Protein Region Important for Regulation of Lipid Metabolism in Angiopoietin-like 3 (ANGPTL3)

Abstract

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiledcoil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg 221 2Ala 222 and Arg 224 2Thr 225 , which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo. Angiopoietin-like 3 (ANGPTL3)1 is a secreted protein that is expressed predominantly in the liver (1). ANGPTL3 is structurally similar to angiopoietins (2-4), which are vascular endothelial growth factors. It also has a signal peptide for secretion, a helical domain predicted to form coiled-coils (CCD), and a C-terminal globular fibrinogen-like domain (FLD). Angiopoietins are ligands that bind to the receptor tyrosine kinase Tie2. The FLDs of angiopoietins bind to Tie2, and the CCDs of angiopoietins mediate ligand homo-oligomerization (5). Although ANGPTL3 does not bind to Tie2, it may also induce angiogenesis by binding to integrin ␣ V ␤ 3 (6).We reported that KK/Snk mice (previously, KK/San) had abnormally low plasma lipid levels and identified a mutation in the Angptl3 gene as the cause of the hypolipidemic phenotype of KK/Snk mice (7,8). Injection of recombinant ANGPTL3 or adenoviruses encoding ANGPTL3 resulted in an increase of plasma lipid levels in mice. It has been speculated that ANGPTL3 was capable of inhibiting lipolysis of triglycerides, because an injection of recombinant ANGPTL3 induced a rapid increase in mouse plasma triglyceride levels (9, 10). We further reported that very low density lipoprotein clearance was enhanced in KK/Snk mice and that recombinant ANGPTL3 directly inhibited lipoprotein lipase (LPL) activity in vitro (11). These observations suggest that ANGPTL3 could be involved in the regulation of lipid metabolism by inhibiting LPL activity.Thus, ANGPTL3 seems to be a multifunctional factor that may regulate both lipid metabolism and angiogenesis. The C-terminal FLD of ANGPTL3 was reported to be involved in angiogenesis via binding t...

The Direct Peroxisome Proliferator-activated Receptor Target Fasting-induced Adipose Factor (FIAF/PGAR/ANGPTL4) Is Present in Blood Plasma as a Truncated Protein That Is Increased by Fenofibrate Treatment

“…Very recently, Ono et al reported that ANGPTL3 is cleaved in vivo, and, similar to our observations for FIAF (or AN-GPTL4), is present in mouse blood plasma in several forms of around 30 kDa (38). Interestingly, it was found that the resulting N-terminal fragment is probably responsible for the plasma triglyceride-raising effect of ANGPTL3.…”

Atypical angiopoietin-like protein that regulates ANGPTL3

“…Ono et al reported that ANGPTL3 must undergo cleavage to increase plasma TAG levels (20). Therefore, a possible explanation for the anomalous effects of ANGPTL8 on ANGPTL3 levels and activity is that ANGPTL8 may promote cleavage of ANGPTL3, a process that would simultaneously increase the activity of the peptide and decrease the amount of full-length ANGPTL3.…”

“…ANGPTL3 is activated by cleavage at a proprotein convertase consensus site ( 221 RAPR 224 ) to release the N-terminal domain (20). Cleavage is essential for ANGPTL3-mediated inhibition of lipases; disruption of the consensus site markedly reduces the effect of the recombinant protein on plasma TAG levels (20).…”

“…Cleavage is essential for ANGPTL3-mediated inhibition of lipases; disruption of the consensus site markedly reduces the effect of the recombinant protein on plasma TAG levels (20).…”

Mice lacking ANGPTL8 (Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis

“…ANGPTL3 is cleaved to yield an N-terminal domain that contains the LPL binding site and a C-terminal fibrinogen-like domain (24). Coexpression of ANGPTL8 and ANGPTL3 in cultured cells increased the appearance of the N-terminal domain of ANGPTL3 in the medium, suggesting that ANGPTL8 activates ANGPTL3 by promoting its cleavage (14).…”