Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

Abstract

We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL2 hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL2, and HDL3 but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL2. We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL2 hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL2, and HDL3 but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL2. Endothelial lipase (EL) belongs to a superfamily of lipases (EC 3.1.1.3) that includes LPL and HL (1Jaye 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: 424-428Crossref PubMed Scopus (412) Google Scholar, 2Hirata 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: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 3Stahnke 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: 45-52Crossref PubMed Scopus (64) Google Scholar, 4Datta S. Luo C.C. Li W.H. Van Tuinen 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 localization, and evolutionary relationships with lipoprotein lipase and pancreatic lipase..J. Biol. Chem. 1988; 263: 1107-1110Abstract Full Text PDF PubMed Google Scholar, 5Martin 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: 10907-10914Abstract Full Text PDF PubMed Google Scholar, 6Hide W.A. Chan L. Li W.H. Structure and evolution of the lipase superfamily..J. Lipid Res. 1992; 33: 167-178Abstract Full Text PDF PubMed Google Scholar). These three lipases have both triglyceride (TG) lipase and phospholipase activity, but EL has relatively more phospholipase activity compared with LPL, which has predominantly TG lipase activity (7McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase..J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). Overexpression of EL in mice was shown to significantly reduce high density lipoprotein cholesterol (HDL-C) (1Jaye 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: 424-428Crossref PubMed Scopus (412) Google Scholar, 8Ishida T. Choi S. Kundu R.K. Hirata K. Rubin E.M. Cooper A.D. Quertermous T. Endothelial lipase is a major determinant of HDL level..J. Clin. Invest. 2003; 111: 347-355Crossref PubMed Scopus (261) Google Scholar, 9Maugeais C. Tietge U.J. Broedl U.C. Marchadier D. Cain W. McCoy M.G. Lund-Katz S. Glick J.M. Rader D.J. Dose-dependent acceleration of high-density lipoprotein catabolism by endothelial lipase..Circulation. 2003; 108: 2121-2126Crossref PubMed Scopus (128) Google Scholar), whereas loss-of-function studies in mice result in significantly elevated plasma HDL-C (8Ishida T. Choi S. Kundu R.K. Hirata K. Rubin E.M. Cooper A.D. Quertermous T. Endothelial lipase is a major determinant of HDL level..J. Clin. Invest. 2003; 111: 347-355Crossref PubMed Scopus (261) Google Scholar, 10Ma K. Cilingiroglu M. Otvos J.D. Ballantyne C.M. Marian A.J. Chan L. Endothelial lipase is a major genetic determinant for high-density lipoprotein concentration, structure, and metabolism..Proc. Natl. Acad. Sci. USA. 2003; 100: 2748-2753Crossref PubMed Scopus (199) Google Scholar, 11Jin W. Millar J.S. Broedl U. Glick J.M. Rader D.J. Inhibition of endothelial lipase causes increased HDL cholesterol levels in vivo..J. Clin. Invest. 2003; 111: 357-362Crossref PubMed Scopus (193) Google Scholar). In vitro and in vivo studies using chimeric proteins of LPL and HL have shown that the differences in substrate specificity between these two lipases are governed by a 22 amino acid loop, or “lid domain,” within the N-terminal domain of the respective lipases that covers the catalytic site (12Dugi K.A. Dichek H.L. Talley G.D. Brewer Jr., H.B. Santamarina-Fojo S. Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates..J. Biol. Chem. 1992; 267: 25086-25091Abstract Full Text PDF PubMed Google Scholar, 13Dugi 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: 25396-25401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 14Kobayashi J. Applebaum-Bowden D. Dugi K.A. Brown D.R. Kashyap V.S. Parrott C. Duarte C. Maeda N. Santamarina-Fojo S. Analysis of protein structure-function in vivo. Adenovirus-mediated transfer of lipase lid mutants in hepatic lipase-deficient mice..J. Biol. Chem. 1996; 271: 26296-26301Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The shorter 19 amino acid lid domain within EL partially contributes to its substrate specificity (15Griffon N. Budreck E.C. Long C.J. Broedl U.C. Marchadier D.H.L. Glick J.M. Rader D.J. Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras..J. Lipid Res. 2006; 47: 1803-1811Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar); other elements affecting substrate specificity remain to be elucidated. Human EL is translated as a 500 amino acid 57 kDa peptide that is processed into a mature 480 amino acid protein with an apparent molecular mass of 68 kDa after the loss of its signal peptide and the addition of N-linked glycosylation (1Jaye 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: 424-428Crossref PubMed Scopus (412) Google Scholar, 2Hirata 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: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). EL has five putative N-linked glycosylation sites [identified by the presence of asparagine-X-serine/threonine (Asn-Xaa-Ser/Thr) motifs]. We previously reported that four of the five sites, specifically Asn-60, Asn-116, Asn-373, and Asn-471, are utilized (16Miller G.C. Long C.J. Bojilova E.D. Marchadier D. Badellino K.O. Blanchard N. Fuki I.V. Glick J.M. Rader D.J. Role of N-linked glycosylation in the secretion and activity of endothelial lipase..J. Lipid Res. 2004; 45: 2080-2087Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Abolishment of N-linked glycosylation at Asn-116 by mutagenesis of Asn to alanine (Ala) resulted in a surprising increase of catalytic activity, whereas removing N-linked glycosylation at other sites produced EL proteins with either decreased or unaffected catalytic activities (16Miller G.C. Long C.J. Bojilova E.D. Marchadier D. Badellino K.O. Blanchard N. Fuki I.V. Glick J.M. Rader D.J. Role of N-linked glycosylation in the secretion and activity of endothelial lipase..J. Lipid Res. 2004; 45: 2080-2087Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Asn-116 of EL is not conserved in either LPL or HL. Thus, the N-linked glycosylation at Asn-116 of EL may play a unique function that regulates enzyme activity. In this study, we confirmed through additional mutagenesis that the N-linked glycosylation at Asn-116 reduces activity against synthetic substrates, and we further explored its role in modulating the ability of EL to hydrolyze native lipoproteins ex vivo and in vivo. Our data support the conclusion that the N-linked glycosylation at Asn-116 of EL affects the substrate specificity of the enzyme by limiting the hydrolysis of substrates in vitro and in vivo. Triolein, dipalmitoylphosphatidyl choline (DPPC), cholesteryl oleate, fatty acid-free BSA, heparin, and FBS were purchased from Sigma. DMEM, antibiotic/antimycotic (A/A), Lipofectamine™, and Nupage™ 10% Bis-Tris gels were purchased from Invitrogen. HRP-conjugated goat anti-rabbit IgG and HRP-conjugated rabbit anti-mouse IgG antibodies were purchased from Jackson Immunoresearch. [3H]triolein was purchased from Perkin-Elmer. [14C]DPPC was purchased from American Radiolabeled Chemicals, Inc. A polyclonal anti-human EL antibody was generated as described previously (17Badellino K.O. Wolfe M.L. Reilly M.P. Rader D.J. Endothelial lipase concentrations are increased in metabolic syndrome and associated with coronary atherosclerosis..PLoS Med. 2006; 3: e22Crossref PubMed Scopus (146) Google Scholar). The monoclonal anti-bovine LPL antibody 5D2, with cross-reactivity to human LPL (18Peterson J. Fujimoto W.Y. Brunzell J.D. Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies..J. Lipid Res. 1992; 33: 1165-1170Abstract Full Text PDF PubMed Google Scholar), was a gift of Dr. John Brunzell (University of Washington). The monoclonal anti-human HL antibody XHL3-6 (19Cheng 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: 10720-10727Abstract Full Text PDF PubMed Google Scholar) was a gift of Dr. André Bensadoun (Cornell University). The cDNAs for human EL (NM006033), human LPL (NM000237), and human HL (NM000236) were inserted into the pcDNA3 mammalian expression vector (Invitrogen). Mutagenesis of Asn-116 from human EL into Ala (N116A) was described previously (16Miller G.C. Long C.J. Bojilova E.D. Marchadier D. Badellino K.O. Blanchard N. Fuki I.V. Glick J.M. Rader D.J. Role of N-linked glycosylation in the secretion and activity of endothelial lipase..J. Lipid Res. 2004; 45: 2080-2087Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Mutagenesis of Asn-116 from EL into Thr (N116T), Asn-117 into Ala (N117A), and Thr-118 into Ala (T118A) was performed using the Quikchange™ mutagenesis kit (Stratagene) with previously described polymerase chain reaction conditions (16Miller G.C. Long C.J. Bojilova E.D. Marchadier D. Badellino K.O. Blanchard N. Fuki I.V. Glick J.M. Rader D.J. Role of N-linked glycosylation in the secretion and activity of endothelial lipase..J. Lipid Res. 2004; 45: 2080-2087Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Complementary sense and antisense oligonucleotides (toward nucleotides 647–677) to generate these mutants are as follows (sense sequence): N116T, 5′-CGGATGCGGTCACTAATACCAGGGTGGTGGG-3′; N117A, 5′-CGGATGCGGTCAATGCTACCAGGGTGGTGGG-3′; and T118A, 5′-CGGATGCGGTCAATAATGCCAGGGTGGTGGG-3′. Mutagenesis of glycine-99 (Gly-99) from LPL into either Asn (G99N) or Ala (G99A) was performed as described above. The sense sequences of the oligonucleotides (toward nucleotides 538–569) to generate the LPL mutants are as follows: G99N, 5′-CCAGTGTCCGCGAACTACACCAAACTGGTGGG-3′; and G99A, 5′-CCAGTGTCCGCGGCCTACACCAAACTGGTG-3′. Mutagenesis of arginine-113 (Arg-113) from HL into either Asn (R113N) or lysine (Lys) (R113K) was performed as described above. The sense sequences of the oligonucleotides (toward nucleotides 441–472) to generate the HL mutants are as follows: R113N, 5′-CCACTACACCATCGCCGTCAACAACACCCGCC-3′; and R113K 5′-CTACACCATCGCCGTCAAGAACACCCGCCTTGTGG-3′. All mutant sequences were verified by DNA sequencing. 293 cells were cultured in DMEM containing 10% FBS and 1% A/A. Cells were grown to 90% confluency (in 60 mm dishes), and 1 μg of plasmid expressing lipase was transfected using Lipofectamine™ according to the manufacturer's instructions. At 24 h after the transfection, media were removed and replaced with serum-free media containing 1% A/A and 10 U/ml heparin. To promote lipase dissociation from cells, at 47.5 h after the transfection, an additional 10 U/ml heparin was added to the media in each plate. At 48 h after the transfection, media were collected and centrifuged at 1,200 rpm for 10 min to remove any cell debris. The supernatant was divided into aliquots and stored at −80°C. The total extracellular EL released from transfected cells over 16 h in the absence versus presence of heparin was determined as described previously for HL (20Brown R.J. Schultz J.R. Ko K.W.S. Hill J.S. Ramsamy T.A. White A.L. Sparks D.L. Yao Z. The amino acid sequences of human and mouse hepatic lipase influence cell surface association..J. Lipid Res. 2003; 44: 1306-1314Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Proteins in conditioned media samples from transfected cells were separated on Nupage™ 10% Bis-Tris gels, and gels were transferred to nitrocellulose membranes. Nitrocellulose membranes were subjected to chemiluminescent immunoblot analyses for EL (using a 1:5,000 dilution of the anti-human EL polyclonal antibody and a 1:5,000 dilution of HRP-conjugated anti-rabbit IgG), LPL (using a 1:3,000 dilution of 5D2 and a 1:5,000 dilution of HRP-conjugated anti-mouse IgG), and HL (using a 1:10,000 dilution of XHL3-6 and a 1:5,000 dilution of HRP-conjugated anti-mouse IgG). The mass of recombinant LPL and HL, expressed as arbitrary units, was determined from standard densitometry curves of immunoblots. Briefly, conditioned media (0–10 μl) of wild-type or mutant lipase were separated on the same Bis-Tris gel and subjected to immunoblot analyses with the appropriate antibodies, as described above. The intensity of detected protein for each dilution was quantified using scanning densitometry of the immunoblot, and standard curves were generated for the lipase pair. The mass of all EL proteins, semiquantified as arbitrary units, was determined using an ELISA (16Miller G.C. Long C.J. Bojilova E.D. Marchadier D. Badellino K.O. Blanchard N. Fuki I.V. Glick J.M. Rader D.J. Role of N-linked glycosylation in the secretion and activity of endothelial lipase..J. Lipid Res. 2004; 45: 2080-2087Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 17Badellino K.O. Wolfe M.L. Reilly M.P. Rader D.J. Endothelial lipase concentrations are increased in metabolic syndrome and associated with coronary atherosclerosis..PLoS Med. 2006; 3: e22Crossref PubMed Scopus (146) Google Scholar) in the same assay. TG lipase and phospholipase assays using the glycerol-stabilized substrates triolein and DPPC, respectively, were performed as described previously (7McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase..J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). LDL, HDL2, and HDL3 were isolated by potassium bromide density gradient ultracentrifugation (21Fuki 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. The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro..J. Clin. Invest. 1997; 100: 1611-1622Crossref PubMed Scopus (205) Google Scholar). Assays of lipoprotein lipid hydrolysis by recombinant lipases were performed as described previously (15Griffon N. Budreck E.C. Long C.J. Broedl U.C. Marchadier D.H.L. Glick J.M. Rader D.J. Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras..J. Lipid Res. 2006; 47: 1803-1811Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The free fatty acids generated by the hydrolysis of lipoproteins were measured using a commercial kit (Waco Pure Chemical Industries) according to the manufacturer's instructions. All activity data were corrected for protein mass (determined as described above) and were normalized to the percentage of wild-type lipase. Adenoviruses expressing wild-type human EL (Ad.EL) and control adenoviruses without a cDNA insert (Ad.null) were generated previously (1Jaye 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: 424-428Crossref PubMed Scopus (412) Google Scholar). Adenoviruses expressing EL-N116A (Ad.EL-N116A) were generated from the mutant cDNA using previously described methods (1Jaye 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: 424-428Crossref PubMed Scopus (412) Google Scholar). Female low density lipoprotein receptor (LDLR)-null mice in a C57BL6/J background (Jackson Laboratories) were maintained on a normal chow diet and a 12 h light/12 h dark cycle. Mice were injected with 3 × 1010 particles of Ad.EL, Ad.EL-N116A, or Ad.null via the tail vein. Before adenovirus injection and at 7 d after injection, blood was collected from the retro-orbital plexus. Postheparin plasma was collected from the retro-orbital plexus at 5 min after tail vein injection of 500 U/kg heparin. Plasma TG, total cholesterol (TC), HDL-C, and phospholipid (PL) were measured using commercially available kits. For separation of lipoproteins, pooled plasma (100 μl) from control, wild-type EL-, and EL-N116A-expressing mice were loaded onto two in-series Superose-6 columns that had been equilibrated with phosphate-buffered saline and fractionated by fast-performance liquid chromatography (FPLC). Five hundred microliter fractions were collected, and TC from each fraction was measured. Where statistical values are provided, the data were analyzed using the paired t-test. Error bars indicate ±SD. We previously reported that EL-N116A abolished N-linked glycosylation at Asn-116 of EL (16Miller G.C. Long C.J. Bojilova E.D. Marchadier D. Badellino K.O. Blanchard N. Fuki I.V. Glick J.M. Rader D.J. Role of N-linked glycosylation in the secretion and activity of endothelial lipase..J. Lipid Res. 2004; 45: 2080-2087Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Additional EL mutants were generated to disrupt the Asn-Asn-Thr sequence of this glycosylation site. The EL mutations N116T and T118A also had reduced molecular masses of EL, consistent with a loss of glycosylation In mutagenesis of Asn-117 to Ala not which was Asn-117 is not to the N-linked glycosylation Consistent with on both and had significantly activities toward triolein and DPPC, whereas was wild-type EL We the catalytic activity of the EL-N116A mutant against native lipoproteins using LDL, HDL2, and HDL3 as substrates The hydrolytic activity toward LDL by EL-N116A was significantly versus wild-type EL (100 A significantly increase of activity by EL-N116A versus wild-type EL (100 was also observed toward HDL2. of activity by EL-N116A was observed versus wild-type EL (100 toward In an to the role of N-linked glycosylation at Asn-116, we assessed the on lipids by wild-type EL and EL-N116A in vivo using adenovirus-mediated expression in shown in 1 by 7 after injection of the TG, HDL-C, and levels were significantly in mice expressing EL-N116A in mice expressing wild-type EL. The mass measured by ELISA of EL-N116A in postheparin plasma arbitrary was to that of wild-type EL arbitrary human EL mass was detected in plasma from of lipoproteins by from pooled plasma of mice at 7 after the injection of that the of lipid levels in mice expressing wild-type EL was in the HDL fraction 3 the expression of EL-N116A a in both the density and HDL we were to in the mass of mouse in vivo are consistent with a activity by EL-N116A toward lipoprotein lipids in levels in mice with adenoviruses expressing wild-type or after versus wild-type versus wild-type versus wild-type versus wild-type versus and wild-type versus and wild-type versus and wild-type versus and wild-type versus and wild-type endothelial HDL-C, high density lipoprotein versus wild-type versus and wild-type in a EL, endothelial HDL-C, high density lipoprotein To that of enhanced activity in vivo by EL-N116A was not to we the cell surface of wild-type EL and EL-N116A using transfected 293 cells analyses that the EL released into in the absence versus the presence of heparin a 16 h was for both wild-type EL and EL-N116A The of EL-N116A into media was from densitometry data of from which was to the of wild-type EL