Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities.

Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing them from triglyceride-rich lipoproteins to release free fatty acids. LPL activity is regulated in a nutritionally responsive manner by macromolecular inhibitors including angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4). However, the mechanism by which ANGPTL3 inhibits LPL is unclear, in part due to challenges in obtaining pure protein for study. We used a new purification protocol for the N-terminal domain of ANGPTL3, removing a DNA contaminant, and found DNA-free ANGPTL3 showed enhanced inhibition of LPL. Structural analysis showed that ANGPTL3 formed elongated, flexible trimers and hexamers that did not interconvert. ANGPTL4 formed only elongated flexible trimers. We compared the inhibition of ANGPTL3 and ANGPTL4 using human very-low-density lipoproteins as a substrate and found both were noncompetitive inhibitors. The inhibition constants for the trimeric ANGPTL3 (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) were only 2-fold different. Heparin has previously been reported to interfere with ANGPTL3 binding to LPL, so we questioned if the negatively charged heparin was acting in a similar fashion to the DNA contaminant. We found that ANGPTL3 inhibition is abolished by binding to low-molecular-weight heparin, whereas ANGPTL4 inhibition is not. Our data show new similarities and differences in how ANGPTL3 and ANGPTL4 regulate LPL and opens new avenues of investigating the effect of heparin on LPL inhibition by ANGPTL3.

Lipoprotein lipase (LPL) strongly links native and oxidized low density lipoprotein particles to decorin-coated collagen. Roles for both dimeric and monomeric forms of LPL.

Low density lipoprotein (LDL) and oxidized LDL are associated with collagen in the arterial intima, where the collagen is coated by the small proteoglycan decorin. When incubated in physiological ionic conditions, decorin-coated collagen bound only small amounts of native and oxidized LDL, the interaction being weak. When decorin-coated collagen was first allowed to bind lipoprotein lipase (LPL), binding of native and oxidized LDL increased dramatically (23- and 7-fold, respectively). This increase depended on strong interactions between LPL that was bound to the glycosaminoglycan chains of the collagen-bound decorin and native and oxidized LDL (kDa 12 and 5.9 nM, respectively). To distinguish between binding to monomeric (inactive) and dimeric (catalytically active) forms of LPL, affinity chromatography on heparin columns was conducted, which showed that native LDL bound to the monomeric LPL, whereas oxidized LDL, irrespective of the type of modification (Cu(2+), 2, 2'-azobis(2-amidinopropane)hydrochloride, hypochlorite, or soybean 15-lipoxygenase), bound preferably to dimeric LPL. However, catalytic activity of LPL was not required for binding to oxidized LDL. Finally, immunohistochemistry of atherosclerotic lesions of human coronary arteries revealed specific areas in which LDL, LPL, decorin, and collagen type I were present. The results suggest that LPL can retain LDL in atherosclerotic lesions along decorin-coated collagen fibers.

Lipoprotein lipase. Molecular model based on the pancreatic lipase x-ray structure: consequences for heparin binding and catalysis.

Lipoprotein lipase and pancreatic lipase have about 30% sequence identity, suggesting a similar tertiary fold. Three-dimensional models of lipoprotein lipase were constructed, based upon two recently determined x-ray crystal structures of pancreatic lipase, in which the active site was in an open and closed conformation, respectively. These models allow us to propose a few hypotheses on the structural determinants of lipoprotein lipase which are responsible for heparin binding, dimer formation, and phospholipase activity. The folding of the protein assembles a number of positive charge clusters at the back of the molecule, opposite the active site. These clusters probably form the heparin binding site, as confirmed by recent site-directed mutagenesis experiments. The active sites of lipoprotein lipase and pancreatic lipase look very similar, except for the lid (a surface loop covering the catalytic serine in the inactive state). A different open (active) conformation of the lid in both enzymes may be responsible for their differing substrate specificities. Predictions of the nature of the lipoprotein lipase dimer remain elusive, although our model enabled us to propose a few possibilities.