A naturally occurring variant of endothelial lipase associated with elevated HDL exhibits impaired synthesis
2009; Elsevier BV; Volume: 50; Issue: 9 Linguagem: Inglês
10.1194/jlr.p900020-jlr200
ISSN1539-7262
AutoresRobert Brown, Andrew C. Edmondson, Nathalie Griffon, Theophelus B. Hill, Ilia V. Fuki, Karen O. Badellino, Mingyao Li, Megan L. Wolfe, Muredach P. Reilly, Daniel J. Rader,
Tópico(s)Diabetes Management and Research
ResumoHuman endothelial lipase (EL) is a member of a family of lipases and phospholipases that are involved in the metabolism of plasma lipoproteins. EL displays a preference to hydrolyze lipids in HDL. We report here that a naturally occurring low frequency coding variant in the EL gene (LIPG), glycine-26 to serine (G26S), is significantly more common in African-American individuals with elevated HDL cholesterol (HDL-C) levels. To test the hypothesis that this variant results in reduced EL function, we extensively characterized and compared the catalytic and noncatalytic functions of the G26S variant and wild-type (WT) EL. While the catalytic-specific activity of G26S EL is similar to WT EL, its secretion is markedly reduced. Consistent with this observation, we found that carriers of the G26S variant had significantly reduced plasma levels of EL protein. Thus, this N-terminal variant results in reduced secretion of EL protein, plausibly leading to increased HDL-C levels. Human endothelial lipase (EL) is a member of a family of lipases and phospholipases that are involved in the metabolism of plasma lipoproteins. EL displays a preference to hydrolyze lipids in HDL. We report here that a naturally occurring low frequency coding variant in the EL gene (LIPG), glycine-26 to serine (G26S), is significantly more common in African-American individuals with elevated HDL cholesterol (HDL-C) levels. To test the hypothesis that this variant results in reduced EL function, we extensively characterized and compared the catalytic and noncatalytic functions of the G26S variant and wild-type (WT) EL. While the catalytic-specific activity of G26S EL is similar to WT EL, its secretion is markedly reduced. Consistent with this observation, we found that carriers of the G26S variant had significantly reduced plasma levels of EL protein. Thus, this N-terminal variant results in reduced secretion of EL protein, plausibly leading to increased HDL-C levels. Endothelial lipase (EL) is a member of a family of lipases that includes LPL and hepatic lipase (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 (426) 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 (266) 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). Overexpression of EL in mouse models significantly reduces plasma HDL 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 (426) Google Scholar, 7Ishida 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 (272) Google Scholar, 8Maugeais 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 (131) Google Scholar), whereas the loss of EL function in mouse models significantly elevates plasma HDL-C (7Ishida 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 (272) Google Scholar, 9Ma 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 (204) Google Scholar, 10Jin 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 (202) Google Scholar). In addition to its catalytic function, EL is capable of "bridging" HDL and other lipoproteins with cell surface proteoglycans (11Fuki I.V. Blanchard N. Jin W. Marchadier D.H. Millar J.S. Glick J.M. Rader D.J. Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway.J. Biol. Chem. 2003; 278: 34331-34338Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The human gene encoding EL, LIPG, has been reported to be associated with variation in HDL-C levels in genome-wide association studies (12Willer 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 (1327) Google Scholar, 13Kathiresan 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 (1135) Google Scholar). In addition, we have recently shown that a previously reported nonsynonymous coding variant of EL that exhibits impaired enzymatic functions in vitro and in vivo is directly associated with elevated HDL-C in multiple cohorts (14Edmondson A.C. Brown R.J. Kathiresan S. Cupples L.A. Demissie S. Manning A.K. Jensen M.K. Rimm E.B. Wang J. Rodrigues A. et al.Loss-of-function variants in endothelial lipase are a cause of elevated HDL cholesterol in humans.J. Clin. Invest. 2009; 119: 1042-1050PubMed Google Scholar). Numerous variants have been identified within EL (14Edmondson A.C. Brown R.J. Kathiresan S. Cupples L.A. Demissie S. Manning A.K. Jensen M.K. Rimm E.B. Wang J. Rodrigues A. et al.Loss-of-function variants in endothelial lipase are a cause of elevated HDL cholesterol in humans.J. Clin. Invest. 2009; 119: 1042-1050PubMed Google Scholar, 15deLemos A.S. Wolfe M.L. Long C.J. Sivapackianathan R. Rader D.J. Identification of genetic variants in endothelial lipase in persons with elevated high-density lipoprotein cholesterol.Circulation. 2002; 106: 1321-1326Crossref PubMed Scopus (128) Google Scholar); however, not all of the mechanisms by which they influence HDL-C levels, if at all, have been elucidated. We previously sequenced a small number of subjects with extremely high levels of HDL-C and reported a low frequency coding variant that results in the substitution of a glycine at residue 26 to serine (G26S) (15deLemos A.S. Wolfe M.L. Long C.J. Sivapackianathan R. Rader D.J. Identification of genetic variants in endothelial lipase in persons with elevated high-density lipoprotein cholesterol.Circulation. 2002; 106: 1321-1326Crossref PubMed Scopus (128) Google Scholar). In the current study, we assessed the relationship of this variant to elevated HDL-C and studied its function to determine whether it reduces EL activity or mass. Our results indicate that this N-terminal G26S variant does not exhibit an impaired biochemical function, but rather it results in reduced secretion of EL protein, leading to increased HDL-C levels in carriers of this variant. Subjects from the University of Pennsylvania High HDL Cholesterol Study (HHDL; n = 854) and the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA; n = 885) were assessed for the presence of either wild-type (WT) EL or the G26S variant of EL by Taqman custom genotyping (Applied Biosystems). The study designs and initial findings of subjects were previously reported from HHDL (14Edmondson A.C. Brown R.J. Kathiresan S. Cupples L.A. Demissie S. Manning A.K. Jensen M.K. Rimm E.B. Wang J. Rodrigues A. et al.Loss-of-function variants in endothelial lipase are a cause of elevated HDL cholesterol in humans.J. Clin. Invest. 2009; 119: 1042-1050PubMed Google Scholar) and SIRCA (16Valdes A.M. Wolfe M.L. Tate H.C. Gefter W. Rut A. Rader D.J. Association of traditional risk factors with coronary calcification in persons with a family history of premature coronary heart disease: the study of the inherited risk of coronary atherosclerosis.J. Investig. Med. 2001; 49: 353-361Crossref PubMed Google Scholar). Subjects identified with the G26S variant were compared with age- and sex-matched control subjects from both SIRCA and HHDL. Subjects from the University of Pennsylvania Coronary Artery Calcification Study (PennCAC; n = 2,616) were assessed for the presence of either WT EL or the G26S variant of EL using the Illumina IBC Candidate Gene array, version 2 (17Keating B.J. Tischfield S. Murray S.S. Bhangale T. Price T.S. Glessner J.T. Galver L. Barrett J.C. Grant S.F. Farlow D.N. et al.Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies.PLoS One. 2008; 3e3583Crossref PubMed Scopus (324) Google Scholar). The PennCAC cohort is composed of subjects from SIRCA, the Penn Diabetes Heart Study (18Reilly M.P. Iqbal N. Schutta M. Wolfe M.L. Scally M. Localio A.R. Rader D.J. Kimmel S.E. Plasma leptin levels are associated with coronary atherosclerosis in type 2 diabetes.J. Clin. Endocrinol. Metab. 2004; 89: 3872-3878Crossref PubMed Scopus (184) Google Scholar, 19Bagheri R. Schutta M. Cumaranatunge R.G. Wolfe M.L. Terembula K. Hoffman B. Schwartz S. Kimmel S.E. Farouk S. Iqbal N. et al.Value of electrocardiographic and ankle-brachial index abnormalities for prediction of coronary atherosclerosis in asymptomatic subjects with type 2 diabetes mellitus.Am. J. Cardiol. 2007; 99: 951-955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), and the Philadelphia Area Metabolic Syndrome Network, which is an ongoing cross-sectional study of individuals with a varying number of the metabolic syndrome criteria. Age, height, mass, and histories of smoking, drinking, cardiovascular disease, type-2 diabetes, and metabolic syndrome were recorded by referring physicians. Total cholesterol, HDL-C, LDL cholesterol, and triglycerides were assessed in clinical laboratories. All studies were approved by the University of Pennsylvania Institutional Review Board and informed consent was obtained from all participants. The cDNA for human EL (NM006033) was inserted into the pcDNA3 expression vector (Invitrogen). Mutagenesis of Gly-26 into Ser was performed using the QuikChangeTM mutagenesis kit (Stratagene). The sense oligonucleotide (toward nucleotides 312–345) to generate the G26S variant is 5′-GAGCCCCGTACCTTTTAGTCCAGAGGGACGGCTG-3′; a complementary antisense oligonucleotide was also used. HEK293 cells were cultured in DMEM (Invitrogen) containing 10% fetal bovine serum (Sigma) and 1% antibiotic/antimycotic (Invitrogen). Cells were grown to 90% confluency (in 12-well plates), and 0.5 μg of EL expression plasmid was transfected per well using LipofectamineTM (Invitrogen) according to the manufacturer's instructions. For analysis of EL expression and catalytic activity, media were replaced at 32 h posttransfection with serum-free media without or with 100 U/ml heparin. At 48 h posttransfection, 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). Cells were lysed to extract total RNA and protein, and samples were stored at −80°C. For inhibition of degradation pathways, media were replaced at 48 h posttransfection with serum-free media containing 100 U/ml heparin and either 75 μM chloroquine or 100 μM N-acetyl-leucinyl-leucinyl-norleucinal (ALLN; Sigma). After a 6 h incubation with chloroquine or ALLN, cells and media were collected as described above. Cells (in 60 mm dishes) were transiently transfected as described above. At 48 h posttransfection, cells were washed three times with PBS and pulse-labeled with 1 ml of 100 µCi/ml [35S]methionine/cysteine (Perkin-Elmer) for 2 h in methionine/cysteine- and serum-free DMEM. Media were replaced with 2 ml serum-free media containing 100 U/ml heparin, and cells were chased for up to 2 h. The media and cells were collected at the end of the pulse and at times 15, 30, 60, 90, and 120 min of chase period. An aliquot (1 ml) of media was mixed with 10 µl of an anti-human EL polyclonal antibody, generated as previously described (21Miller 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 (28) Google Scholar), 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 EL was likewise immunoprecipitated from the cell lysates. The antibody-EL complexes were adsorbed to Protein A and washed six times with PBS (for medium samples) or RIPA buffer (for cell samples). The EL was eluted from Protein A with 200 µl of a 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) at 100°C for 10 min and separated by electrophoresis on 10% polyacrylamide gels containing 0.1% SDS. Gels were exposed to film and bands were excised from the gel and counted for radioactivity. Data from four pulse-chase experiments were normalized based on percentage of cell 35S-EL after pulse. Proteins in conditioned media samples from transfected cells were separated on NupageTM 10% Bis-Tris gels (Invitrogen), 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 horseradish peroxidase-conjugated anti-rabbit IgG). Total RNA from cells transfected with EL was subjected to real-time PCR analyses for human EL and β-actin using commercially available primers (Applied Biosystems). The mass of all EL proteins used in lipase activity assays and lipoprotein binding assays, semiquantified as arbitrary units, was determined using an ELISA in the same assay (21Miller 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 (28) Google Scholar, 22Badellino 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 (154) Google Scholar). The mass of EL in preheparin plasma from human subjects was quantified by ELISA as ng/ml using a human EL protein standard (kindly provided by Dr. Karen Badellino, University of Pennsylvania). Triglyceride lipase and phospholipase assays using glycerol-stabilized substrates of triolein and dipalmitoylphosphatidyl choline (DPPC), respectively, were performed as described previously (23McCoy 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 and HDL3 were isolated by potassium bromide density gradient ultracentrifugation (24Fuki 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 (206) Google Scholar). Assays of the kinetics of lipoprotein lipid hydrolysis by EL were performed as described previously (25Griffon 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 WT EL. HEK293 cells in 12-well plates were transfected with EL as described above. At 32 h posttransfection, media were changed to 0.5 ml of serum-free medium containing 0.2% BSA. At 48 h, the serum-free media with BSA were replaced with fresh serum-free media with BSA containing either 5 μg/ml [125I]LDL or 5 μg/ml [125I]HDL3 ± 100 U/ml heparin. LDL and HDL3 were radiolabeled using the iodine monochloride method (26McFarlane A.S. Efficient trace-labelling of proteins with iodine.Nature. 1958; 182: 53Crossref PubMed Scopus (1497) Google Scholar). Cells were incubated at 4°C for 1 h, and cell-associated lipoproteins were measured. Additional wells were transfected to assess cell surface-bound EL within experiments. To assess cell surface-bound EL, at 48 h, the serum-free media with BSA was replaced with serum-free media containing only 100 U/ml heparin. Cells were incubated at 4°C for 1 h, and conditioned media were assessed for EL by immunoblot analyses as described above. EL-mediated binding of lipoproteins was calculated as the amount of lipoprotein bound per cell protein above mock-transfected background. Multiple experiments were normalized based on a percentage of WT. The expression plasmids for EL and empty vector (which also contain the T7 promoter) were used to express EL by in vitro transcription/translation using a rabbit reticulocyte system (Promega) in the presence of [35S]methionine according to manufacturer's instructions. Reactions were halted at various time points for up to 60 min, and proteins were separated on NupageTM 10% Bis-Tris gels. Gels were exposed to film, and protein bands were excised from the gel and counted for radioactivity. Error bars indicate ±SD. A nonparametric version of the t-test (Wilcoxon's Rank-Sum) was used for comparisons of plasma lipid levels among African-American probands in the HHDL cohort. Plasma lipid levels among subjects from the PennCAC cohort were analyzed using multivariable linear regression after adjustment for age, gender, diabetes, body mass index, and alcohol use. Plasma EL levels were compared using a two-tailed t-test for unequal variance. Rate constants for pulse-chase analyses and in vitro translation were calculated using GraphPad Prism software [assuming one-phased kinetics with the formula Y = (Y0−Y∞) × exp(−k × t) + Y∞, where Y represents amount of radiolabeled protein at time t, Y0 represents amount of radiolabeled protein at time zero, Y∞ represents maximal or minimal amount of radiolabeled protein at infinite time, and k represents the rate constant in reciprocal units of time]. All biochemical studies were analyzed using a two-tailed paired t-test. We genotyped 854 unrelated subjects from the HHDL cohort for the G26S variant of EL. Of the 68 African-Americans in the cohort who were genotyped, 8 (11.8%) were identified as carriers for the G26S variant. In contrast, of the 767 Caucasians in the HHDL cohort who were genotyped, none were found to be carriers of the G26S variant. The G26S probands within the HHDL cohort had significantly higher levels of HDL-C versus noncarriers within the same cohort (Table 1). Genotyping of family members from 3 of the G26S probands within the HHDL cohort has revealed 6 additional subjects with the G26S variant.TABLE 1Clinical characteristics of African-American noncarriers and carriers of the G26S variant of EL from the High HDL cohortNoncarriersG26S Carriersn608Age (y)58.4 ± 13.858.1 ± 15.4Height (cm)164.7 ± 9.5165.1 ± 11.7Mass (kg)73.7 ± 18.468.8 ± 15.7BMI (kg/m2)26.9 ± 5.325.1 ± 4.2TC (mg/dl)224.8 ± 45.1240.3 ± 66.9TG (mg/dl)69.5 ± 23.669.0 ± 23.7LDL-C (mg/dl)124.5 ± 38.4121.7 ± 41.0HDL-C (mg/dl)86.4 ± 16.2104.8 ± 27.7*% smoke11.70% drink51.725.0% family Hx CAD5.012.5BMI, Body mass index; TC, total cholesterol; TG, triglyceride; LDL-C, LDL cholesterol; Hx CAD, history of coronary artery disease. Data represent the mean ±SD. *, p=0.03 versus noncarriers. Open table in a new tab BMI, Body mass index; TC, total cholesterol; TG, triglyceride; LDL-C, LDL cholesterol; Hx CAD, history of coronary artery disease. Data represent the mean ±SD. *, p=0.03 versus noncarriers. We also genotyped 2,616 unrelated subjects from the PennCAC cohort for the G26S variant of EL. Of the 521 African-Americans who were genotyped, we identified 55 (10.6%) subjects as carriers for the variant. None of the 2,095 Caucasians were found to be carriers of the G26S variant. Furthermore, we failed to identify any carriers for the G26S variant in Caucasians (n=851/885) from the SIRCA cohort, thus strengthening the likelihood that the G26S variant is specific to African-Americans. The G26S probands within the PennCAC cohort exhibited a small but significant increase of HDL-C versus noncarriers within the same cohort (Table 2).TABLE 2Clinical characteristics of African-American noncarriers and carriers of the G26S variant of EL from the PennCAC cohortNoncarriersG26S Carriersn46655Age (y)55.4 ± 10.155.9 ± 10.7Height (cm)169.2 ± 10.7169.9 ± 8.1Mass (kg)93.0 ± 19.194.7 ± 16.8BMI (kg/m2)32.6 ± 6.532.9 ± 6.3TC (mg/dl)187.9 ± 42.1178.4 ± 34.0TG (mg/dl)109.6 ± 75.2101.7 ± 59.9LDL-C (mg/dl)110.7 ± 34.499.5 ± 26.0HDL-C (mg/dl)52.0 ± 15.255.3 ± 15.1*% smoke14.416.7% drink EtOH41.244.4% Type-2 diabetes75.588.8% Metabolic syndrome59.661.1BMI, Body mass index; EtOH, alcohol; TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol. Data represent the mean ±SD. *, P = 0.04 versus noncarriers. Open table in a new tab BMI, Body mass index; EtOH, alcohol; TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol. Data represent the mean ±SD. *, P = 0.04 versus noncarriers. We suspected that the G26S variant of EL may have an impaired function leading to elevated HDL-C levels. In transient transfections of the G26S EL and WT cDNAs, we consistently observed a profoundly reduced level of both cell-associated and secreted G26S EL protein (full-length 68 kDa protein, plus the 40 kDa and 28 kDa cleavage products of full-length EL) versus WT EL, despite identical levels of mRNA (Fig. 1). We confirmed that an epitope recognized by our antibody was not disrupted with the G26S variant of EL by comparing in vitro translated G26S EL and WT EL through immunoblot analyses (Supplemental Fig. I). The specific hydrolytic activity of recombinant G26S EL toward synthetic substrates triolein and DPPC was comparable to WT EL (Fig. 2). We also tested the kinetics of catalytic activity by the G26S EL variant using HDL3 as substrate, and we found that both the apparent KM and Vmax values between WT and G26S EL were similar (appKM: WT, 464 ± 51 μM HDL3 phospholipid vs. G26S, 363 ± 119 μM HDL3 phosoholipid; appVmax: WT, 272 ± 18 nmol free fatty acid/EL mass/h vs. G26S, 349 ± 61 nmol free fatty acid/EL mass/h).Fig. 2Activities of WT and G26S EL. Media from cells transiently expressing WT or G26S EL in the presence of heparin were collected and assayed for hydrolysis of triolein and DPPC. Free fatty acids released were normalized for EL protein and quantified as described under "Experimental Procedures." The specific activity data for the EL variants were normalized to 100% of WT EL. Assays were performed at least in triplicate. Error bars indicate ±SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess the bridging function of G26S EL, we first addressed the cell surface association of the variant. As shown in Fig. 1B, immunoblot analyses of the media from transfected cells in the presence of heparin show that the protein mass of both WT and G26S EL was greater than the WT and G26S EL protein mass of the media from transfected cells in the absence of heparin during a 16 h incubation period. We determined that the release of uncleaved full-length (68 kDa) G26S EL into heparin-free media was 22 ± 11% (calculated from densitometry data of immunoblots), which was comparable to the 14 ± 7% release of uncleaved WT EL (Fig. 3A). Having ascertained that the cell surface association of G26S EL and WT EL are comparable, we determined the ability of cells expressing each EL to bridge 125I-labeled LDL and HDL3 to the cell surface at 4°C. We show that transfected cells expressing WT and G26S EL can equally bind LDL (Fig. 3B) and HDL3 (Fig. 3C) to the cell surface. However, the amount of uncleaved full-length (68 kDa) G26S EL on the cell surface in our bridging assays is 50% lower (calculated from densitometry data of immunoblot) than WT EL (Fig. 3D); thus, normalizing the bridging data to EL expression would suggest that G26S EL has a 2-fold greater ability to bind lipoproteins to the cell surface. We next focused our attention on the markedly reduced G26S EL protein mass in transfected cells by addressing the possibility that G26S EL may be subjected to intracellular degradation. The lysosomal degradation inhibitor chloroquine failed to raise the cell-associated (Fig. 4A) or media (Fig. 4B) G26S EL mass to levels comparable to WT EL. We also assessed whether G26S EL was degraded via the ubiquitin-proteosomal pathway by incubating cells in the presence of ALLN. Like the lysosomal inhibition, ubiquitin-proteosomal inhibition failed to raise both the cell-associated G26S EL (Fig. 4C) and media G26S EL (Fig. 4D) to levels comparable to WT EL. We confirmed the effectiveness of our chloroquine and ALLN treatments by assessing the lysosomal degradation of LDL apolipoprotein B and the accumulation of polyubiquitinated proteins, respectively (Supplemental Fig. II). To address whether newly synthesized G26S EL was being degraded through an alternate mechanism, we assessed the trafficking of newly synthesized EL using pulse-chase analyses. From quadruplicate experiments with cells transiently transfected with G26S or WT EL, following a 2 h pulse with [35S]methionine/cysteine, we consistently observed a ∼20% reduction of total (cell and media) immunoprecipitated newly synthesized 35S-G26S EL versus newly synthesized 35S-WT EL at all time points throughout a 2 h chase (Supplemental Fig. III). Despite the reduced mass of 35S-G26S EL versus 35S-WT EL throughout the chase, the rate of disappearance from cells (Fig. 5A), the rate of appearance into media (Fig. 5B), and the stability throughout the chase (Fig. 5C) of both 35S-G26S EL and 35S-WT EL were comparable. The rate constants for the disappearance of EL from cells (WT: 0.013 ± 0.003 min−1; G26S: 0.020 ± 0.017 min−1, errors represent ±SD) and the appearance of EL into media (WT, 0.031 ± 0.015 min−1; G26S, 0.019 ± 0.010 min−1) were not significantly different. These data show that there was no difference in trafficking between G26S EL and WT EL, but it suggests that a defect exists in the translation of G26S EL. Using an in vitro transcription/translation rabbit reticulocyte system in the presence of [35S]methionine, we compared the rates of translation between G26S EL and WT EL. Under these conditions, we failed to observe any difference in the rate of protein production between G26S EL (with a rate constant of 0.035 ± 0.005 min−1) and WT EL (with a rate constant of 0.040 ± 0.008 min−1) (Fig. 6).Fig. 6In vitro translation of WT and G26S EL. WT and the G26S variant of EL were expressed in a rabbit reticulocyte in vitro transcription/translation system through the T7 promoter. Completely and incompletelysynthesized EL proteins from three separate experiments we
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