Angiopoietin-like protein 4/8 complex-mediated plasmin generation leads to cleavage of the complex and restoration of LPL activity

Figures

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  Fig. 1.. Mass spectrometry demonstrates ANGPTL4/8 binds human serum plasminogen. Recombinant ANGPTL4/8 was serially diluted (D1 to D4: 0.125 to 1.0 µg, with 0 µg as a control), immobilized, and incubated with human serum. Interacting proteins were eluted and identified by mass spectrometry after enzymatic digestion. (A) Plots show fold-changes of the mean ion intensities of ANGPTL4, ANGPTL8, and plasminogen for each dilution compared to corresponding control ion intensities. Each condition was analyzed in triplicate. All P values were <0.001 for D1 to D4 with anti-FLAG and anti-ANGPTL4/8 antibodies versus the isotype-matched control antibody. (B) Graphs show correlations of the mean log2AUC ion intensities between plasminogen, ANGPTL4, and ANGPTL8 in samples with anti-FLAG and anti-ANGPTL4/8 antibodies. Dashed lines represent 95% CIs. Results are representative of two independent experiments.

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  Fig. 2.. Western blotting confirms ANGPTL4/8 binds plasminogen and tPA. (A) Immobilized FLAG-tagged ANGPTL4/8, FLAG-tagged ANGPTL3/8, His-tagged ANGPTL4, or His-tagged ANGPTL3 were incubated with human serum. Bound proteins were eluted, separated electrophoretically, and transferred to PVDF membrane probed with rabbit polyclonal anti-plasminogen primary antibody, with Alexa 680-labeled anti-rabbit antibody used for detection. Results are representative of two independent experiments. (B) The above experiment was repeated except immobilized proteins were incubated with phosphate buffered saline (PBS) containing tPA. Bound proteins were eluted, separated electrophoretically, and transferred to PVDF membrane probed with mouse monoclonal anti-tPA primary antibody, with Alexa 680-labeled anti-mouse antibody used for detection. Results are representative of two independent experiments.

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  Fig. 3.. ANGPTL4/8 and fibrin increase tPA-mediated plasmin generation. (A) Activation of plasminogen to plasmin was monitored using the chromogenic plasmin substrate D-Val-Leu-Lys-pNA. Plasminogen alone, tPA + plasminogen, ANGPTL4 + tPA + plasminogen, ANGPTL4/8 + tPA + plasminogen, ANGPTL3 + tPA + plasminogen, ANGPTL3/8 + tPA + plasminogen, or fibrin + tPA + plasminogen were added to wells containing chromogenic substrate. Samples were incubated at 37 °C for 60 min, and absorbance at 405 nm was recorded every 2 min. Results are representative of three independent experiments. (B) Increasing concentrations of plasminogen (0 to 1,200 nM) were incubated with tPA alone, ANGPTL4 + tPA, ANGPTL4/8 + tPA, ANGPTL3 + tPA, ANGPTL3/8 + tPA, or fibrin + tPA in buffer containing chromogenic plasmin substrate for 60 min at 37 °C. Wells containing only plasminogen and chromogenic substrate served controls. Maximal absorbance was normalized to 100% plasmin activity. Results are representative of three independent experiments.

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  Fig. 4.. LPL-bound ANGPTL4/8 is rapidly degraded by tPA + plasminogen. Recombinant ANGPTL4/8 was incubated with LPL stable-expressing cells for 1 h at 37 °C. The incubation was continued for 0 to 120 min at 37 °C in the absence or presence of tPA + plasminogen. Aliquots were removed at times indicted, and reactions stopped for ANGPTL4/8 and LPL analyses. Samples were separated on different sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to separate PVDF membranes. All results are representative of two independent experiments. (A) The membrane was probed with biotinylated anti-ANGPTL4 antibodies, followed by Alexa 680-labeled streptavidin. (B) The membrane was probed with biotinylated anti-ANGPTL8 antibodies, followed by Alexa 680-labeled streptavidin. (C) The membrane was probed with goat anti-LPL antibody, followed by Alexa 680-labeled anti-goat antibody.

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  Fig. 5.. The combination of tPA + plasminogen specifically reverses ANGPTL4/8-mediated LPL inhibition. The ability of 30 nM ANGPTL3 (A), ANGPTL3/8 (B), ANGPTL4 (C), or ANGPTL4/8 (D) to inhibit LPL activity in the absence or presence of tPA + plasminogen was assessed using LPL-stable expression cells with fluorescent lipase substrate. Fluorescence was monitored continuously every minute for 60 min. In each case, cells preincubated in the absence of ANGPTL proteins served as the vehicle control. All results are representative of at least two independent experiments.

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  Fig. 6.. Plasminogen activation is required for reversal of ANGPTL4/8 mediated LPL inhibition. The ability of recombinant ANGPTL4/8 to reduce LPL activity in the presence of plasminogen, tPA, tPA + plasminogen, plasmin, PAI-1, tPA + plasminogen + PAI-1, plasminogen (S760A), or tPA + plasminogen (S760A) was assessed using LPL-stable expression cells with fluorescent lipase substrate and VLDL substrate. LPL activity in the absence of ANGPTL4/8 served as the maximum activity reference. All results are shown as the mean ± SD (n = 8 from two independent experiments) (*P < 0.0001 versus ANGPTL4/8 alone). (A) LPL activity with fluorescent lipase substrate. (B) LPL activity with VLDL substrate and NEFA measurement.

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  Fig. 7.. Human serum ANGPTL4 may come from plasmin-mediated ANGPTL4/8 cleavage rather than furin-mediated ANGPTL4/8 or ANGPTL4 cleavage. (A) Endogenous ANGPTL4 in human serum was immunoprecipitated using a polyclonal anti-ANGPTL4 antibody. Recombinant ANGPTL4/8 digests with tPA and plasminogen in assay buffer and LPL cell culture media were included for comparison. Proteins were separated electrophoretically and transferred to a PVDF membrane. (B) Recombinant ANGPTL4/8 was incubated with tPA + plasminogen or furin. Aliquots were removed at different time points, and the reaction was stopped by adding SDS sample buffer. Samples were separated electrophoretically and transferred to a PVDF membrane. (C) Recombinant ANGPTL4/8 complex or ANGPTL4 was incubated with furin for various periods of time. At each time point, the reaction was stopped, and proteins were separated electrophoretically and transferred to a PVDF membrane. All membranes were probed with biotinylated anti-ANGPTL4 antibodies followed by Alexa 680-labeled streptavidin. Results are representative of two independent experiments.

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  Fig. 8.. A model for how adipose tissue LPL activity is controlled during fasting and feeding. (A) During fasting, ANGPTL8 expression is low, while ANGPTL4 levels are high. ANGPTL4 binds LPL intracellularly to decrease its secretion by adipocytes, binds LPL in the subendothelial space, and irreversibly inactivates LPL. Therefore, LPL cannot be translocated across the capillary endothelium by GPIHBP1. Therefore, LPL levels on the luminal surfaces of capillaries are very low. The endothelium secretes tPA into the circulation, where it can catalyze plasmin-mediated cleavage of any fibrin clots present. (B) After feeding, ANGPTL8 increases, causing increased ANGPTL4/8 secretion from adipocytes (a). ANGPTL4/8 binds LPL in the subendothelial space and protects LPL from inactivation by ANGPTL4 but also partially inhibits LPL as shown by the lighter blue color (b). The partially active ANGPTL4/8–LPL complex binds GPIHBP1 on the abluminal surface of the endothelium (c) and is translocated across the endothelium (d). As this occurs, tPA secreted by the endothelium binds ANGPTL4/8, and circulating plasminogen binds the LPL–ANGPTL4/8-tPA complex (e) as it is positioned on the luminal capillary…