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

Abstract

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. 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) plays a central role in the regulation of whole-body energy balance by hydrolyzing triglycerides from circulating lipoproteins to provide free fatty acids to tissues. LPL activity is differentially regulated in adipose versus muscle tissue depending on nutritional state in order to ensure appropriate distribution of available energy throughout the body (1Dijk W. Kersten S. Regulation of lipid metabolism by angiopoietin-like proteins.Curr. Opin. Lipidol. 2016; 27: 249-256Crossref PubMed Scopus (109) Google Scholar). Specifically, LPL activity is downregulated in adipose tissue during fasting to direct triglyceride-rich lipoproteins to muscle tissue for fatty acid oxidation (2Fried S.K. Hill J.O. Nickel M. DiGirolamo M. Prolonged effects of fasting-refeeding on rat adipose tissue lipoprotein lipase activity: Influence of caloric restriction during refeeding.J. Nutr. 1983; 113: 1861-1869Crossref PubMed Scopus (40) Google Scholar, 3Quig D.W. Layman D.K. Bechtel P.J. Hackler L.R. The influence of starvation and refeeding on the lipoprotein lipase activity of skeletal muscle and adipose tissue of lean and obese Zucker rats.J. Nutr. 1983; 113: 1150-1156Crossref PubMed Scopus (22) Google Scholar). Conversely, in the postprandial state, LPL activity increases in adipose tissue but decreases in muscle tissue. This allows utilization of glucose by muscle tissue and fat storage in adipose tissue (3Quig D.W. Layman D.K. Bechtel P.J. Hackler L.R. The influence of starvation and refeeding on the lipoprotein lipase activity of skeletal muscle and adipose tissue of lean and obese Zucker rats.J. Nutr. 1983; 113: 1150-1156Crossref PubMed Scopus (22) Google Scholar). This tissue-specific regulation is facilitated by three members of the angiopoietin-like protein (ANGPTL) family of proteins: ANGPTL3, ANGPTL4, and ANGPTL8 (4Zhang R. The ANGPTL3-4-8 model, a molecular mechanism for triglyceride trafficking.Open Biol. 2016; 6: 150272Crossref PubMed Scopus (133) Google Scholar). Each protein is an inhibitor of LPL, and each has a different pattern of expression. ANGPTL3 is highly expressed in the liver and is largely insensitive to nutritional state (5Conklin D. Gilbertson D. Taft D.W. Maurer M.F. Whitmore T.E. Smith D.L. Walker K.M. Chen L.H. Wattler S. Nehls M. Lewis K.B. Identification of a mammalian angiopoietin-related protein expressed specifically in liver.Genomics. 1999; 62: 477-482Crossref PubMed Scopus (114) Google Scholar). Upon fasting, ANGPTL4 is upregulated in the adipose tissue (6Kersten S. Mandard S. Tan N.S. Escher P. Metzger D. Chambon P. Gonzalez F.J. Desvergne B. Wahli W. Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene.J. Biol. Chem. 2000; 275: 28488-28493Abstract Full Full PubMed Scopus Google Scholar). ANGPTL8 is upregulated and expressed in the liver and adipose R. a novel factor that triglyceride PubMed Scopus Google with ANGPTL3 to LPL S.K. W. R. ANGPTL8 the of ANGPTL3 to and lipoprotein 6: PubMed Scopus Google Scholar, P. D. S. S. 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Full Full PubMed Scopus Google Scholar). we found that N-terminal ANGPTL3 with a acid as by The acid was to but to that ANGPTL3 was to DNA We in the of the that ANGPTL3 We and the ANGPTL3 from each and in LPL inhibition using human as a showed that ANGPTL3 a LPL inhibition activity order to the DNA from the ANGPTL3 we an purification which to a in DNA and ANGPTL3 as from the from the DNA-free ANGPTL3 in from the and pure ANGPTL3 ANGPTL3 from and were for inhibition of LPL acting on showed that protein from both were as LPL of DNA from ANGPTL3 as an LPL We to the state of N-terminal ANGPTL3 in and a showed that ANGPTL3 from a as the of was not regulation and of angiopoietin-like proteins 3 and Full Full PubMed Scopus Google Scholar). both and were and by that a molecular of which to a of ANGPTL3 The a molecular of which to an ANGPTL3 is that we found that the ANGPTL3 hexamers and trimers not interconvert. we each we only a on the and that hexamers not trimers and trimers not that N-terminal ANGPTL3 not which for the the state, we ANGPTL3 to a purification and the protein and compared the from with that of ANGPTL3. This that ANGPTL3 trimers order to the state of ANGPTL3 in mammalian we compared N-terminal ANGPTL3 in with ANGPTL3 The ANGPTL3 from as a the ANGPTL3 from which was to the of in mammalian We on N-terminal ANGPTL4 and found ANGPTL4 in a with a molecular of which to a of ANGPTL4 the differences ANGPTL3 and ANGPTL4, we ANGPTL3 hexamers and trimers were by and for of ANGPTL3 hexamers and trimers elongated with the the of the and The of is with proteins of similar ANGPTL4 was found to an elongated and highly flexible by both the ANGPTL3 and and ANGPTL4 of highly flexible proteins is of the N-terminal of ANGPTL3, so we did not to in the D. highly the domain of angiopoietin-like protein 4 effects on lipoprotein lipase by the Biol. Chem. Full Full PubMed Scopus Google Scholar). are of that both ANGPTL3 and ANGPTL4 elongated in and that the data the state of the We ANGPTL3 and ANGPTL4 inhibition of LPL acting on of we a previously S. D. S. R. lipoprotein protein triglycerides in for Biol. Chem. Full Full PubMed Scopus Google that fatty acids using an in with a This allows of so that of LPL with and inhibitors. We used to the a of triglycerides This on LPL, the ANGPTL3 ± nM) and ANGPTL4 ± nM) similar inhibition whereas the ANGPTL3 is a inhibitor ± We used the data to of both the inhibitor and to a of for each We each and the data by and This to that both ANGPTL3 and ANGPTL4 show the of pure noncompetitive inhibition and of and Scholar, M. for inhibition for and PubMed Scopus Google Scholar). we used to noncompetitive of the to the inhibition We the from three to a for each from the ANGPTL3 the ± ANGPTL3 (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) were as with the ANGPTL4 was as as the ANGPTL3 both are inhibitors. using acid as an LPL substrate that ANGPTL3 inhibition of LPL is by the of heparin, whereas ANGPTL4 inhibition is not The angiopoietin-like proteins ANGPTL3 and ANGPTL4 lipoprotein lipase activity Biol. Chem. Full Full PubMed Scopus Google Scholar). that the ANGPTL3 was a inhibitor of LPL, we that both heparin and DNA a in ANGPTL3 which is to the LPL and LPL inhibition M. M. M. R. R. plasma of Biol. 27: PubMed Scopus Google Scholar). We by the effects of heparin and on LPL inhibition by ANGPTL3 and ANGPTL4 in We used different for with The angiopoietin-like proteins ANGPTL3 and ANGPTL4 lipoprotein lipase activity Biol. Chem. Full Full PubMed Scopus Google and as a and found that both the ANGPTL3 LPL in the of heparin However, ANGPTL3 inhibition of LPL was in the of heparin and ANGPTL4 showed of inhibition from heparin and ANGPTL3 activity was in the of heparin, and ANGPTL3 inhibition similar to the heparin We that the of ANGPTL3 inhibition in the of was the of heparin binding to ANGPTL3 and inhibition by the on LPL, as previously been The angiopoietin-like proteins ANGPTL3 and ANGPTL4 lipoprotein lipase activity Biol. Chem. Full Full PubMed Scopus Google Scholar). we and We LPL, ANGPTL3, and ANGPTL4 binding to both of heparin Each protein was with of the heparin and using The were and and the protein to the heparin was by We found that ANGPTL3 to but binding to This that ANGPTL3 binding to to LPL, heparin binding to LPL, that LPL to both and ANGPTL4 has previously been to heparin M. S. P. S. angiopoietin-like 4 inhibits and and PubMed Scopus Google and we to low-molecular-weight of binding to ANGPTL4, the low-molecular-weight did not ANGPTL4 inhibition of LPL Our of ANGPTL3 with ANGPTL4 has of ANGPTL3 and ANGPTL4 inhibition of LPL. Our that ANGPTL3 with a DNA the differences in the reported of ANGPTL3 The angiopoietin-like proteins ANGPTL3 and ANGPTL4 lipoprotein lipase activity Biol. Chem. Full Full PubMed Scopus Google Scholar, D. W. B. S. M. lipoprotein lipase and inhibition by angiopoietin-like 3 and angiopoietin-like Full Full PubMed Scopus Google Scholar, M. Lipoprotein lipase activity and in human plasma by Full Full PubMed Scopus Google Scholar). ANGPTL3 is to both DNA and heparin the of in ANGPTL3 to ANGPTL3. ANGPTL3 was we found that was a inhibitor of LPL, in the of This has for the ANGPTL8 ANGPTL3 ANGPTL3 LPL but the has not been S.K. W. R. ANGPTL8 the of ANGPTL3 to and lipoprotein 6: PubMed Scopus Google Scholar, M. the mechanism of angiopoietin-like protein for of lipoprotein lipase Full Full PubMed Scopus Google we that ANGPTL8 to ANGPTL3. The mechanism a the of heparin from the of ANGPTL3. This the of the N-terminal of ANGPTL3 and both and we found that N-terminal ANGPTL3 in hexamers and that not interconvert. However, and ANGPTL3 a trimeric state that the trimeric is the of the We that ANGPTL3 in mammalian a trimeric ANGPTL4 was to which from that ANGPTL4 hexamers W. S. S. in ANGPTL4 protein and Biol. Chem. 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We used analysis to a mechanism of pure noncompetitive the of which are a with inhibitor and in with inhibitor and We found that the of the data on the and that the were and of and Scholar). inhibition that ANGPTL3 and ANGPTL4 to both LPL and LPL in a We that inhibition with both and in of of inhibition in for the to a protocol that used to of different and inhibitor previously ANGPTL3 and ANGPTL4 is with N-terminal ANGPTL3 a to LPL whereas N-terminal ANGPTL4 heparin an M. M. M. R. R. plasma of Biol. 27: PubMed Scopus Google Scholar, M. S. P. S. angiopoietin-like 4 inhibits and and PubMed Scopus Google Scholar). was that ANGPTL3 inhibition of LPL was by heparin binding to LPL The angiopoietin-like proteins ANGPTL3 and ANGPTL4 lipoprotein lipase activity Biol. Chem. Full Full PubMed Scopus Google Scholar). Our data show that LPL to both and heparin, but ANGPTL3 only to ANGPTL3 is not an LPL which we with both and and We found that ANGPTL4 both and heparin, but inhibition of LPL and the to if the of to LPL inhibitor binding in the of of activity binding to heparin opens new to ANGPTL3 activity in This new the and LPL inhibition of ANGPTL3 and is that ANGPTL3 and ANGPTL4 including elongated trimeric and noncompetitive We an which is that the binding of ANGPTL3 to heparin LPL This in the of to LPL The N-terminal domain of human ANGPTL3 to was to a and a The molecular of the protein was The was a the human tissue ANGPTL3 to with a was in the was and in to an of was using and the were for 3 by for 4 were in on and were and were 4 were using an the was by for The was with a and to a in the was in by and was using ANGPTL3 were and using were a using an was a using ANGPTL3 were and was using a The the and the of ANGPTL3, ANGPTL3 and were in the of both ANGPTL3 and were and using The protein were by acid and DNA was by the with a the ANGPTL3 were as and by for in The was by for The was with a and to a in was used to the in a from to to the The ANGPTL3 was from the as and by using a ANGPTL3 was human using the ANGPTL3 were in with and of ANGPTL3 was by the of with and The was and with new for The was and the was and to 4 ANGPTL3 was as for expression. 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