The amino acid sequences of the carboxyl termini of human and mouse hepatic lipase influence cell surface association
2003; Elsevier BV; Volume: 44; Issue: 7 Linguagem: Inglês
10.1194/jlr.m200374-jlr200
ISSN1539-7262
AutoresRobert Brown, Joshua R. Schultz, Kerry W.S. Ko, John S. Hill, Tanya A. Ramsamy, Ann L. White, Daniel L. Sparks, Zemin Yao,
Tópico(s)Cannabis and Cannabinoid Research
ResumoHuman hepatic lipase (hHL) mainly exists cell surface bound, whereas mouse HL (mHL) circulates in the blood stream. Studies have suggested that the carboxyl terminus of HL mediates cell surface binding. We prepared recombinant hHL, mHL, and chimeric proteins (hHLmt and mHLht) in which the carboxyl terminal 70 amino acids of hHL were exchanged with the corresponding sequence from mHL. The hHL, mHL, and hHLmt proteins were catalytically active using triolein and tributyrin as substrates. In transfected cells, the majority of hHLs bound to the cell surface, with only 4% of total extracellular hHL released into heparin-free media, whereas under the same conditions, 61% of total extracellular mHLs were released. Like mHL, hHLmt showed decreased cell surface binding, with 68% of total extracellular hHLmt released. To determine the precise amino acid residues involved in cell surface binding, we prepared a truncated hHL mutant (hHL471) by deleting the carboxyl terminal five residues (KRKIR). The hHL471 also retained hydrolytic activity with triolein and tributyrin, and showed decreased cell surface binding, with 40% of total extracellular protein released into the heparin-free media.These data suggest that the determinants of cell surface binding exist within the carboxyl terminal 70 amino acids of hHL, of which the last five residues play an important role. Human hepatic lipase (hHL) mainly exists cell surface bound, whereas mouse HL (mHL) circulates in the blood stream. Studies have suggested that the carboxyl terminus of HL mediates cell surface binding. We prepared recombinant hHL, mHL, and chimeric proteins (hHLmt and mHLht) in which the carboxyl terminal 70 amino acids of hHL were exchanged with the corresponding sequence from mHL. The hHL, mHL, and hHLmt proteins were catalytically active using triolein and tributyrin as substrates. In transfected cells, the majority of hHLs bound to the cell surface, with only 4% of total extracellular hHL released into heparin-free media, whereas under the same conditions, 61% of total extracellular mHLs were released. Like mHL, hHLmt showed decreased cell surface binding, with 68% of total extracellular hHLmt released. To determine the precise amino acid residues involved in cell surface binding, we prepared a truncated hHL mutant (hHL471) by deleting the carboxyl terminal five residues (KRKIR). The hHL471 also retained hydrolytic activity with triolein and tributyrin, and showed decreased cell surface binding, with 40% of total extracellular protein released into the heparin-free media. These data suggest that the determinants of cell surface binding exist within the carboxyl terminal 70 amino acids of hHL, of which the last five residues play an important role. Mature human hepatic lipase (hHL) contains 476 amino acids, and its apparent molecular mass varies from 55 to 69 kDa (1Stahnke G. Sprengel R. Augustin J. Will H. Human hepatic triglyceride lipase: cDNA, cloning, amino acid sequence and expression in a cultured cell line.Differentiation. 1987; 35: 45-52Google Scholar, 2Datta S. Luo C.C. Li W.H. VanTuinen P. Ledbetter D.H. Brown M.A. Chen S.H. Liu S.W. Chan L. Human hepatic lipase. Cloned cDNA sequence, restriction fragment length polymorphisms, chromosomal locations, and evolutionary relationships with lipoprotein lipase and pancreatic lipase.J. Biol. Chem. 1988; 263: 1107-1110Google Scholar, 3Martin G.A. Busch S.J. Meredith G.D. Cardin A.D. Blankenship D.T. Mao S.J. Rechtin A.E. Woods C.W. Racke M.M. Schafer M.P. Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase.J. Biol. Chem. 1988; 263: 10907-10914Google Scholar), presumably owing to variation in the magnitude of glycosylation at its four N-linked glycosylation sites. hHL is a member of a superfamily of lipases and phospholipases (EC 3.1.1.3) that share the GxSxG motif at the active site and a catalytic Asp-His-Ser charge relay triad found in a typical serine hydrolase (4Davis R.C. Stahnke G. Wong H. Doolittle M.H. Ameis D. Will H. Schotz M.C. Hepatic lipase: site-directed mutagenesis of a serine residue important for catalytic activity.J. Biol. Chem. 1990; 265: 6291-6295Google Scholar). Known members of this superfamily include lipoprotein lipase (LPL), endothelial lipase, pancreatic lipase, and HL (1Stahnke G. Sprengel R. Augustin J. Will H. Human hepatic triglyceride lipase: cDNA, cloning, amino acid sequence and expression in a cultured cell line.Differentiation. 1987; 35: 45-52Google Scholar, 2Datta S. Luo C.C. Li W.H. VanTuinen P. Ledbetter D.H. Brown M.A. Chen S.H. Liu S.W. Chan L. Human hepatic lipase. Cloned cDNA sequence, restriction fragment length polymorphisms, chromosomal locations, and evolutionary relationships with lipoprotein lipase and pancreatic lipase.J. Biol. Chem. 1988; 263: 1107-1110Google Scholar, 3Martin G.A. Busch S.J. Meredith G.D. Cardin A.D. Blankenship D.T. Mao S.J. Rechtin A.E. Woods C.W. Racke M.M. Schafer M.P. Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase.J. Biol. Chem. 1988; 263: 10907-10914Google Scholar, 5Hide W.A. Chan L. Li W.H. Structure and evolution of the lipase superfamily.J. Lipid Res. 1992; 33: 167-178Google Scholar, 6Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Google Scholar, 7Hirata K. Dichek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T. Cloning of a unique lipase from endothelial cells extends the lipase gene family.J. Biol. Chem. 1999; 274: 14170-14175Google Scholar). The active hHL is a homodimer (8Hill J.S. Davis R.C. Yang D. Wen J. Philo J.S. Poon P.H. Phillips M.L. Kempner E.S. Wong H. Human hepatic lipase subunit structure determination.J. Biol. Chem. 1996; 271: 22931-22936Google Scholar) with broad substrate specificity and is involved in the metabolism of HDLs and triacylglycerol (TG)-rich lipoproteins (9Busch S.J. Barnhart R.L. Martin G.A. Fitzgerald M.C. Yates M.T. Mao S.J. Thomas C.E. Jackson R.L. Human hepatic triglyceride lipase expression reduces high density lipoprotein and aortic cholesterol in cholesterol-fed transgenic mice.J. Biol. Chem. 1994; 269: 16376-16382Google Scholar, 10Huff M.W. Sawyez C.G. Connelly P.W. Maguire G.F. Little J.A. Hegele R.A. Beta-VLDL in hepatic lipase deficiency induces apoE-mediated cholesterol ester accumulation in macrophages.Arterioscler. Thromb. 1993; 13: 1282-1290Google Scholar, 11Jensen G.L. Daggy B. Bensadoun A. Triacylglycerol lipase, monoacylglycerol lipase, and phospholipase activities of highly purified rat hepatic lipase.Biochim. Biophys. Acta. 1982; 710: 464-470Google Scholar, 12Shafi S. Brady S.E. Bensadoun A. Havel R.J. Role of hepatic lipase in the uptake and processing of chylomicron remnants in rat liver.J. Lipid Res. 1994; 35: 709-720Google Scholar, 13Homanics G.E. Silva H.V.de Osada J. Zhang S.H. Wong H. Borensztajn J. Maeda N. Mild dyslipidemia in mice following targeted inactivation of the hepatic lipase gene.J. Biol. Chem. 1995; 270: 2974-2980Google Scholar). A series of studies have been conducted to compare heparin binding, enzyme activity, and substrate specificities between HL and LPL (14Wong H. Davis R.C. Nikazy J. Seebart K.E. Schotz M.C. Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase.Proc. Natl. Acad. Sci. USA. 1991; 88: 11290-11294Google Scholar –18Davis R.C. Wong H. Nikazy J. Wang K. Han Q. Schotz M.C. Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.J. Biol. Chem. 1992; 267: 21499-21504Google Scholar). The amino acid sequences of HL and LPL show close sequence homology to that of pancreatic lipase. Thus, based on the known structure of pancreatic lipase showing a two-domain structure, HL and LPL have been modeled into two domains with distinct functions. The active sites of HL and LPL reside within the respective N-terminal domains. Relative to TG hydrolysis, HL displays higher phospholipase activity than LPL. The substrate specificity of HL and LPL is governed by a 22-amino acid loop (“lid”) within the N-terminal domain of the respective lipases (15Dugi K.A. Dichek H.L. Santamarina-Fojo S. Human hepatic and lipoprotein lipase: the loop covering the catalytic site mediates lipase substrate specificity.J. Biol. Chem. 1995; 270: 25396-25401Google Scholar). The activity of LPL requires the cofactor apolipoprotein C-II (apoC-II) and is sensitive to high salt (i.e., 1 M NaCl), whereas the activity of HL is independent of apoC-II and remains active at 1 M NaCl. Studies with a chimeric protein composed of amino acid sequences derived from human LPL and hHL have shown that the catalytic properties and high-salt sensitivity of LPL are determined by the N-terminal domain (17Dichek H.L. Parrott C. Ronan R. Brunzell J.D. Brewer H.B. Santamarina-Fojo S. Functional characterization of a chimeric lipase genetically engineered from human lipoprotein lipase and human hepatic lipase.J. Lipid Res. 1993; 34: 1393-1401Google Scholar), whereas the apoC-II activation of LPL probably involves both the N-terminal (16Hill J.S. Yang D. Nikazy J. Curtiss L.K. Sparrow J.T. Wong H. Subdomain chimeras of hepatic lipase and lipoprotein lipase. Localization of heparin and cofactor binding.J. Biol. Chem. 1998; 273: 30979-30984Google Scholar, 18Davis R.C. Wong H. Nikazy J. Wang K. Han Q. Schotz M.C. Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.J. Biol. Chem. 1992; 267: 21499-21504Google Scholar) and carboxyl terminal domains of LPL (16Hill J.S. Yang D. Nikazy J. Curtiss L.K. Sparrow J.T. Wong H. Subdomain chimeras of hepatic lipase and lipoprotein lipase. Localization of heparin and cofactor binding.J. Biol. Chem. 1998; 273: 30979-30984Google Scholar). Studies with chimeric proteins have also suggested that the carboxyl terminal domains of HL and LPL play a role in determining their heparin affinity. The affinity of HL and LPL toward heparin can be estimated from the concentration of NaCl required to elute the lipases from immobilized heparin: 1.1 M NaCl to elute LPL and 0.75 M NaCl to elute HL (18Davis R.C. Wong H. Nikazy J. Wang K. Han Q. Schotz M.C. Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.J. Biol. Chem. 1992; 267: 21499-21504Google Scholar). The heparin affinity of LPL is thus higher than that of HL. For chimeric proteins in which the carboxyl terminal sequences of human LPL were substituted with either rat (18Davis R.C. Wong H. Nikazy J. Wang K. Han Q. Schotz M.C. Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.J. Biol. Chem. 1992; 267: 21499-21504Google Scholar) or hHL (17Dichek H.L. Parrott C. Ronan R. Brunzell J.D. Brewer H.B. Santamarina-Fojo S. Functional characterization of a chimeric lipase genetically engineered from human lipoprotein lipase and human hepatic lipase.J. Lipid Res. 1993; 34: 1393-1401Google Scholar) sequences, reduced heparin binding affinity of the chimeric proteins was observed. From these studies, it has been suggested that the major determinant of heparin binding resides within the carboxyl terminal domain of hHL. The majority of hHL activity and mass is found in the liver. Immunocytochemistry studies have revealed that hHL is located on the sublumenal extracellular matrix component of endothelium, the microvillar surface of hepatocytes in the space of Disse, interhepatocyte space, and lumenal surface of sinusoidal endothelium (19Sanan D.A. Fan J. Bensadoun A. Taylor J.M. Hepatic lipase is abundant on both hepatocyte and endothelial cell surfaces in the liver.J. Lipid Res. 1997; 38: 1002-1013Google Scholar). Infusing heparin in vivo increases HL activity in the serum by 1,000-fold, suggesting an interaction of hHL with heparan sulfate proteoglycans (HSPGs) (20Peterson J. Bengtsson-Olivecrona G. Olivecrona T. Mouse preheparin plasma contains high levels of hepatic lipase with low affinity for heparin.Biochim. Biophys. Acta. 1986; 878: 65-70Google Scholar). In contrast to that of hHL (21Schoonderwoerd K. Verhoeven A.J. Jansen H. Rat liver contains a limited number of binding sites for hepatic lipase.Biochem. J. 1994; 302: 717-722Google Scholar), the major proportion of mouse HL (mHL) mass and activity (60–70% of total) circulates in the plasma (20Peterson J. Bengtsson-Olivecrona G. Olivecrona T. Mouse preheparin plasma contains high levels of hepatic lipase with low affinity for heparin.Biochim. Biophys. Acta. 1986; 878: 65-70Google Scholar). The low affinity of mHL to the cell surface could be attributable to the lack of glycosaminoglycans specific for HL binding that are present in humans but not in mice. Alternatively, the low affinity of mHL could be due to the lack of amino acid sequence elements that are present in hHL but not in mHL. Two pieces of evidence suggest that the determinants of HSPG binding lie within the amino acid sequence of HL. First, infusion of hHL into mice resulted in nearly a 100% association of hHL mass and activity with the cell surface (20Peterson J. Bengtsson-Olivecrona G. Olivecrona T. Mouse preheparin plasma contains high levels of hepatic lipase with low affinity for heparin.Biochim. Biophys. Acta. 1986; 878: 65-70Google Scholar). Second, mHL has a lower affinity for heparin versus hHL, as demonstrated by its earlier elution from heparin-Sepharose; 0.7–0.8 M NaCl was required to elute hHL, and 0.48 M NaCl for mHL (20Peterson J. Bengtsson-Olivecrona G. Olivecrona T. Mouse preheparin plasma contains high levels of hepatic lipase with low affinity for heparin.Biochim. Biophys. Acta. 1986; 878: 65-70Google Scholar). The interaction of heparin with proteins containing HSPG binding motifs is presumably mediated by an electrostatic interaction (22Cardin A.D. Weintraub H.J. Molecular modeling of protein-glycosaminoglycan interactions.Arteriosclerosis. 1989; 9: 21-32Google Scholar) that can be interrupted by increasing salt concentrations. In addition to electrostatic forces, it is thought that a characteristic steric fit is also essential for specific binding to heparin (23Cole G.J. Akeson R. Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides.Neuron. 1989; 2: 1157-1165Google Scholar, 24Marcum J.A. Rosenberg R.D. Role of endothelial cell surface heparin-like polysaccharides.Ann. N. Y. Acad. Sci. 1989; 556: 81-94Google Scholar, 25Lellouch A.C. Lansbury Jr., P.T. A peptide model for the heparin binding site of antithrombin III.Biochemistry. 1992; 31: 2279-2285Google Scholar). Common structural motifs were identified when sequences of a number of heparin binding proteins were compared (22Cardin A.D. Weintraub H.J. Molecular modeling of protein-glycosaminoglycan interactions.Arteriosclerosis. 1989; 9: 21-32Google Scholar); they are xBBxBx, xBxxBBBx, and xBBxxBBBxxBBx, respectively (B: basic amino acids). In the case of antithrombin III and apoE, a distinct distribution of two basic amino acids located about 20 Å apart (xBxxxxxxxxxxxxBx in α-helix and xBxxxxxxBx in β-strand) has been suggested to be critical in HSPG binding (26Margalit H. Fischer N. Ben-Sasson S.A. Comparative analysis of structurally defined heparin binding sequences reveals a distinct spacial distribution of basic residues.J. Biol. Chem. 1993; 268: 19228-19231Google Scholar). In the current study, we have tested the hypothesis that the carboxyl terminal region of hHL confers high-affinity cell surface binding activity through interaction with HSPG using chimeric HL proteins. Our data support the notion that the difference in cell surface association between hHL and mHL is caused by the divergence in the carboxyl terminal amino acids between the two proteins. Triolein, tributyrin, fatty acid-free BSA, heparin, and fetal bovine serum (FBS) were purchased from Sigma. Dulbecco's modified Eagle medium (DMEM), Ham's F12 medium, and G418 were purchased from Gibco BRL. The Hi-Trap heparin-Sepharose columns, HRP-conjugated goat anti-mouse IgG antibody, HRP-conjugated goat anti-rabbit IgG antibody, and [35S]methionine/cysteine were purchased from Amersham Pharmacia Biotech. [3H]triolein was purchased from Dupont. [14C]tributyrin was purchased from American Radiolabeled Chemicals, Inc. Restriction endonucleases were purchased from New England Biolabs. A polyclonal anti-hHL antibody (27Boedeker J.C. Doolittle M. Santamarina-Fojo S. White A.L. Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion.J. Lipid Res. 1999; 40: 1627-1635Google Scholar) was a generous gift of Dr. Ann White (University of Texas Southwestern Medical Center). The monoclonal anti-hHL antibody XHL3-6 (28Cheng C.F. Bensadoun A. Bersot T. Hsu J.S. Melford K.H. Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase. Reactivity with monoclonal antibodies to hepatic triglyceride lipase.J. Biol. Chem. 1985; 260: 10720-10727Google Scholar) was a gift of Dr. André Bensadoun (Cornell University). A polyclonal anti-rat HL antibody was a gift of Dr. Howard Wong (University of California, Los Angeles). The hHL was isolated from postheparin human plasma as described previously (29Ramsamy T.A. Neville T.A-M. Chauhan B.M. Aggarwal D. Sparks D.L. Apolipoprotein A-I regulates lipid hydrolysis by hepatic lipase.J. Biol. Chem. 2000; 275: 33480-33486Google Scholar). The hHL cDNA was excised from the pLiv10.hHL vector (provided by Dr. John Taylor at The Gladstone Institute) (30Fan J. Wang J. Bensadoun A. Lauer S.J. Dang Q. Mahley R.W. Taylor J.M. Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins.Proc. Natl. Acad. Sci. USA. 1994; 91: 8724-8728Google Scholar) by digestion with KpnI and HindIII, and the KpnI-HindIII fragment was inserted into the polylinker region of the pCMV5 expression vector (31Blackhart B.D. Yao Z. McCarthy B.J. An expression system for human apolipoprotein B100 in a rat hepatoma cell line.J. Biol. Chem. 1990; 265: 8358-8360Google Scholar) to create pCMV5.hHL. Similarly, the mHL cDNA was excised from the pSP64.mHL vector (provided by Dr. Hans Will at Hamburg University) (32Chang S.F. Netter H.J. Will H. Characterization of cDNA encoding the mouse hepatic triglyceride lipase and expression by in vitro translation.FEBS Lett. 1991; 289: 69-72Google Scholar) by digestion with HindIII and XbaI, and the HindIII-XbaI fragment was inserted into pCMV5 to create pCMV5.mHL. To create the chimeric construct hHLmt encoding amino acids 1−406 of hHL and amino acids 409−488 of mHL, the respective hHL and mHL cDNAs were excised from pCMV5.hHL and pCMV5.mHL by digestion with KpnI and XbaI, and subcloned into pBluescript (pBlue.hHL and pBlue.mHL). A PflmI-XbaI fragment was excised from pBlue.hHL and replaced with a PflmI-XbaI fragment from pBlue.mHL, generating an in-frame chimeric HL construct (pBlue.hHLmt). The KpnI-XbaI fragment was excised from pBlue.hHLmt and inserted into pCMV5 to create pCMV5.hHLmt. A chimeric construct encoding the amino acids 1−408 of mHL and the amino acids 407−476 of hHL, designated pCVM5.mHLht, was similarly constructed. To generate the plasmid encoding hHL471, forward (ACGTAAGCTTGCCACCATGGACACAAGTCCCCTGTGT) and reverse (ACGTGGATCCTCATCTGATCTTTCGCTATGATGT) PCR primers were designed such that the carboxyl terminal five residues of wild-type hHL were eliminated. The PCR product was inserted into pCMV5 using the HindIII and BamHI restriction sites that were encoded within the primers (underlined). All wild-type and mutant-HL constructs were sequence verified to ensure no errors were generated. Chinese hamster ovary (CHO) 13-5-1 cells deficient in LDL receptor-related protein (LRP) expression (33FitzGerald D.J. Fryling C.M. Zdanovsky A. Saelinger C.B. Kounnas M. Winkles J.A. Strickland D. Leppla S. Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein.J. Cell Biol. 1995; 129: 1533-1541Google Scholar) were cultured in Ham's F-12 medium containing 10% FBS. Cell lines were generated by cotransfecting 10 μg of the corresponding pCMV5 plasmid with 0.10 μg pSV2neo using the calcium precipitation method (34Chen C. Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA.Mol. Cell. Biol. 1987; 7: 2745-2752Google Scholar). Stable cell lines were selected with 500 μg/ml G418 and screened for HL expression by immunoblot analysis (details are described in figure legends). Stably transfected cells were grown to confluency (T175 flasks), washed three times with phosphate buffered saline (PBS), and incubated overnight at 37°C with Ham's F-12 medium containing 1% FBS, 500 μg/ml G418, and 10 U/ml heparin. Media (up to 400 ml) were collected, centrifuged at 1,200 rpm for 10 min at 4°C to remove any cell debris, and the supernatant was adjusted with glycerol to a final concentration of 20%. The media was cooled to 4°C, then applied to a Hi-Trap heparin-Sepharose column at 4°C preequilibrated with 90% Buffer A (10 mM sodium phosphate, 20% glycerol, pH 7.2) and 10% Buffer B (10 mM sodium phosphate, 1.5 M sodium chloride, 20% glycerol, pH 7.2). Following reequilibration of the column, 33% Buffer A and 67% Buffer B (final NaCl concentration of 1.0 M) was used to elute bound proteins. The fractions (1 ml) were collected, and those containing protein (as detected using the absorbance at 280 nm) were stored at −80°C. In other experiments, confluent cells (T175 flasks) were washed three times with PBS and incubated with 20 ml serum-free media containing 100 U/ml heparin. Following 4 h incubation at 37°C, the media were treated as above and applied to a Hi-Trap heparin-Sepharose column at 4°C preequilibrated with 90% Buffer A and 10% Buffer B. Following reequilibration of the column, a gradient of Buffer A and Buffer B (NaCl concentration from 0.15−1.5 M) was used to elute the bound proteins. The fractions (1 ml each) were collected and stored at −80°C. The activity of HL obtained by chromatography was determined using a [3H]triolein emulsion (35Ehnholm C. Kuusi T. Preparation, characterization, and measurement of hepatic lipase.Methods Enzymol. 1986; 129: 716-738Google Scholar) and [14C]tributyrin (36Shirai K. Matsuoka N. Saito Y. Yoshida S. Post-heparin plasma hepatic triacylglycerol lipase-catalyzed hydrolysis of tributyrin. Effect of lipid interface.Biochim. Biophys. Acta. 1984; 795: 1-8Google Scholar), as previously described. The protein content of HL samples was determined using a modified Lowry assay (37Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). Stably transfected cells (100 mm dishes) were grown to confluency, washed three times with PBS, and incubated with either 4 ml serum-free Ham's F-12 medium supplemented with G418 or serum-free media containing 100 U/ml heparin. After 4 h incubation at 37°C, the media were collected, centrifuged at 1,200 rpm for 10 min to remove any cell debris, and the HL proteins in the supernatant were concentrated with fumed silica (50 mg) by overnight incubation at 4°C (38Ji Z-S. Dichek H.L. Miranda R.D. Mahley R.W. Heparan sulfate proteoglycans participate in hepatic lipase and apolipoprotein E-mediated binding and uptake of plasma lipoproteins, including high density lipoproteins.J. Biol. Chem. 1997; 272: 31285-31292Google Scholar). The absorbed HL proteins were eluted from fumed silica with 200 μl of lysis/gel-loading buffer (38.5 mM Tris-HCL, 0.1% EDTA, 2% SDS, 6 M urea, 0.1% dithiothreitol, 0.05% reduced glutathione, 0.001% bromophenol blue) by occasional mixing at 90°C for 20 min, then stored at −80°C until needed for immunoblot analysis. Confluent cells (60 mm dishes) were washed three times with PBS and pulse-labeled with [35S]methionine/cysteine (200 μCi/ml) for 2 h in 2 ml methionine/cysteine- and serum-free DMEM. Media were replaced and cells were chased for 2 or 4 h in 2 ml serum-free media ±100 U/ml heparin. The media were collected at the end of chase, and an aliquot (1 ml) was mixed with 5 μl of the anti-hHL polyclonal antibody overnight at 4°C. The cells were lysed with RIPA buffer (50 mM Tris-HCL, pH 8.0, 1 mM EDTA, 1% Triton X-100, 1% deoxycholic acid, 1 mM dithiothreitol, 150 mM NaCl, 0.015% phenylmethylsulfonyl fluoride, 0.1% SDS), and cell-associated HL was likewise immunoprecipitated from the cell lysates. The antibody-HL complexes were adsorbed to Protein A and washed six times with PBS (for medium samples) or RIPA buffer (for cell samples). The HL was eluted from Protein A with 200 μl of gel loading buffer at 100°C for 10 min, separated by electrophoresis on 10% polyacrylamide gel containing 0.1% SDS (SDS-PAGE), and analyzed by fluorography. Data where statistical values were provided were analyzed using the paired t-test. The error bars on data are ±SD. The amino acid sequence of the carboxyl terminus of hHL differs significantly from that of mHL. Not only does hHL have 10 fewer carboxyl terminal amino acid residues than mHL, but also the putative heparin binding domains (HBDs) (22Cardin A.D. Weintraub H.J. Molecular modeling of protein-glycosaminoglycan interactions.Arteriosclerosis. 1989; 9: 21-32Google Scholar, 26Margalit H. Fischer N. Ben-Sasson S.A. Comparative analysis of structurally defined heparin binding sequences reveals a distinct spacial distribution of basic residues.J. Biol. Chem. 1993; 268: 19228-19231Google Scholar) found in hHL are absent in mHL (Fig. 1A). Furthermore, the amino acids in mHL corresponding to the six basic amino acid residues (R or K) located within the putative HBD of hHL (underlined in Fig. 1A) were found to be either acidic or neutral. Notably, four of the basic amino acid residues are present in the carboxyl terminus of hHL. The sequence divergence between hHL and mHL led us to postulate that the carboxyl terminal amino acids govern cell surface association. To test this hypothesis, we prepared the chimeric proteins hHLmt and mHLht, in which the carboxyl terminal regions of hHL and mHL, respectively, were exchanged. We chose a convenient in-frame PflmI restriction endonuclease site within the hHL and mHL cDNAs that allowed us to swap 70 amino acids of hHL with the corresponding 80 amino acids derived from the carboxyl terminus of mHL. To more closely examine the amino acid residues involved in heparin binding, we prepared an hHL mutant (designated hHL471) in which the carboxyl terminal five residues (KRKIR) were deleted. Expression of hHL, mHL, hHLmt, and hHL471 was achieved in CHO 13-5-1 cells that are deficient in LRP expression (33FitzGerald D.J. Fryling C.M. Zdanovsky A. Saelinger C.B. Kounnas M. Winkles J.A. Strickland D. Leppla S. Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein.J. Cell Biol. 1995; 129: 1533-1541Google Scholar). The use of LRP-null cells circumvented complications of potential interaction between HL and LRP (38Ji Z-S. Dichek H.L. Miranda R.D. Mahley R.W. Heparan sulfate proteoglycans participate in hepatic lipase and apolipoprotein E-mediated binding and uptake of plasma lipoproteins, including high density lipoproteins.J. Biol. Chem. 1997; 272: 31285-31292Google Scholar, 39Krapp A. Ahle S. Kersting S. Hua Y. Kneser K. Nielsen M. Gliemann J. Beisiegel U. Hepatic lipase mediates the uptake of chylomicrons and β-VLDL into cells via the LDL receptor-related protein (LRP).J. Lipid Res. 1996; 37: 926-936Google Scholar). Immunoblots of whole-cell extracts from representative hHL, mHL, hHLmt, and hHL471 stable cells are shown in Fig. 1B. The doublet of hHL and hHL471 (53 kDa and 66 kDa), similar to what was seen for rat HL in primary hepatocytes (40Laposata E.A. Laboda H.M. Glick J.M. Strauss III, J.F. Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes.J. Biol. Chem. 1987; 262: 5333-5338Google Scholar), represented differently glycosylated species. Likewise, the doublet of hHLmt (57 kDa and 72 kDa) was seen in the whole-cell extracts. The molecular mass of mHL was observed to be 55 kDa. Expression of hHL, mHL, hHLmt, and hHL471 was also achieved in COS-7 cells, human epithelial kidney 293 cells, and CHO-K1 cells (data not shown). The chimeric protein mHLht transiently expressed in COS-7 cells was unstable and showed multiple degradation products in addition to the full-length protein (Fig. 1C). For this reason, mHLht was not further studied in the following experiments. To ascertain whether or not the recombinant HL proteins were catalytically active, we semi-purified the hHL, mHL, hHLmt, and hHL471 proteins from conditioned media (in the presence of heparin) by heparin-Sepharose affinity chromatography. All four of the recombinant HL proteins showed catalytic activity toward the short-chained triglyceride tributyrin (Fig. 2A) and the long-chained triglyceride triolein (Fig. 2C). No salt-insensitive hydrolytic activity was detected from control mock-transfected cells. The rates of butyrate production and oleate production per microgram of semi-purified hHL, mHL, hHLmt, and hHL471 were assessed at different concentrations of tributyrin (Fig. 2A) and triolein (Fig. 2C). The apparent Km and Vmax values for the semi-purified HLs (Table 1) were obtained from Lineweaver-Burk plots (Fig. 2B, D) using the data shown in Figs. 2A and C, respectively. The apparent Km values for hHL, hHLmt, and hHL471 were comparable, being 0.63 ± 0.17 mM, 0.59 ± 0.13 mM, and 0.57 ± 0.11 mM tributyrin, respectively. These data suggest that the mutant HL proteins had cata
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