A highly conserved motif within the NH2-terminal coiled-coil domain of angiopoietin-like protein 4 confers its inhibitory effects on lipoprotein lipase by disrupting the enzyme dimerization

Abstract

Lipoprotein lipase (LPL) is a principal enzyme responsible for the clearance of chylomicrons and very low density lipoproteins from the bloodstream. Two members of the Angptl (angiopoietin-like protein) family, namely Angptl3 and Angptl4, have been shown to inhibit LPL activity in vitro and in vivo. Here, we further investigated the structural basis underlying the LPL inhibition by Angptl3 and Angptl4. By multiple sequence alignment analysis, we have identified a highly conserved 12-amino acid consensus motif that is present within the coiled-coil domain (CCD) of both Angptl3 and Angptl4, but not other members of the Angptl family. Substitution of the three polar amino acid residues (His(46), Gln(50), and Gln(53)) within this motif with alanine abolishes the inhibitory effect of Angptl4 on LPL in vitro and also abrogates the ability of Angptl4 to elevate plasma triglyceride levels in mice. The CCD of Angptl4 interacts with LPL and converts the catalytically active dimers of LPL to its inactive monomers, whereas the mutant protein with the three polar amino acids being replaced by alanine loses such a property. Furthermore, a synthetic peptide consisting of the 12-amino acid consensus motif is sufficient to inhibit LPL activity, although the potency is much lower than the recombinant CCD of Angptl4. In summary, our data suggest that the 12-amino acid consensus motif within the CCD of Angptl4, especially the three polar residues within this motif, is responsible for its interaction with and inhibition of LPL by blocking the enzyme dimerization.

FIGURE 1.

NH₂-terminal CCDs of Angptl4 and Angptl3 inhibit the activities of LPL with different potencies. A, SDS-PAGE analysis of recombinant hAngptl3-CCD and hAngptl4-CCD. 3 μg of proteins per lane were separated under either reducing or nonreducing conditions by 12% SDS-PAGE. For DTT treatment, purified recombinant hAngptl4-CCD was incubated with 20 mm DTT for 1 h at room temperature to disrupt all the disulfide linkages. B, measurement of LPL activities in the presence of recombinant hAngptl3-CCD, hAngptl4-CCD, or DTT-treated hAngptl4-CCD. Note that DTT was completely removed by extensive dialysis before use. C, effects of hAngptl4-CCD on LPL activities stimulated with different concentrations of apoCIII. D, percentage of LPL activity remained after addition of different concentrations of hAngptl4-CCD. n = 4–5 in each group.

FIGURE 2.

Role of the three polar amino acid residues within the conserved motif in mediating the inhibitory activity of hAngptl4-CCD on LPL. A, SDS-PAGE analysis of the purified mutant hAngptl4-CCD with a single alanine substitution (H46A, Q50A, and Q53A). Each lane contains 3 μg of protein separated by 12% polyacrylamide gel under either reducing or non-reducing conditions. B, comparison of the inhibitory effects of wild type hAngptl4-CCD and the three mutants on LPL activity. Different concentrations of the wild type hAngptl4-CCD or the mutant proteins were included in the LPL activity assay mixture as indicated. The results are expressed as the percentage of LPL activity in the absence of hAngptl4-CCD or its mutants. *, p < 0.05; **, p < 0.01 versus wild type Angptl4-CCD treated group (n = 4–5).

FIGURE 3.

Effects of simultaneous alanine substitutions at His⁴⁶, Gln⁵⁰, and Gln⁵³ on LPL inhibition by hAngptl4-CCD. A, SDS-PAGE analysis of the purified hAngptl4-CCD mutant, A4-465053A, with His⁴⁶, Gln⁵⁰, and Gln⁵³ being substituted with alanine residues. An equal amount of protein (3 μg) was loaded onto each lane and separated by 12% polyacrylamide gel under either reducing or nonreducing conditions. B, comparison of the effects between wild type hAngptl4-CCD and A4-465053A on purified bovine LPL activity. **, p < 0.01 versus wild type recombinant hAngptl4-CCD (n = 4). C, comparison of the effects between wild type hAngptl4-CCD and A4-465053A on LPL activity in differentiated 3T3-L1 adipocytes. Fully differentiated adipocytes were incubated with different concentrations of the recombinant proteins for 4 h at 37 °C. Free LPL in medium and heparin-releasable LPL fractions were collected to determine LPL activity. ••, p < 0.01 versus medium control; ++, p < 0.01 versus heparin-releasable control (n = 4–5). D, effects of wild type hAngptl4-CCD and A4-465053A on plasma TG levels of C57BL/6J mice. 50 μg of proteins were injected intravenously into C57BL/6J mice. Control mice were injected with PBS only. Blood samples were collected at the indicated time points and analyzed for TG contents. n = 4–5, #, p < 0.05; ##, p < 0.01 versus control mice (n = 4–5).

FIGURE 4.

Determination of the interactions between LPL and wild type or mutated hAngptl4-CCD using SELDI-TOF MS analysis. The protein chips were coated with bovine LPL and incubated with hAngptl4-CCD (A), its mutant A4-212528A (B), or BSA as control (C). The bound proteins were detected by mass spectrometry as described under “Experimental Procedures.” Note that the molecular mass of 20996.78, 42132.82, 63288.33 and 84419.64 represents the monomeric, dimeric, trimeric and tetrameric forms of hAngptl4-CCD, respectively.

FIGURE 5.

MALDI-TOF MS analysis of LPL monomers and dimers. LPL was incubated with BSA (A), hAngptl4-CCD (B), or its A4-212528A (C) as described under “Experimental Procedures,” and the protein mixtures were analyzed by MALDI-TOF MS. Note that LPL dimeric form is not present in the sample treated with wild type hAngptl4-CCD.

FIGURE 6.

Conversion of LPL dimers to monomers by both hAngptl4 and hAngptl3 are dependent on the three polar residues within the conserved motif of the coiled-coil domain. Bovine LPL was biotinylated and re-purified by sucrose density gradient ultracentrifugation to obtain LPL dimers. 5 μg of biotinylated LPL dimers were added to a tube containing 1 ml of heparin-Sepharose beads. The beads were collected by centrifugation, washed, and incubated further with 15 μg of hAngptl4-CCD or its mutant A4-212528A and 75 μg of hAngptl3-CCD or its mutant in which the three polar amino acid residues were replaced by alanine. Afterward, LPL bound to the beads were eluted by a linear gradient of NaCl (from 0.20 to 1.8 m) as described under “Experimental Procedures.” Protein in each fraction was precipitated by 10% trichloroacetic acid, transferred to nylon membrane, and then probed with horseradish peroxidase-conjugated streptavidin to detect biotinylated LPL. The representative blots were shown from three independent experiments. Note that LPL monomers and dimers were eluted by 0.4–0.7 m NaCl and 1.0–1.3 m NaCl, respectively.

FIGURE 7.

Effect of the synthetic peptide containing the conserved sequence motif on LPL activity. A, synthetic peptides consisting of the wild type (NH₂-VLAHGLLQLGQGLRE-COOH) or the mutated motif (NH₂-VLAAGLLALGAGLRE-COOH) within the CCD of hAngptl4 were added to the LPL assay mixtures to a final concentration as indicated, and the reactions were performed at 30 °C for 1 h. B, time-dependent effect of the synthetic wild type peptide on the LPL activity. C, plasma triglyceride levels in C57/B6 mice at various time points after intravenous injection of wild type or mutated synthetic peptides (500 μg/mouse) or saline as control. *, p < 0.05; **, p < 0.01 versus control group (n = 5).