proximal α-helix forms extensive hydrophobic interactions with the lid.

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  Fig. 8.. Model for the regulation of LPL activity in the capillary lumen ANGPTL3/8. LPL is tethered to the luminal surface of capillary endothelial cells by binding to GPIHBP1 and HSPGs in the glycocalyx. In this polyanionic microenvironment (orange bar) LPL is stabilized and is less sensitive to inhibition by ANGPTL3 thus shifting its regulation toward ANGPTL3/8 inhibition. The unbalanced concentrations of LPL (8–16 nM) and its inhibitor ANGPTL3/8 (0.1 nM) call for an untraditional mechanism of inhibition—that of permanent LPL inhibition by catalyzed unfolding. Unbridled unfoldase activity of ANGPTL3/8 is controlled by its inhibitor APOA5.