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• W1969352719 abstract "Endothelial lipase (EL) is a new member of the triglyceride lipase gene family, which includes lipoprotein lipase (LpL) and hepatic lipase (HL). Enzymatic activity of EL has been studied before. Here we characterized the ability of EL to bridge lipoproteins to the cell surface. Expression of EL in wild-type Chinese hamster ovary (CHO)-K1 but not in heparan sulfate proteoglycan (HSPG)-deficient CHO-677 cells resulted in 3–4.4-fold increases of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein 3 binding (HDL3). Inhibition of proteoglycan sulfation by sodium chlorate or incubation of cells with labeled lipoproteins in the presence of heparin (100 μg/ml) abolished bridging effects of EL. An enzymatically inactive EL, EL-S149A, was equally effective in facilitating lipoprotein bridging as native EL. Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound 125I-LDL was destined for internalization and degradation, whereas about 70% of the surface-bound 125I-HDL3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering. Endothelial lipase (EL) is a new member of the triglyceride lipase gene family, which includes lipoprotein lipase (LpL) and hepatic lipase (HL). Enzymatic activity of EL has been studied before. Here we characterized the ability of EL to bridge lipoproteins to the cell surface. Expression of EL in wild-type Chinese hamster ovary (CHO)-K1 but not in heparan sulfate proteoglycan (HSPG)-deficient CHO-677 cells resulted in 3–4.4-fold increases of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein 3 binding (HDL3). Inhibition of proteoglycan sulfation by sodium chlorate or incubation of cells with labeled lipoproteins in the presence of heparin (100 μg/ml) abolished bridging effects of EL. An enzymatically inactive EL, EL-S149A, was equally effective in facilitating lipoprotein bridging as native EL. Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound 125I-LDL was destined for internalization and degradation, whereas about 70% of the surface-bound 125I-HDL3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering. Lipoprotein lipase (LpL) 1The abbreviations used are: LpL, lipoprotein lipase; anti-apoA-I, polyclonal goat antibody against human apolipoprotein A-I; AdGFP, adenovirus encoding GFP; adEL, adenovirus encoding endothelial lipase; adEL-S149A, adenovirus encoding catalytically inactive endothelial lipase; AdHL, adenovirus encoding hepatic lipase; adLpL, adenovirus encoding lipoprotein lipase; apo, apolipoprotein; CHO, Chinese hamster ovary; EL, endothelial lipase; HL, hepatic lipase; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; HDL, high density lipoprotein; LDL, low-density lipoprotein; VLDL, very low density lipoprotein; TG, triglyceride; GFP, green fluorescent protein; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine.1The abbreviations used are: LpL, lipoprotein lipase; anti-apoA-I, polyclonal goat antibody against human apolipoprotein A-I; AdGFP, adenovirus encoding GFP; adEL, adenovirus encoding endothelial lipase; adEL-S149A, adenovirus encoding catalytically inactive endothelial lipase; AdHL, adenovirus encoding hepatic lipase; adLpL, adenovirus encoding lipoprotein lipase; apo, apolipoprotein; CHO, Chinese hamster ovary; EL, endothelial lipase; HL, hepatic lipase; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; HDL, high density lipoprotein; LDL, low-density lipoprotein; VLDL, very low density lipoprotein; TG, triglyceride; GFP, green fluorescent protein; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine. and hepatic lipase (HL), two members of the triglyceride lipase gene family, have well established roles in regulating lipid and lipoprotein metabolism and are implicated in atherosclerosis (1Murthy V. Julien P. Gagne C. Pharmacol. Ther. 1996; 70: 101-135Crossref PubMed Scopus (155) Google Scholar, 2Santamarina-Fojo S. Haudenschild C. Amar M. Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar, 3Wong H. Schotz M.C. J. Lipid Res. 2002; 43: 993-999Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). LpL is synthesized primarily in adipose and skeletal muscle and is transported to the endothelial surface, where it is bound to heparan sulfate proteoglycans (HSPGs). LpL is predominantly a triacylglycerol hydrolase, and its enzymatic action is mainly related to hydrolysis of triglycerides (TG) in TG-rich apolipoprotein B-containing lipoproteins, chylomicrons and very low density lipoproteins (VLDL) (reviewed in Ref. 4Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar). HL is synthesized primarily in hepatocytes (5Jansen H. Kalkman C. Zonneveld A.J. Hulsmann W.C. FEBS Lett. 1979; 98: 299-302Crossref PubMed Scopus (53) Google Scholar, 6Semenkovich C.F. Chen S.H. Wims M. Luo C.C. Li W.H. Chan L. J. Lipid Res. 1989; 30: 423-431Abstract Full Text PDF PubMed Google Scholar) and is bound mostly to hepatic and endothelial HSPGs in the hepatic sinusoids (7Sanan D.A. Fan J. Bensadoun A. Taylor J.M. J. Lipid Res. 1997; 38: 1002-1013Abstract Full Text PDF PubMed Google Scholar). Like LpL, HL has substantial TG lipase activity, but unlike LpL, HL also has significant phospholipase activity (8Deckelbaum R.J. Ramakrishnan R. Eisenberg S. Olivecrona T. Bengtsson-Olivecrona G. Biochemistry. 1992; 31: 8544-8551Crossref PubMed Scopus (65) Google Scholar). This increased phospholipase activity may play an important role in the ability of HL, as opposed to LpL, to directly modulate HDL metabolism (2Santamarina-Fojo S. Haudenschild C. Amar M. Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar).In addition to their lipolytic activities, LpL and HL have been shown to mediate “bridging” between lipoproteins and HSPGs on the cell surface, which results in increased cellular uptake and degradation of lipoproteins (9Robinson D.S. Borensztajn J. Lipoprotein Lipase. Evener, Chicago, IL1987: 1-13Google Scholar, 10Williams K.J. Fless G.M. Petrie K.A. Snyder M.L. Brocia R.W. Swenson T.L. J. Biol. Chem. 1992; 267: 13284-13292Abstract Full Text PDF PubMed Google Scholar, 11Rumsey S.C. Obunike J.C. Arad Y. Deckelbaum R.J. Goldberg I.J. J. Clin. Invest. 1992; 90: 1504-1512Crossref PubMed Scopus (154) Google Scholar, 12Eisenberg S. Sehayek E. Olivecrona T. Vlodavsky I. J. Clin. Invest. 1992; 90: 2013-2021Crossref PubMed Scopus (192) Google Scholar, 13Mulder M. Lombardi P. Jansen H. van Berkel T.J. Frants R.R. Havekes L.M. Biochem. Biophys. Res. Commun. 1992; 185: 582-587Crossref PubMed Scopus (87) Google Scholar, 14Fernandez-Borja M. Bellido D. Vilella E. Olivecrona G. Vilaro S. J. Lipid Res. 1996; 37: 464-481Abstract Full Text PDF PubMed Google Scholar, 15Ji Z.S. Lauer S.J. Fazio S. Bensadoun A. Taylor J.M. Mahley R.W. J. Biol. Chem. 1994; 269: 13429-13436Abstract Full Text PDF PubMed Google Scholar, 16Komaromy M. Azhar S. Cooper A.D. J. Biol. Chem. 1996; 271: 16906-16914Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In several studies LpL dramatically enhanced binding, internalization, and degradation of VLDL and LDL by cultured cells (10Williams K.J. Fless G.M. Petrie K.A. Snyder M.L. Brocia R.W. Swenson T.L. J. Biol. Chem. 1992; 267: 13284-13292Abstract Full Text PDF PubMed Google Scholar, 11Rumsey S.C. Obunike J.C. Arad Y. Deckelbaum R.J. Goldberg I.J. J. Clin. Invest. 1992; 90: 1504-1512Crossref PubMed Scopus (154) Google Scholar, 12Eisenberg S. Sehayek E. Olivecrona T. Vlodavsky I. J. Clin. Invest. 1992; 90: 2013-2021Crossref PubMed Scopus (192) Google Scholar, 13Mulder M. Lombardi P. Jansen H. van Berkel T.J. Frants R.R. Havekes L.M. Biochem. Biophys. Res. Commun. 1992; 185: 582-587Crossref PubMed Scopus (87) Google Scholar, 14Fernandez-Borja M. Bellido D. Vilella E. Olivecrona G. Vilaro S. J. Lipid Res. 1996; 37: 464-481Abstract Full Text PDF PubMed Google Scholar, 17Weaver A.M. Lysiak J.J. Gonias S.L. J. Lipid Res. 1997; 38: 1841-1850Abstract Full Text PDF PubMed Google Scholar, 18Seo T. St. Clair R.W. J. Lipid Res. 1997; 38: 765-779Abstract Full Text PDF PubMed Google Scholar). In contrast, LpL had relatively small, if any, effects on HDL binding and holoparticle uptake, but significantly increased selective uptake of cholesterol esters from HDL particles (19Panzenboeck U. Wintersberger A. Levak-Frank S. Zimmermann R. Zechner R. Kostner G.M. Malle E. Sattler W. J. Lipid Res. 1997; 38: 239-253Abstract Full Text PDF PubMed Google Scholar, 20Schorsch F. Malle E. Sattler W. FEBS Lett. 1997; 414: 507-513Crossref PubMed Scopus (13) Google Scholar, 21Rinninger F. Kaiser T. Mann W.A. Meyer N. Greten H. Beisiegel U. J. Lipid Res. 1998; 39: 1335-1348Abstract Full Text Full Text PDF PubMed Google Scholar, 22Rinninger F. Brundert M. Brosch I. Donarski N. Budzinski R.M. Greten H. J. Lipid Res. 2001; 42: 1740-1751Abstract Full Text Full Text PDF PubMed Google Scholar). Like LpL, HL has been shown to enhance binding and/or uptake of chylomicrons, chylomicron remnants, VLDL, and LDL by different cell types in vitro (15Ji Z.S. Lauer S.J. Fazio S. Bensadoun A. Taylor J.M. Mahley R.W. J. Biol. Chem. 1994; 269: 13429-13436Abstract Full Text PDF PubMed Google Scholar, 16Komaromy M. Azhar S. Cooper A.D. J. Biol. Chem. 1996; 271: 16906-16914Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Diard P. Malewiak M.I. Lagrange D. Griglio S. Biochem. J. 1994; 299: 889-894Crossref PubMed Scopus (84) Google Scholar, 24Krapp A. Ahle S. Kersting S. Hua Y. Kneser K. Nielsen M. Gliemann J. Beisiegel U. J. Lipid Res. 1996; 37: 926-936Abstract Full Text PDF PubMed Google Scholar) via a HSPG-dependent process (15Ji Z.S. Lauer S.J. Fazio S. Bensadoun A. Taylor J.M. Mahley R.W. J. Biol. Chem. 1994; 269: 13429-13436Abstract Full Text PDF PubMed Google Scholar, 16Komaromy M. Azhar S. Cooper A.D. J. Biol. Chem. 1996; 271: 16906-16914Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Diard P. Malewiak M.I. Lagrange D. Griglio S. Biochem. J. 1994; 299: 889-894Crossref PubMed Scopus (84) Google Scholar). In addition, in several studies HL efficiently increased holoparticle cellular uptake of HDL as well as selective uptake of cholesterol esters (16Komaromy M. Azhar S. Cooper A.D. J. Biol. Chem. 1996; 271: 16906-16914Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Ji Z.S. Dichek H.L. Miranda R.D. Mahley R.W. J. Biol. Chem. 1997; 272: 31285-31292Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 26Rinninger F. Mann W.A. Kaiser T. Ahle S. Meyer N. Greten H. Atherosclerosis. 1998; 141: 273-285Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). These bridging effects of LpL and HL require HSPGs and heparin-binding domains of the lipases but do not depend on their catalytical activities. There is also evidence for nonenzymatic effects of LpL and HL on lipoprotein metabolism in vivo (27Windler E. Greeve J. Robenek H. Rinninger F. Greten H. Jackle S. Hepatology. 1996; 24: 344-351Crossref PubMed Google Scholar, 28Merkel M. Kako Y. Radner H. Cho I.S. Ramasamy R. Brunzell J.D. Goldberg I.J. Breslow J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13841-13846Crossref PubMed Scopus (137) Google Scholar, 29Dichek H.L. Brecht W. Fan J. Ji Z.S. McCormick S.P. Akeefe H. Conzo L. Sanan D.A. Weisgraber K.H. Young S.G. Taylor J.M. Mahley R.W. J. Biol. Chem. 1998; 273: 1896-1903Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 30Dugi K.A. Amar M.J. Haudenschild C.C. Shamburek R.D. Bensadoun A. Hoyt Jr., R.F. Fruchart-Najib J. Madj Z. Brewer Jr., H.B. Santamarina-Fojo S. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 793-800Crossref PubMed Scopus (59) Google Scholar, 31Zambon A. Deeb S.S. Bensadoun A. Foster K.E. Brunzell J.D. J. Lipid Res. 2000; 41: 2094-2099Abstract Full Text Full Text PDF PubMed Google Scholar, 32Merkel M. Heeren J. Dudeck W. Rinninger F. Radner H. Breslow J.L. Goldberg I.J. Zechner R. Greten H. J. Biol. Chem. 2002; 277: 7405-7411Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).Endothelial lipase (EL), a 480-amino acid protein (M r ∼68,000), is a new member of the triglyceride lipase gene family (33Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (416) Google Scholar, 34Hirata K. Dichek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T. J. Biol. Chem. 1999; 274: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 35Rader D.J. Jaye M. Curr. Opin. Lipidol. 2000; 11: 141-147Crossref PubMed Scopus (84) Google Scholar, 36Cohen J.C. J. Clin. Invest. 2003; 111: 318-321Crossref PubMed Scopus (28) Google Scholar). Many typical features of this gene family are conserved in EL: the catalytic triad residues, the lid that controls access of substrate to the hydrolytic pocket, and the cysteine residues that form intramolecular disulfide bonds. EL is unique in the triglyceride lipase family in that it is synthesized by endothelial cells, however, a number of other cell types also express EL (33Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (416) Google Scholar, 34Hirata K. Dichek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T. J. Biol. Chem. 1999; 274: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). EL has detectable triglyceride lipase activity, but this activity is significantly less relative to its phospholipase activity compared with HL and especially LpL (33Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (416) Google Scholar, 37McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). In vitro, conditioned medium containing EL had the ability to substantially hydrolyze HDL phospholipids but had little activity toward LDL phospholipids, suggesting relative selectivity for HDL (37McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). In vivo, even low levels of EL overexpression in the livers of wild-type and human apoA-I transgenic mice dramatically reduced HDL cholesterol and apoA-I levels and increased HDL catabolism (33Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (416) Google Scholar). As a result of EL overexpression, plasma levels of apoB-containing lipoproteins were also reduced, although to a lesser extent (33Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (416) Google Scholar). Recent studies in EL knockout mice (38Ishida T. Choi S. Kundu R.K. Hirata K. Rubin E.M. Cooper A.D. Quertermous T. J. Clin. Invest. 2003; 111: 347-355Crossref PubMed Scopus (265) Google Scholar) and in mice injected with specific antibody against EL (39Jin W. Millar J.S. Broedl U. Glick J.M. Rader D.J. J. Clin. Invest. 2003; 111: 357-362Crossref PubMed Scopus (197) Google Scholar) provided additional evidence for a physiological importance of EL for lipid and lipoprotein metabolism in vivo.The clusters of positively charged residues in LpL and HL have been implicated in binding of these molecules to heparin and HSPGs. Because the putative heparin-binding and lipoprotein-binding sites present in LpL and HL are highly conserved in EL, we hypothesized that EL may also serve as a bridging molecule between lipoproteins and cell surface and matrix HSPGs. Therefore, the primary focus of this study was to examine the ability of EL to mediate binding and holoparticle uptake of plasma lipoproteins and to compare EL-dependent metabolism of apoB-versus apoA-I-containing lipoproteins in vitro. We also compared EL with HL and LpL in their abilities to facilitate bridging of different major classes of plasma lipoproteins with cells. In this study, we demonstrated that EL can function as an efficient bridging molecule between plasma lipoproteins and cells in a process that requires intact cell surface HSPGs, but does not depend on EL enzymatic activity. Moreover, we found that compared with LpL and HL, EL has distinct preferences in bridging individual classes of lipoproteins.EXPERIMENTAL PROCEDURESPreparation of Reagents—Unless otherwise indicated, chemicals of analytical grade were purchased from Sigma. VLDL (d < 1.006 g/ml), LDL (1.019 < d < 1.063 g/ml), and HDL3 (1.125 < d < 1.21 g/ml) were isolated from fresh human plasma by ultracentrifugation as described previously (40Fuki I.V. Kuhn K.M. Lomazov I.R. Rothman V.L. Tuszynski G.P. Iozzo R.V. Swenson T.L. Fisher E.A. Williams K.J. J. Clin. Invest. 1997; 100: 1611-1622Crossref PubMed Scopus (206) Google Scholar). 125I-Labeled VLDL (125I-VLDL), 125I-labeled LDL (125I-LDL), and 125I-labeled HDL3 (125I-HDL) were iodinated using the iodine monochloride method (41Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1277) Google Scholar). DiI-labeled HDL3 was purchased from Intracel Corp. (Rockville, MD). Polyclonal goat antibody against apolipoprotein A-I (anti-apoA-I), which are able to cross-link human HDL, were obtained from Wako Chemical USA, Inc. (Richmond, VA).Cultured Cells—COS-7 and two types of Chinese hamster ovary cell lines, wild-type CHO-K1 and the CHO mutant line pgs d-677 (CHO-677), which is specifically deficient in both N-acetylglucosaminyltransferase and glucuronosyltransferase activities and hence lacks heparan sulfate (42Esko J.D. Curr. Opin. Cell Biol. 1991; 3: 805-816Crossref PubMed Scopus (183) Google Scholar, 43Lidholt K. Weinke J.L. Kiser C.S. Lugemwa F.N. Bame K.J. Cheifetz S. Massague J. Lindahl U. Esko J.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2267-2271Crossref PubMed Scopus (234) Google Scholar), were obtained from the American Type Culture Collection (Manassas, VA). COS-7 cells were grown and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Both CHO cell lines were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum. These three cell lines were chosen because they are readily infected with recombinant adenoviral constructs and were successfully used in the past to characterize effects of LpL and HL on lipoprotein metabolism (16Komaromy M. Azhar S. Cooper A.D. J. Biol. Chem. 1996; 271: 16906-16914Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 37McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar, 44Berryman D.E. Bensadoun A. J. Biol. Chem. 1995; 270: 24525-24531Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 45Fuki I.V. Iozzo R.V. Williams K.J. J. Biol. Chem. 2000; 275: 25742-25750Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar).Expression of Enzymes—Wild-type EL or enzymatically inactive EL, in which the active site serine was substituted with alanine (EL-S149A), LpL, and HL were expressed using recombinant adenoviruses. Recombinant adenoviruses encoding human EL (AdEL), human EL-S149A (adEL-S149A), human HL (AdHL), or GFP (AdGFP), used as a negative control in all experiments, were constructed as previously described (37McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). A recombinant adenovirus encoding human LpL (AdLpL) was a generous gift from Dr. Nicolas Duverger (Aventis Pharmaceuticals).Adenoviral infections of cells were performed as described (37McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, prior to infection cells were grown to 80–90% confluence in 12-well plates (22 mm/well). Cells were then incubated with recombinant adenoviruses encoding EL, EL-S149A, LpL, HL, or GFP (control virus) in 0.3 ml of serum-free medium at a multiplicity of infection of 3000 particles/cell. Two hours later, 0.7 ml of fresh growth medium containing serum was added to each well and incubations were continued for 2 days. At 48 h post-infection, cells were washed twice with phosphate-buffered saline and used for ligand binding experiments with 125I-labeled lipoproteins.Expression of lipases by infected cells was confirmed by Western blotting and by assay of triglyceride lipase activity of conditioned medium according to the protocols described previously (37McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). Antibody against human EL was generated to a peptide in the N-terminal region of EL, as previously described (33Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (416) Google Scholar). Antibody to rat HL that cross-reacts with human HL was also previously described (46Laposata E.A. Laboda H.M. Glick J.M. Strauss III, J.F. J. Biol. Chem. 1987; 262: 5333-5338Abstract Full Text PDF PubMed Google Scholar). A polyclonal antibody to human LpL was a generous gift from Dr. Mark H. Doolittle.Cellular Metabolism of 125I-VLDL, 125I-LDL, and 125I-HDL3—For binding studies, cells grown in 12-well plates were incubated with 0.5 ml of the serum-free medium supplemented with 0.2% bovine serum albumin (Sigma number A-8806) and 125I-labeled lipoproteins (5 μg of protein/ml) unless otherwise stated. To assess the effects of endogenous expression of lipases on surface binding of lipoproteins, cells were incubated with labeled lipoproteins for 1 h at 4 °C. To measure cellular uptake and degradation of lipoproteins, cells were incubated for 3 h at 37 °C in the presence of labeled lipoproteins, then surface-bound (heparin-releasable), intracellular (heparin-resistant), and degraded (assessed by trichloroacetic acid-soluble, CHCl3-insoluble radioactivity in medium) ligand was measured as described (40Fuki I.V. Kuhn K.M. Lomazov I.R. Rothman V.L. Tuszynski G.P. Iozzo R.V. Swenson T.L. Fisher E.A. Williams K.J. J. Clin. Invest. 1997; 100: 1611-1622Crossref PubMed Scopus (206) Google Scholar, 41Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1277) Google Scholar).To examine the time course of cellular processing of 125I-LDL versus 125I-HDL3, labeled lipoproteins were incubated with cells in serum-free medium for 1 h at 4 °C to allow surface binding without further catabolism. Cells were washed at 4 °C to remove unbound material. Fresh medium at 37 °C with no ligands was then added, and incubations were continued at 37 °C for the indicated times. Assays for surface-bound, intracellular, and degraded ligands were then performed as above. Additionally, trichloroacetic acid-precipitable radioactivity in the medium was quantified as an indication of lipoproteins released into the medium via retroendocytosis or desorption from the cell surface during the incubation at 37 °C.To test potential effects of ligand clustering on cellular metabolism of 125I-HDL3, cells were incubated with 125I-HDL3 at 4 °C for 1 h, to allow cell-surface binding. Cells were rinsed at 4 °C to remove unbound ligand and incubated for an additional 30 min at 4 °C with or without antibody against human apoA-I (10 μg/ml final concentration). Cells were then washed briefly and incubated with pre-warmed media at 37 °C for the indicated times followed by measurements of surface-bound, intracellular, degraded and medium released radioactivity.All results for 125I-lipoprotein metabolism were normalized to cellular protein. Lipase-dependent catabolism was calculated by subtracting the values obtained in control cells infected with adGFP from those obtained in cells expressing lipases (total catabolism). HSPG-mediated catabolism was calculated by subtracting the values obtained in the presence of heparin (100 μg/ml) from those obtained in its absence.Assay of EL Protein—In parallel with ligand binding experiments, a separate set of cells was used to measure EL or EL-S149A protein available for bridging of labeled lipoproteins. For this purpose, cells were not exposed to radiolabeled lipoproteins, but instead were incubated in the presence of heparin (100 μg/ml) to release surface-bound lipases into the medium. EL protein concentration was evaluated by densitometric analysis of Western blots of the conditioned medium using a Bio-Rad imager (model GS-700) and Quantity One program (Bio-Rad). Protein concentration was defined in arbitrary units as determined by densitometric analysis. Several dilutions of conditioned medium were used to make sure the intensity of the bands was proportional to the amount of protein applied on the gel.Role of HSPGs in Lipoprotein Metabolism—We used three approaches to test the contribution of HSPGs in EL-mediated processing of lipoproteins. First, we blocked sulfation of cellular proteoglycans by preincubating cells for 18 h at 37 °C in sodium chlorate (50 mm), an inhibitor of sulfate adenyltransferase (47Hoogewerf A.J. Cisar L.A. Evans D.C. Bensadoun A. J. Biol. Chem. 1991; 266: 16564-16571Abstract Full Text PDF PubMed Google Scholar), thus preventing sulfation of glycosaminoglycan side chains of HSPGs. Control cells were exposed to 50 mm sodium chloride. Second, we compared lipoprotein metabolism in wild-type versus HSPG-deficient CHO cells. Third, we incubated cells with labeled lipoproteins in the presence or absence of heparin at 100 μg/ml, a concentration that specifically blocks interactions with HSPGs (40Fuki I.V. Kuhn K.M. Lomazov I.R. Rothman V.L. Tuszynski G.P. Iozzo R.V. Swenson T.L. Fisher E.A. Williams K.J. J. Clin. Invest. 1997; 100: 1611-1622Crossref PubMed Scopus (206) Google Scholar), but not lipoprotein binding to the members of the LDL receptor family or other lipoprotein receptors (48Goldstein J.L. Basu S.K. Brunschede G.Y. Brown M.S. Cell. 1976; 7: 85-95Abstract Full Text PDF PubMed Scopus (357) Google Scholar).Statistical Analyses—All results are displayed as mean ± S.E., n = 3. Error bars that appear absent indicate S.E. values smaller than the drawn symbols.RESULTSEffects of EL Expression on Binding of 125I-LDL and 125I-HDL3—We examined the effects of EL on the binding of 125I-LDL and 125I-HDL3 in CHO-K1 cells. For these experiments CHO-K1 cells were infected with control (GFP) or EL-encoding adenoviral constructs. 48 h after infection, cells were incubated with radiolabeled 125I-LDL or 125I-HDL3 for1hat4 °C, and cell binding of labeled lipoproteins was measured as described under “Experimental Procedures.” Expression of EL was assayed in parallel wells by Western blotting with anti-EL antibody and by measuring triglyceride lipase activity in the conditioned medium. Infection with the control virus (adGFP) did not affect binding of either 125I-LDL or 125I-HDL3 compared with uninfected cells (91.3 ± 4.8 and 95.6 ± 7.4% of values for uninfected cells, respectively). In contrast, cells infected with adEL demonstrated 4.4-fold increase in binding of 125I-LDL (Fig. 1A) and 3.0-fold increase in binding of 125I-HDL3 (Fig. 1B) compared with control, adGFP-infected cells. Of interest, although 125I-LDL or 125I-HDL3 were added to the cells at equal protein concentrations, about 2.3 times more 125I-LDL protein was bound to the cells compared with 125I-HDL3 via the EL-mediated process (114.3 ± 7.6 versus 49.4 ± 2.3 ng/mg of cell protein, respectively) indicating that a higher percentage of LDL particles was bound to the cells compared with HDL.Role of Cell Surface HSPGs in EL-mediated Increase of 125I-LDL and 125I-HDL3 Metabolism—We next tested if cell surface HSPGs are responsible for the observed effects of EL on binding of lipoproteins by incubating cells with labeled lipoproteins in the presence of 100 μg of heparin/ml. At this concentration heparin selectively blocks ligand binding to heparan sulfate side chains of HSPGs (40Fuki I.V. Kuhn K.M. Lomazov I.R. Rothman V.L. Tuszynski G.P. Iozzo R.V. Swenson T.L. Fisher E.A. Williams K.J. J. Clin. Invest. 1997; 100: 1611-1622Crossref PubMed Scopus (206) Google Scholar) but not lipoprotein binding to the members of the LDL receptor family or other lipoprotein receptors (48Goldstein J.L. Basu S.K. Brunschede G.Y. Brown M.S. Cell. 19" @default.
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• W1969352719 title "Endogenously Produced Endothelial Lipase Enhances Binding and Cellular Processing of Plasma Lipoproteins via Heparan Sulfate Proteoglycan-mediated Pathway" @default.
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