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

Ming-Hon Yau,Yu Wang,Karen S.L Lam,Jialiang Zhang,Donghai Wu,Aimin Xu

doi:10.1074/jbc.m809802200

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.

Highlights

Lipoprotein lipase (LPL)3 is an endothelium-bound enzyme that catalyzes the hydrolysis of plasma triglyceride (TG) asso
When the LPL activity was expressed as the percentage remaining in comparison with those without hAngptl4-coiled-coil domain (CCD) treatment (Fig. 1D), we found that the presence of apoCII did not rescue hAngptl4-CCDinduced suppression of LPL activity
These data exclude the possibility that hAngptl4-CCD inhibits LPL activity through competing

Summary

EXPERIMENTAL PROCEDURES

Materials—Restriction endonucleases were from New England Biolabs (Ipswich, MA). T4 DNA ligase and ImProm-IITM Reverse Transcription System were purchased from Promega (Madison, WI). Differentiated 3T3-L1 adipocytes were treated with different concentrations of wild type or mutant hAngptl4-CCD proteins for 4 h, and LPL activity in both secreted and surface-bound fractions was measured as above. The cell surface-bound LPL was released by incubating the sample with 0.5 ml of Dulbecco’s modified Eagle’s medium containing 0.2% BSA, 100 units/ml heparin for 30 min at 4 °C. Bovine LPL (0.3 ␮g, Sigma) was incubated at room temperature with 1.5 ␮g of BSA, hAngptl4-CCD, or its mutant protein in a total volume of 10 ␮l of 20 mM Tris-HCl, 0.15 M NaCl (TBS) (pH 7.4), for 1 h. The samples were subjected to a brief centrifugation, followed by washing three times with 10 ml of buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 2% BSA to remove unbound LPL. Pressed as the percentage remaining in comparison with those without hAngptl4-CCD treatment (Fig. 1D), we found that the presence of apoCII did not rescue hAngptl4-CCDinduced suppression of LPL activity

RESULTS

CCD on LPL was also abrogated following substitution of the three

Both our in vitro and in vivo findings

DISCUSSION