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• W2149487645 abstract "Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) binds both LPL and chylomicrons, suggesting that GPIHBP1 is a platform for LPL-dependent processing of triglyceride (TG)-rich lipoproteins. Here, we investigated whether GPIHBP1 affects LPL activity in the absence and presence of LPL inhibitors angiopoietin-like (ANGPTL)3 and ANGPTL4. Like heparin, GPIHBP1 stabilized but did not activate LPL. ANGPTL4 potently inhibited nonstabilized LPL as well as heparin-stabilized LPL but not GPIHBP1-stabilized LPL. Like ANGPTL4, ANGPTL3 inhibited nonstabilized LPL but not GPIHBP1-stabilized LPL. ANGPTL3 also inhibited heparin-stabilized LPL but with less potency than nonstabilized LPL. Consistent with these in vitro findings, fasting serum TGs of Angptl4−/−/Gpihbp1−/− mice were lower than those of Gpihbp1−/− mice and approached those of wild-type littermates. In contrast, serum TGs of Angptl3−/−/Gpihbp1−/− mice were only slightly lower than those of Gpihbp1−/− mice. Treating Gpihbp1−/− mice with ANGPTL4- or ANGPTL3-neutralizing antibodies recapitulated the double knockout phenotypes. These data suggest that GPIHBP1 functions as an LPL stabilizer. Moreover, therapeutic agents that prevent LPL inhibition by ANGPTL4 or, to a lesser extent, ANGPTL3, may benefit individuals with hyperlipidemia caused by gene mutations associated with decreased LPL stability. Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) binds both LPL and chylomicrons, suggesting that GPIHBP1 is a platform for LPL-dependent processing of triglyceride (TG)-rich lipoproteins. Here, we investigated whether GPIHBP1 affects LPL activity in the absence and presence of LPL inhibitors angiopoietin-like (ANGPTL)3 and ANGPTL4. Like heparin, GPIHBP1 stabilized but did not activate LPL. ANGPTL4 potently inhibited nonstabilized LPL as well as heparin-stabilized LPL but not GPIHBP1-stabilized LPL. Like ANGPTL4, ANGPTL3 inhibited nonstabilized LPL but not GPIHBP1-stabilized LPL. ANGPTL3 also inhibited heparin-stabilized LPL but with less potency than nonstabilized LPL. Consistent with these in vitro findings, fasting serum TGs of Angptl4−/−/Gpihbp1−/− mice were lower than those of Gpihbp1−/− mice and approached those of wild-type littermates. In contrast, serum TGs of Angptl3−/−/Gpihbp1−/− mice were only slightly lower than those of Gpihbp1−/− mice. Treating Gpihbp1−/− mice with ANGPTL4- or ANGPTL3-neutralizing antibodies recapitulated the double knockout phenotypes. These data suggest that GPIHBP1 functions as an LPL stabilizer. Moreover, therapeutic agents that prevent LPL inhibition by ANGPTL4 or, to a lesser extent, ANGPTL3, may benefit individuals with hyperlipidemia caused by gene mutations associated with decreased LPL stability. Our understanding of how triglyceride (TG) metabolism is regulated is essential for designing avenues of therapeutic intervention for diseases such as atherosclerosis, pancreatitis, or dyslipidemia associated with metabolic syndrome or type II diabetes (1Wang J. Ban M.R. Zou G.Y. Cao H. Lin T. Kennedy B.A. Anand S. Yusuf S. Huff M.W. Pollex R.L. et al.Polygenic determinants of severe hypertriglyceridemia.Hum. Mol. Genet. 2008; 17: 2894-2899Crossref PubMed Scopus (109) Google Scholar, 2Lusis A.J. Pajukanta P. A treasure trove for lipoprotein biology.Nat. Genet. 2008; 40: 129-130Crossref PubMed Scopus (71) Google Scholar, 3Mead J.R. Irvine S.A. Ramji D.P. Lipoprotein lipase: structure, function, regulation, and role in disease.J. Mol. Med. 2002; 80: 753-769Crossref PubMed Scopus (678) Google Scholar). Central to triglyceride metabolism is lipoprotein lipase (LPL), an extracellular enzyme primarily located in the vascular beds of many tissues (3Mead J.R. Irvine S.A. Ramji D.P. Lipoprotein lipase: structure, function, regulation, and role in disease.J. Mol. Med. 2002; 80: 753-769Crossref PubMed Scopus (678) Google Scholar, 4Merkel M. Eckel R.H. Goldberg I.J. Lipoprotein lipase: genetics, lipid uptake, and regulation.J. Lipid Res. 2002; 43: 1997-2006Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). LPL catalyzes the hydrolysis of the triglyceride component of chylomicrons (CM) and VLDL, which constitute the major forms of triglycerides in plasma (3Mead J.R. Irvine S.A. Ramji D.P. Lipoprotein lipase: structure, function, regulation, and role in disease.J. Mol. Med. 2002; 80: 753-769Crossref PubMed Scopus (678) Google Scholar, 5Li C. Genetics and regulation of angiopoietin-like proteins 3 and 4.Curr. Opin. Lipidol. 2006; 17: 152-156Crossref PubMed Scopus (62) Google Scholar). Although LPL is expressed in many different tissues, the enzyme is expressed at high levels in metabolically active tissues, such as adipose, cardiac muscle, and skeletal muscle, where fatty acids released by the action of LPL are stored or used (4Merkel M. Eckel R.H. Goldberg I.J. Lipoprotein lipase: genetics, lipid uptake, and regulation.J. Lipid Res. 2002; 43: 1997-2006Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). LPL appears to be regulated by a variety of mechanisms. Several apolipoproteins associated with CM and VLDL, including apolipoprotein CII (APOC2) and apolipoprotein AV (APOA5), stimulate LPL activity (6Dorfmeister B. Zeng W.W. Dichlberger A. Nilsson S.K. Schaap F.G. Hubacek J.A. Merkel M. Cooper J.A. Lookene A. Putt W. et al.Effects of six APOA5 variants, identified in patients with severe hypertriglyceridemia, on in vitro lipoprotein lipase activity and receptor binding.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1866-1871Crossref PubMed Scopus (57) Google Scholar, 7Jong M.C. Hofker M.H. Havekes L.M. Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar, 8Merkel M. Loeffler B. Kluger M. Fabig N. Geppert G. Pennacchio L.A. Laatsch A. Heeren J. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase.J. Biol. Chem. 2005; 280: 21553-21560Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 9Priore Oliva C. Pisciotta L. Li Volti G. Sambataro M.P. Cantafora A. Bellocchio A. Catapano A. Tarugi P. Bertolini S. Calandra S. Inherited apolipoprotein A-V deficiency in severe hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 411-417Crossref PubMed Scopus (170) Google Scholar) apparently by increasing its Vmax (10McIlhargey T.L. Yang Y. Wong H. Hill J.S. Identification of a lipoprotein lipase cofactor-binding site by chemical cross-linking and transfer of apolipoprotein C–II-responsive lipolysis from lipoprotein lipase to hepatic lipase.J. Biol. Chem. 2003; 278: 23027-23035Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 11Olivecrona G. Beisiegel U. Lipid binding of apolipoprotein CII is required for stimulation of lipoprotein lipase activity against apolipoprotein CII-deficient chylomicrons.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1545-1549Crossref PubMed Scopus (52) Google Scholar). In contrast, apolipoproteins CI (APOC1) and CIII (APOC3) can inhibit LPL activity (7Jong M.C. Hofker M.H. Havekes L.M. Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar, 12Wang C.S. McConathy W.J. Kloer H.U. Alaupovic P. Modulation of lipoprotein lipase activity by apolipoproteins. Effect of apolipoprotein C–III.J. Clin. Invest. 1985; 75: 384-390Crossref PubMed Scopus (383) Google Scholar). LPL is inherently unstable and proteins or other factors that either stabilize or destabilize LPL are likely to play a role in regulating its in vivo activity (13Zhang L. Lookene A. Wu G. Olivecrona G. Calcium triggers folding of lipoprotein lipase into active dimers.J. Biol. Chem. 2005; 280: 42580-42591Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The active form of LPL exists as a head-to-tail homodimer, which dissociates into metastable monomers. These monomers can reassociate to form catalytically active LPL or they can undergo conformational changes, forming inactive, stable monomers. The spontaneous inactivation of LPL is mostly irreversible (14Kobayashi Y. Nakajima T. Inoue I. Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain.Eur. J. Biochem. 2002; 269: 4701-4710Crossref PubMed Scopus (64) Google Scholar, 15Lookene A. Zhang L. Hultin M. Olivecrona G. Rapid subunit exchange in dimeric lipoprotein lipase and properties of the inactive monomer.J. Biol. Chem. 2004; 279: 49964-49972Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Wong H. Yang D. Hill J.S. Davis R.C. Nikazy J. Schotz M.C. A molecular biology-based approach to resolve the subunit orientation of lipoprotein lipase.Proc. Natl. Acad. Sci. USA. 1997; 94: 5594-5598Crossref PubMed Scopus (59) Google Scholar). Heparin oligomers greatly increase the in vitro half-life of LPL, suggesting that the association of LPL with vascular heparan sulfate proteoglycans is a factor that regulates in vivo LPL activity (8Merkel M. Loeffler B. Kluger M. Fabig N. Geppert G. Pennacchio L.A. Laatsch A. Heeren J. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase.J. Biol. Chem. 2005; 280: 21553-21560Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 14Kobayashi Y. Nakajima T. Inoue I. Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain.Eur. J. Biochem. 2002; 269: 4701-4710Crossref PubMed Scopus (64) Google Scholar, 15Lookene A. Zhang L. Hultin M. Olivecrona G. Rapid subunit exchange in dimeric lipoprotein lipase and properties of the inactive monomer.J. Biol. Chem. 2004; 279: 49964-49972Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 17Lutz E.P. Merkel M. Kako Y. Melford K. Radner H. Breslow J.L. Bensadoun A. Goldberg I.J. Heparin-binding defective lipoprotein lipase is unstable and causes abnormalities in lipid delivery to tissues.J. Clin. Invest. 2001; 107: 1183-1192Crossref PubMed Scopus (49) Google Scholar). More recently, angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), two proteins of the angiopoietin gene family, have been shown to inhibit LPL activity and to regulate triglyceride metabolism (5Li C. Genetics and regulation of angiopoietin-like proteins 3 and 4.Curr. Opin. Lipidol. 2006; 17: 152-156Crossref PubMed Scopus (62) Google Scholar, 18Desai U. Lee E.C. Chung K. Gao C. Gay J. Key B. Hansen G. Machajewski D. Platt K.A. Sands A.T. et al.Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice.Proc. Natl. Acad. Sci. USA. 2007; 104: 11766-11771Crossref PubMed Scopus (152) Google Scholar, 19Kathiresan S. Melander O. Guiducci C. Surti A. Burtt N.P. Rieder M.J. Cooper G.M. Roos C. Voight B.F. Havulinna A.S. et al.Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans.Nat. Genet. 2008; 40: 189-197Crossref PubMed Scopus (1142) Google Scholar, 20Kersten S. Regulation of lipid metabolism via angiopoietin-like proteins.Biochem. Soc. Trans. 2005; 33: 1059-1062Crossref PubMed Scopus (110) Google Scholar, 21Oike Y. Akao M. Kubota Y. Suda T. Angiopoietin-like proteins: potential new targets for metabolic syndrome therapy.Trends Mol. Med. 2005; 11: 473-479Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 22Romeo S. Pennacchio L.A. Fu Y. Boerwinkle E. Tybjaerg-Hansen A. Hobbs H.H. Cohen J.C. Population-based resequencing of ANGPTL4 uncovers variations that reduce triglycerides and increase HDL.Nat. Genet. 2007; 39: 513-516Crossref PubMed Scopus (429) Google Scholar, 23Willer C.J. Sanna S. Jackson A.U. Scuteri A. Bonnycastle L.L. Clarke R. Heath S.C. Timpson N.J. Najjar S.S. Stringham H.M. et al.Newly identified loci that influence lipid concentrations and risk of coronary artery disease.Nat. Genet. 2008; 40: 161-169Crossref PubMed Scopus (1330) Google Scholar). ANGPTL3 and ANGPTL4 are secreted proteins that contain a signal peptide, a coiled-coil domain, and a fibrinogen-like domain. The coiled-coil domain mediates the formation of higher order oligomers, which appear to be required for the LPL-inhibitory activity of ANGPTL3 and ANGPTL4. The mature protein is proteolytically cleaved between the coiled-coil domain and the fibrinogen-like domain to form an N-terminal fragment that is involved in LPL inhibition. The by which ANGPTL4 inhibits LPL involves the conversion of LPL from the active dimeric form to the inactive monomeric form, a process that appears to be irreversible. This inactivation process requires association of ANGPTL4 with LPL and is not appreciably inhibited by stabilizing concentrations of heparin (24Sukonina V. Lookene A. Olivecrona T. Olivecrona G. Angiopoietin-like protein 4 converts lipoprotein lipase to inactive monomers and modulates lipase activity in adipose tissue.Proc. Natl. Acad. Sci. USA. 2006; 103: 17450-17455Crossref PubMed Scopus (320) Google Scholar). ANGPTL4 is expressed primarily in adipose tissue and liver but is also expressed in cardiac muscle, skeletal muscle, and intestine under the control of peroxisome proliferator-activated receptors. ANGPTL3, in contrast, is expressed in the liver under the control of liver X receptors. ANGPTL3 is likely to function as an endocrine regulator that suppresses triglyceride clearance primarily in the fed state. ANGPTL4, in contrast, is likely to function as an autocrine or paracrine regulator as well as an endocrine regulator, preventing uptake of fatty acids from plasma triglyceride sources, particularly in the fasted state (5Li C. Genetics and regulation of angiopoietin-like proteins 3 and 4.Curr. Opin. Lipidol. 2006; 17: 152-156Crossref PubMed Scopus (62) Google Scholar, 20Kersten S. Regulation of lipid metabolism via angiopoietin-like proteins.Biochem. Soc. Trans. 2005; 33: 1059-1062Crossref PubMed Scopus (110) Google Scholar, 21Oike Y. Akao M. Kubota Y. Suda T. Angiopoietin-like proteins: potential new targets for metabolic syndrome therapy.Trends Mol. Med. 2005; 11: 473-479Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Together, these two proteins likely play a role in regulating triglyceride metabolism largely by inhibiting LPL. Recently, Beigneux et al. (25Beigneux A.P. Davies B.S. Gin P. Weinstein M.M. Farber E. Qiao X. Peale F. Bunting S. Walzem R.L. Wong J.S. et al.Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.Cell Metab. 2007; 5: 279-291Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar) and Young et al. (26Young S.G. Davies B.S. Fong L.G. Gin P. Weinstein M.M. Bensadoun A. Beigneux A.P. GPIHBP1: an endothelial cell molecule important for the lipolytic processing of chylomicrons.Curr. Opin. Lipidol. 2007; 18: 389-396Crossref PubMed Scopus (74) Google Scholar) have shown that LPL and the endogenous substrate CM associate with glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) (27Ioka R.X. Kang M.J. Kamiyama S. Kim D.H. Magoori K. Kamataki A. Ito Y. Takei Y.A. Sasaki M. Suzuki T. et al.Expression cloning and characterization of a novel glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein, GPI-HBP1.J. Biol. Chem. 2003; 278: 7344-7349Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). This protein attaches to the surface of endothelial cells of adipose tissue, cardiac muscle, and skeletal muscle by a glycosylphosphatidylinositol (GPI) anchor and has been proposed to function as a platform for LPL and its substrates, presumably increasing the efficiency of substrate hydrolysis and uptake of fatty acids by underlying tissues. This proposed function is consistent with the phenotype of Gpihbp1−/− mice, which have elevated plasma triglycerides, largely in the form of CM (25Beigneux A.P. Davies B.S. Gin P. Weinstein M.M. Farber E. Qiao X. Peale F. Bunting S. Walzem R.L. Wong J.S. et al.Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.Cell Metab. 2007; 5: 279-291Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). A mutant form of human GPIHBP1 that contains a Q115P substitution has been shown to be associated with chylomicronemia. This mutation alters the function of GPIHBP1, affecting its ability to bind with LPL and CM (28Beigneux A.P. Franssen R. Bensadoun A. Gin P. Melford K. Peter J. Walzem R.L. Weinstein M.M. Davies B.S. Kuivenhoven J.A. et al.Chylomicronemia with a mutant GPIHBP1 (Q115P) that cannot bind lipoprotein lipase.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 956-962Crossref PubMed Scopus (137) Google Scholar). Although LPL associates with GPIHBP1, it is not clear whether GPIHBP1 affects LPL activity. In this study, we examined whether GPIHBP1 affects LPL catalytic activity or interacts with LPL to alter its inhibition by ANGPTL3 or ANGPTL4. Bovine LPL and lipase substrate 1,2-O-dilauryl-rac-glycero-3-glutaric acid – (6’-methylresorufin) ester (DGGR) were purchased from Sigma-Aldrich (St. Louis, MO). Primers for PCR amplification and production of polyclonal antisera reactive with mouse GPIHBP1 were purchased from Sigma-Genosys (St. Louis, MO). cDNA for PCR amplification was purchased from CLONTECH Laboratories (Mountain View, CA). Ni-NTA agarose and HiSpeed Plasmid Maxi Kit were purchased from Qiagen (Valencia, CA). Pierce TMB substrate and Pierce Microplate BCA™ Protein Assay Kit–Reducing Agent Compatible were purchased from Thermo Scientific (Rockford, IL). HRP-conjugated goat anti-rabbit IgGs were purchased from Bethyl Laboratories (Montgomery, TX). Mouse anti-human LPL monoclonal IgG1 (clone A00090.01) was purchased from GeneScript (Piscataway, NJ). PCR amplification reactions were performed with Phusion DNA polymerase according to the recommendations supplied by the vendor (New England Biolabs, Ipswich, MA). A549 cells (CCL-185) were purchased from ATCC (Manassas, VA). HyQSFM4CHO serum-free medium was purchased from Hyclone (Logan, UT). F-12K medium was purchased from MediaTech (Manassas, VA). All other molecular biology techniques, such as restriction endonuclease digests, ligations, and agarose gel electrophoresis, were performed essentially as described in common manuals (29Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology.John Wiley & Sons New York, NY. 1998; Google Scholar, 30Sambrook J.E. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Recombinant human LPL was produced in 293F cells and catalytically active LPL was partially purified and concentrated from conditioned medium by heparin Sepharose affinity chromatography as described previously (31Lee E.C. Desai U. Gololobov G. Hong S. Feng X. Yu X.C. Gay J. Wilganowski N. Gao C. Du L.L. et al.Identification of a new functional domain in angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) involved in binding and inhibition of lipoprotein lipase (LPL).J. Biol. Chem. 2009; 284: 13735-13745Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Fractions containing active LPL were pooled, assayed for protein concentration, and stored at −80°C. Recombinant human ANGPTL3 (amino acids 17-221 [RefSeq accession NP_055310.1]) and ANGPTL4 (amino acids 26-176 [RefSeq accession NP_647475.1)], both possessing an N-terminal six-histidine tail, were expressed in Escherichia coli and subsequently purified by Ni-NTA affinity chromatography as described previously (31Lee E.C. Desai U. Gololobov G. Hong S. Feng X. Yu X.C. Gay J. Wilganowski N. Gao C. Du L.L. et al.Identification of a new functional domain in angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) involved in binding and inhibition of lipoprotein lipase (LPL).J. Biol. Chem. 2009; 284: 13735-13745Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The recombinant proteins were dialyzed against 50 mM sodium phosphate, 500 mM sodium chloride, 10% glycerol, 5 mM β-mercaptoethanol, pH 7.8, assayed for protein concentration, and stored at −80°C. A cDNA encoding mouse GPIHBP1 amino acids 1–198 (RefSeq accession NP_081006.1) with a C-terminal six-histidine tail was generated by PCR amplification with sense 39mer 5′-GCCACCAAGCTTAGCCACCATGAAGGCTCTCAGGGCTGT-3′ and antisense 54mer 5′-ACTAGTGGATCCTCAATGGTGATGGTGATGGTGTCCCTGGGGCTG GTTAGCCTT-3′. The cDNA was inserted into the Ad5 E1-deleted region of pAd5 under the control of hCMV promoter. Recombinant soluble GPIHBP1 adenovirus was generated, amplified, and purified as described previously (32Hitt M. Bett A.J. Prevec L. Graham F.L. Cell Biology: A Laboratory Handbook.in: Celis F.E. Academic Press. San Diego, CA.1998: 500-512Google Scholar). The purified recombinant adenovirus was confirmed by sequencing the cloning region and tested for infectious unit titer by plaque formation in HEK293 cells. A549 cells were infected with recombinant adenovirus at a multiplicity of infection of 100 in F-12K medium containing 10% fetal bovine serum. After incubating at 37°C overnight, the medium was replaced with HyQSFM4CHO serum-free medium. After incubating at 37°C for about 24 h, the condition medium containing mouse soluble GPIHBP1 protein was harvested, filtered through a 0.22-micron filter unit, and stored at −20°C. All procedures for purifying recombinant mouse GPIHBP1 were performed at 4°C. Conditioned medium containing GPIHBP1 was concentrated approximately 2-fold and then diafiltered against 8 vols of Buffer E (50 mM sodium phosphate, 500 mM sodium chloride, pH 7.8) using a GE Kvick Lab System fitted with three 0.11 m2 PES cassettes (5000 Da MWCO). The retentate was supplemented with imidazole to a concentration of 10 mM and applied to a 1-ml Ni-NTA column at a rate of 1 ml/min. The column was washed with 50 ml of Buffer E containing 20 mM imidazole and then with 12 ml of Buffer E containing 50 mM imidazole. GPIHBP1 was eluted from the column with Buffer E containing 250 mM imidazole. Eluate fractions were analyzed by SDS-PAGE and the fractions containing the peak recombinant protein were pooled, dialyzed against phosphate-buffered saline, and stored at −80°C. Production of affinity-purified rabbit polyclonal IgGs reactive with mouse GPIHBP1 amino acids 27–38 (DADPEPENYNYD) was ordered from Sigma-Genosys. Mouse monoclonal antibodies (mAbs) reactive with keyhole limpet hemocyanin (KLH) (control mAb KLH), ANGPTL4 (mAb 14D12) (18Desai U. Lee E.C. Chung K. Gao C. Gay J. Key B. Hansen G. Machajewski D. Platt K.A. Sands A.T. et al.Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice.Proc. Natl. Acad. Sci. USA. 2007; 104: 11766-11771Crossref PubMed Scopus (152) Google Scholar), and ANGPTL3 (mAb 5.50.3) (31Lee E.C. Desai U. Gololobov G. Hong S. Feng X. Yu X.C. Gay J. Wilganowski N. Gao C. Du L.L. et al.Identification of a new functional domain in angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) involved in binding and inhibition of lipoprotein lipase (LPL).J. Biol. Chem. 2009; 284: 13735-13745Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) were prepared as described previously. A 96-well Nunc MaxiSorp plate was coated with 1 μg/ml mouse anti-LPL monoclonal antibody in 0.2 M sodium carbonate buffer (pH 9.4) at 4°C overnight. The wells were washed three times with phosphate-buffered saline, 0.05% Tween-20 (PBST) and blocked with 5% human serum albumin in PBST at room temperature for 1 h. Bovine LPL (5.8 nM) was added to the wells and incubated at room temperature for 1 h. After washing the wells three times with PBST, purified mouse soluble GPIHBP1 at concentrations ranging between 0 and 40 nM was added to the wells and incubated at room temperature for 1 h. After washing the wells three times with PBST, rabbit anti-mouse GPIHBP1 peptide antibodies (10 μg/ml) were added and incubated at room temperature for 1 h. After washing three times with PBST, HRP-conjugated goat anti-rabbit antibodies (10 μg/ml) were added and incubated at room temperature for 1 h. The wells were washed three times with PBST and HRP activity was quantitated with TMB substrate according to the manufacturer's protocol. GPIHBP1 binding with LPL is expressed as absorbance at 450 nm. LPL activity was assayed with the fluorogenic substrate DGGR (33Panteghini M. Bonora R. Pagani F. Measurement of pancreatic lipase activity in serum by a kinetic colorimetric assay using a new chromogenic substrate.Ann. Clin. Biochem. 2001; 38: 365-370Crossref PubMed Scopus (86) Google Scholar) as described previously (31Lee E.C. Desai U. Gololobov G. Hong S. Feng X. Yu X.C. Gay J. Wilganowski N. Gao C. Du L.L. et al.Identification of a new functional domain in angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) involved in binding and inhibition of lipoprotein lipase (LPL).J. Biol. Chem. 2009; 284: 13735-13745Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). All experiments in which LPL activity was measured were performed in LPL assay buffer (50 mM Tris-HCl, 0.12 M sodium chloride, 0.5% Triton X-100, 10 mg/ml BSA, 1.5 mM calcium chloride, pH 7.4). Reactions were performed at room temperature in triplicate and were initiated by adding 90 ul of sample to 10 ul of 0.24 mM DGGR substrate. Hydrolysis of DGGR was measured at 30 s intervals over 10 min with a Cytofluor 4000 Fluorescence Multi-well Plate Reader (Applied Biosystems, Foster City, CA) fitted with a 530/25 nm excitation filter and a 620/40 nm emission filter. The rate of product formation is expressed as the change in relative fluorescence units (RFU) per minute. Under these assay conditions, LPL activity was linear up to at least 120 RFU/min. The initial concentration of active LPL (monomer) in the reaction mixture was approximately 10 nM. Generation of the Angptl4−/−, Angpt13−/−, and Gpihbp1−/− mouse lines4 has been described previously (18Desai U. Lee E.C. Chung K. Gao C. Gay J. Key B. Hansen G. Machajewski D. Platt K.A. Sands A.T. et al.Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice.Proc. Natl. Acad. Sci. USA. 2007; 104: 11766-11771Crossref PubMed Scopus (152) Google Scholar, 25Beigneux A.P. Davies B.S. Gin P. Weinstein M.M. Farber E. Qiao X. Peale F. Bunting S. Walzem R.L. Wong J.S. et al.Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.Cell Metab. 2007; 5: 279-291Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 31Lee E.C. Desai U. Gololobov G. Hong S. Feng X. Yu X.C. Gay J. Wilganowski N. Gao C. Du L.L. et al.Identification of a new functional domain in angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) involved in binding and inhibition of lipoprotein lipase (LPL).J. Biol. Chem. 2009; 284: 13735-13745Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Angptl3−/− mice were bred with Gpihbp1−/− mice to generate Angptl3−/−/Gpihbp1−/− mice. Angptl4−/− mice were bred with Gpihbp1−/− mice to generate Angptl4−/−/Gpihbp1−/− mice. All procedures involving animals were conducted in conformity with Institutional Animal Care and Use Committee guidelines in compliance with state and federal laws and the standards outlined in the Guide for the Care and Use of Laboratory Animals (35Institute of Laboratory Animal Research. C. o. L. S., National Research Council. 1996. Guide for the Care and Use of Laboratory Animals The National Academic Press, Washington, D.C.Google Scholar). Mice were housed at 24°C on a fixed 12 h light/12 h dark cycle and had free access to water and diet. All mice were maintained on regular chow (Cat# 5021, Purina, St. Louis, MO). Serum samples for lipid analysis were prepared from blood obtained from the retro-orbital plexus. Total TG levels were measured by kit (Serum TG determination kit, Cat# TR0100; Sigma-Aldrich). Calculations for determining EC50 values, IC50 values, and confidence intervals were determined by nonlinear regression analysis with sigmoidal dose-response (variable slope) equation. Binding constants (Kd) were determined by nonlinear regression analysis with one-site binding (hyperbola) equation. LPL activity decay was determined by nonlinear regression analysis with one-phase exponential decay equation (GraphPad Prism version 4.03 for Windows, GraphPad Software, San Diego, CA). Values for GPIHBP1 binding or LPL activity are expressed as the mean (± SEM) of triplicate determinations. Comparisons between two mouse groups were analyzed by unpaired Student's t-test. Comparisons among multiple mouse groups were analyzed by Kruskal-Wallis test followed by a posthoc test if statistical significance was less than 0.05. To investigate the interaction of GPIHBP1 with LPL activity in a chemically defined system, we produced a recombinant GPIHPB1 protein that was freely soluble in aqueous buffers. Mouse GPIHBP1 amino acids 199–228 and human GPIHBP1 amino acids 152–184 contain a C-terminal signal sequence for GPI anchoring (35Poisson G. Chauve C. Chen X. Bergeron A. FragAnchor: a large-scale predictor of glycosylphosphatidylinositol anchors in eukaryote protein sequences by qualitative scoring.Genomics Proteomics Bioinformat" @default.
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• W2149487645 title "GPIHBP1 stabilizes lipoprotein lipase and prevents its inhibition by angiopoietin-like 3 and angiopoietin-like 4" @default.
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