Targeting of neutral cholesterol ester hydrolase to the endoplasmic reticulum via its N-terminal sequence

Abstract

Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membrane-spanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn270, Asn367, and Asn389) of NCEH are glycosylated. Glycosylation at Asn270, which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for efflux out of the cells. Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membrane-spanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn270, Asn367, and Asn389) of NCEH are glycosylated. Glycosylation at Asn270, which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for efflux out of the cells. The hydrolysis of intracellular cholesterol ester (CE) is the initial step of reverse cholesterol transport (1Brown M.S. Goldstein J.L. Krieger M. Ho Y.K. Anderson R.G. Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins.J. Cell Biol. 1979; 82: 597-613Google Scholar). Since this step is rate-limiting particularly in macrophage foam cells (2Kritharides L. Christian A. Stoudt G. Morel D. Rothblat G.H. Cholesterol metabolism and efflux in human THP-1 macrophages.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1589-1599Google Scholar, 3Graham A. Angell A.D. Jepson C.A. Yeaman S.J. Hassall D.G. Impaired mobilisation of cholesterol from stored cholesteryl esters in human (THP-1) macrophages.Atherosclerosis. 1996; 120: 135-145Google Scholar, 4Ishii I. Oka M. Katto N. Shirai K. Saito Y. Hirose S. Beta-VLDL-induced cholesterol ester deposition in macrophages may be regulated by neutral cholesterol esterase activity.Arterioscler. Thromb. 1992; 12: 1139-1145Google Scholar, 5Rothblat G.H. de la Llera-Moya M. Favari E. Yancey P.G. Kellner-Weibel G. Cellular cholesterol flux studies: methodological considerations.Atherosclerosis. 2002; 163: 1-8Google Scholar), a hallmark of atherosclerosis, it is important to clarify the mechanisms that mediate the hydrolysis of CE in foam cells (2Kritharides L. Christian A. Stoudt G. Morel D. Rothblat G.H. Cholesterol metabolism and efflux in human THP-1 macrophages.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1589-1599Google Scholar, 3Graham A. Angell A.D. Jepson C.A. Yeaman S.J. Hassall D.G. Impaired mobilisation of cholesterol from stored cholesteryl esters in human (THP-1) macrophages.Atherosclerosis. 1996; 120: 135-145Google Scholar, 4Ishii I. Oka M. Katto N. Shirai K. Saito Y. Hirose S. Beta-VLDL-induced cholesterol ester deposition in macrophages may be regulated by neutral cholesterol esterase activity.Arterioscler. Thromb. 1992; 12: 1139-1145Google Scholar, 5Rothblat G.H. de la Llera-Moya M. Favari E. Yancey P.G. Kellner-Weibel G. Cellular cholesterol flux studies: methodological considerations.Atherosclerosis. 2002; 163: 1-8Google Scholar). As the hydrolysis of CE preceding reverse cholesterol transport takes place at neutral pH, the enzymes catalyzing it have been collectively called neutral CE hydrolases (nCEHs) to distinguish them from lysosomal acid lipase, the CE hydrolase in lysosome whose optimal pH is acidic. Since carboxyl ester lipase (also called cholesterol esterase or bile salt-dependent lipase) is a secreted protein, it is not involved in the hydrolysis of intracellular CE (6Li F. Hui D.Y. Modified low density lipoprotein enhances the secretion of bile salt-stimulated cholesterol esterase by human monocyte-macrophages. Species-specific difference in macrophage cholesteryl ester hydrolase.J. Biol. Chem. 1997; 272: 28666-28671Google Scholar). To date, three enzymes have been proposed to serve as nCEHs in macrophages: hormone-sensitive lipase (HSL) (7Holm C. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis.Biochem. Soc. Trans. 2003; 31: 1120-1124Google Scholar, 8Yeaman S.J. Hormone-sensitive lipase–new roles for an old enzyme.Biochem. J. 2004; 379: 11-22Google Scholar); cholesterol ester hydrolase (CEH) (9Ghosh S. Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing, and expression of full-length cDNA.Physiol. Genomics. 2000; 2: 1-8Google Scholar), which is identical to a human liver carboxylesterase 1 (hCE-1) (10Long R.M. Calabrese M.R. Martin B.M. Pohl L.R. Cloning and sequencing of a human liver carboxylesterase isoenzyme.Life Sci. 1991; 48: PL43-PL49Google Scholar) or macrophage serine esterase1 (11Munger J.S. Shi G.P. Mark E.A. Chin D.T. Gerard C. Chapman H.A. A serine esterase released by human alveolar macrophages is closely related to liver microsomal carboxylesterases.J. Biol. Chem. 1991; 266: 18832-18838Google Scholar), also known as a human ortholog of porcine triacylglycerol hydrolase (TGH) (12Lehner R. Vance D.E. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol.Biochem. J. 1999; 343: 1-10Google Scholar); and neutral cholesterol ester hydrolase (NCEH) (13Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Google Scholar), which is also known as KIAA1363 or arylacetamide deacetylase-like 1 (14Nomura D.K. Leung D. Chiang K.P. Quistad G.B. Cravatt B.F. Casida J.E. A brain detoxifying enzyme for organophosphorus nerve poisons.Proc. Natl. Acad. Sci. USA. 2005; 102: 6195-6200Google Scholar). However, it is not clear which enzyme is of primary importance in the hydrolysis of CE in macrophages. In mice, the ablation of HSL did not significantly reduce nCEH activity in murine peritoneal macrophages (MPMs) (15Contreras J.A. Hormone-sensitive lipase is not required for cholesteryl ester hydrolysis in macrophages.Biochem. Biophys. Res. Commun. 2002; 292: 900-903Google Scholar, 16Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Google Scholar). Hence, HSL is not the major enzyme mediating nCEH activity in MPMs. Ghosh reported CEH as a promising candidate for an nCEH because its macrophage-specific overexpression under the promoter of scavenger receptor protected against diet-induced atherosclerosis in LDL receptor knockout mice (17Zhao B. Song J. Chow W.N. St Clair R.W. Rudel L.L. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice.J. Clin. Invest. 2007; 117: 2983-2992Google Scholar). However, the effects of loss of function of CEH on nCEH activity in macrophages have not been reported. Furthermore, a mouse ortholog of CEH, TGH, was expressed at a low level in MPMs (13Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Google Scholar) and possessed a negligible nCEH activity compared with its hydrolase activity toward p-nitrophenyl butyrate (PNPB), triolein, and monoolein (18Okazaki H. Igarashi M. Nishi M. Tajima M. Sekiya M. Okazaki S. Yahagi N. Ohashi K. Tsukamoto K. Amemiya-Kudo M. et al.Identification of a novel member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte lipolysis.Diabetes. 2006; 55: 2091-2097Google Scholar). In contrast, NCEH is robustly expressed in MPMs as well as in macrophage foam cells in atherosclerotic lesions, and the knockdown of NCEH by short interference RNA significantly reduced nCEH activity in MPMs (18Okazaki H. Igarashi M. Nishi M. Tajima M. Sekiya M. Okazaki S. Yahagi N. Ohashi K. Tsukamoto K. Amemiya-Kudo M. et al.Identification of a novel member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte lipolysis.Diabetes. 2006; 55: 2091-2097Google Scholar). Therefore, NCEH is more likely to be involved in the hydrolysis of CE in MPMs. Recently, it has become clear that NCEH is a mouse ortholog of KIAA1363. KIAA1363 was initially identified as a membrane-bound serine hydrolase whose expression is upregulated in invasive cancer cells (19Jessani N. Liu Y. Humphrey M. Cravatt B.F. Enzyme activity profiles of the secreted and membrane proteome that depict cancer Natl. Acad. Sci. USA. 2002; Scholar). in the of Cravatt and Casida have that KIAA1363 is protein (14Nomura D.K. Leung D. Chiang K.P. Quistad G.B. Cravatt B.F. Casida J.E. A brain detoxifying enzyme for organophosphorus nerve poisons.Proc. Natl. Acad. Sci. USA. 2005; 102: 6195-6200Google Scholar), which has hydrolase activity and serves as a in an that and acid K.P. S. A. Cravatt B.F. enzyme that in cancer by Biol. 2006; Scholar). the novel function of KIAA1363 not the that KIAA1363 also serves as NCEH because enzymes on of it is where and NCEH is with and its In this we that NCEH is a single-membrane-spanning endoplasmic reticulum (ER) protein that to the ER membrane an N-terminal catalytic and lipid-binding are located in the ER lumen. N-linked at is for its enzymatic activity. and from and from was from was from was from an was from to LDL and from the by density H.A. The and of in human Clin. Invest. Scholar). LDL was acetylated by of J.L. M.S. of lipoprotein in Scholar). and cells in with or 1 MPMs as (18Okazaki H. Igarashi M. Nishi M. Tajima M. Sekiya M. Okazaki S. Yahagi N. Ohashi K. Tsukamoto K. Amemiya-Kudo M. et al.Identification of a novel member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte lipolysis.Diabetes. 2006; 55: 2091-2097Google Scholar). was from and for to the of the of the of or NCEH cDNA was or and as NCEH or NCEH. Furthermore, NCEH cDNA or NCEH cDNA was and as protein expression was or as Asn270, Asn367, and by and by was the to an 1997; Scholar). The to the are as and A NCEH that the for the N-terminal of NCEH was by as a The full-length NCEH cDNA was as a The was the or and In the for the N-terminal and of NCEH was by and the and The as and reverse and reverse the or HSL that been in the (18Okazaki H. Igarashi M. Nishi M. Tajima M. Sekiya M. Okazaki S. Yahagi N. Ohashi K. Tsukamoto K. Amemiya-Kudo M. et al.Identification of a novel member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte lipolysis.Diabetes. 2006; 55: 2091-2097Google Scholar). acid of human NCEH was expressed in as a protein, which was by and for of to the as H. J. Tamura Y. Yahagi N. S. Shionoiri F. Y. Ohashi K. K. S. et in the of hormone-sensitive for a regulating 2002; Scholar). with a protein of was as a of human NCEH human and mouse NCEH. Furthermore, we of mouse which acid catalytic domain of mouse NCEH was in (18Okazaki H. Igarashi M. Nishi M. Tajima M. Sekiya M. Okazaki S. Yahagi N. Ohashi K. Tsukamoto K. Amemiya-Kudo M. et al.Identification of a novel member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte lipolysis.Diabetes. 2006; 55: 2091-2097Google Scholar). which was in the J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Google Scholar). was also in the H. T. J. Tozawa R. Y. Oka T. S. Tamura Y. Ohashi K. et of atherosclerosis but and in mice with Biol. Chem. 2000; Scholar). was from was from The mouse was from The was from in A pH 1 and and at for at Furthermore, the was as The was at for at The was under the and as the The of the and at for at The and as a microsomal cells in A and at for at The was in of of to and to a of the the incubated with the at a of in a with a and by on a cells in A and at for at The was to enzymatic activity. nCEH activity was as by and S. neutral cholesteryl A hormone-sensitive enzyme from lysosomal cholesteryl Biol. Chem. Scholar), a cholesterol 1 activity was determined by the of of at at by a K. M. J.A. hydrolysis of of low density and of and of on the catalytic Res. Scholar). cells in The was on the with S. expressed in membrane and Biol. 2003; Scholar, Goldstein J.L. M.S. of human a protein of Biol. Chem. 2004; Scholar). MPMs in to with and with 1 in or with in at for of pH and was to was by the of K. The at for The by of at a of in A and at for at The was for in The and cells in in for at by to 1 or in for at with in for at at and incubated with primary at was to neutral cells incubated with or in in for 1 at in and with a a and a or with an and a The with MPMs in A and was by and to the In cells with with incubated for and A The by for in the to the The by of cells or with NCEH or HSL by of mouse which was to by mouse was to the of cells in and the with 1 of the cells in and for and incubated in 1 of for 1 and in the or of a protein A for in A and at for at The of protein in the was determined a The was by of mouse with a and are as was to the All with for the of NCEH. of three domains: an N-terminal a catalytic and a lipid-binding and the domain in The N-terminal of NCEH a a of the acid sequence of mouse NCEH. on this NCEH is a type II membrane protein with a The of NCEH is in In with this of NCEH in the microsomal MPMs by of subcellular is to that of an ER protein that the membrane R.G. K. Anderson Rudel L.L. and membrane a serine for activity to of the endoplasmic reticulum Biol. 2000; Scholar, S. D. Liu M.S. J. in the endoplasmic reticulum Biol. Chem. 1999; Scholar) but is from that of a ER protein, or a protein C. of in of hormone-sensitive lipase to the Natl. Acad. Sci. USA. 1992; Scholar). was in the and microsomal because a the HSL in macrophages was as as (13Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Google Scholar). The was in the the was in the microsomal To that NCEH is to the we for NCEH in MPMs for NCEH a the cells, a of an the the for HSL was that the of NCEH in the ER by the ER protein Cholesterol loading by incubation with the of the of NCEH protein in the or microsomal A in the ER localization was NCEH was in cells The of for with the of the ER Cholesterol loading by incubation with did not change the of To NCEH is on as a for the hydrolysis of neutral we to of NCEH the of and the of NCEH on the ER the cholesterol To that of NCEH its catalytic domain and lipid-binding domain is in the ER as by the we MPMs with and and an against the catalytic domain of NCEH and against or HSL as with with and ER with with three that NCEH and are located in the ER and that HSL is in the results of by an in which to by was compared of MPMs with NCEH was to K. However, with NCEH was to the To NCEH is a we the membrane with with or the and of NCEH to a the of NCEH as may from the of glycosylation in to that it is located in the ER and does not the where N-linked become to by II in the A in was cells with the NCEH expression incubated with which N-linked glycosylation NCEH does not of because the protein by the in of NCEH was identical in to NCEH with NCEH three potential N-linked glycosylation sites or Asn270, Asn367, and To clarify sites was to by The of to is as or on this acid and NCEH or of the expressed in cells A a of was not mutants of the three and that three are glycosylated. the three mutants with a that a the the three with the and results that glycosylation at the to The NCEH three the as the NCEH with that three are sites for N-linked To which N-linked glycosylation is important for the enzymatic we the hydrolase activity of NCEH The expressed as hydrolase activity as the NCEH. the nCEH of the and mutants significantly reduced compared with NCEH. In the hydrolase activity of the was reduced to Hence, N-linked glycosylation in an important role in A of Asn270, Asn367, and to the subcellular was not To that the or the serine of serine esterase is for enzymatic we mutants in which the catalytic in or in was by and The hydrolase activity of of mutants was reduced to To the role of the N-terminal of we a NCEH that the N-terminal and expressed it in cells of the expressed significantly reduced compared with of NCEH Furthermore, the expressed was a whose was as as that of NCEH with or the NCEH three potential N-linked glycosylation that the did not N-linked with this did not change the of the was not in the where the cells incubated not results that the N-terminal of NCEH the of the protein and that is not glycosylated. we of of to the subcellular of NCEH within cells with an ER protein, is from the expression of and that of the protein to the of NCEH did not the subcellular the was the and did not with comprising the N-terminal of NCEH with with In to the was also in the where it was not with comprising the N-terminal of NCEH with with results that the N-terminal of NCEH serves as a type II signal anchor sequence which NCEH to the ER with its within the M. R. T. K. T. of signal and are determined by the the and the N-terminal Natl. Acad. Sci. USA. 1992; Scholar). the results of that the NCEH and in the microsomal it in the that the N-terminal of NCEH is a As from the with NCEH a domain that potential sites for the of enzymatic To that NCEH is not we HSL or NCEH in cells and them in low the cells incubated in a with The in which HSL or NCEH was by an against the and to for HSL or NCEH or of the HSL protein, which was by with a was In contrast, NCEH was not the with we HSL or NCEH in cells by and the effects of of on the nCEH activity of the expressed enzymes with 1 significantly the nCEH activity of HSL by as reported J. Yeaman S.J. cholesterol ester hydrolase from and relationship to hormone-sensitive Biophys. Scholar, J.L. H.A. K. Hormone-sensitive lipase overexpression cholesteryl ester hydrolysis in macrophage foam Thromb. Vasc. Biol. 1998; 18: Scholar). However, did not the nCEH activity of that NCEH was not at under where is of the nCEH activity in the from MPMs may from the of not NCEH. In this we that NCEH is to the ER membrane with its catalytic domain in the ER lumen. The N-terminal are required for to the NCEH three potential N-linked glycosylation sites that are glycosylated. In the N-linked glycosylation at Asn270, which is located closest to the catalytic is for the enzymatic activity. NCEH to a family of ER that are and in the ER lumen. The this are and the sequence The signal for of the endoplasmic Sci. Scholar, S. A signal secretion of ER 48: Scholar). NCEH to of that are membrane and the ER ER involved in have a are and In contrast, NCEH has a it is a type II membrane protein with its to the ER lumen. The N-terminal is the domain that NCEH to the ER In to the protein, the N-terminal the as a the ER because NCEH of this does not in the which is with the loss of N-linked glycosylation of A of the is required for the as NCEH. are with the that ER are for membrane by a signal at the G. protein Natl. Acad. Sci. USA. Scholar). The of is determined by the of the membrane and the of the the membrane G. M. H. G. for membrane protein in Biol. Chem. 1997; 272: Scholar). and proposed the of signal on a of the N-terminal of 1 and esterase H. G. Gerard C. J. to the of endoplasmic reticulum N-terminal of and the Biol. Chem. 1999; Scholar). to an acid sequence of short or neutral at the by a of with is for of The acid sequence of the of NCEH is with this II of NCEH a of that are by and NCEH is by that NCEH is to the ER and not to the that three putative sites of NCEH (Asn270, Asn367, and Asn389) are In N-linked glycosylation at an important role in The of is required for the of of glycosylation in the of in Biol. Chem. Scholar). N-linked glycosylation at may a more important role in the of NCEH. was important for NCEH to of the is and of the is is not important for the enzymatic activity of NCEH the of NCEH in glycosylation at may have enzymatic activity. A difference is unknown. the CE in macrophage foam cells is in and its hydrolysis the of the CE hydrolases with HSL and CEH are et C. of in of hormone-sensitive lipase to the Natl. Acad. Sci. USA. 1992; Scholar) reported that HSL from the to with in et B. St Clair R.W. Rudel L.L. Ghosh S. of macrophage cholesteryl ester hydrolase from to Res. 2005; Scholar) reported that CEH to cholesterol loading in macrophages. and TGH, of CEH, have been reported to to the ER its ER in D. M. Vance D.E. R. hydrolase is to the endoplasmic reticulum by an sequence where it in as Biol. 2005; Scholar). is subcellular localization of the protein is of cells. In to it is to an ER protein with its catalytic domain in the ER which in in foam cells the efflux of cholesterol from THP-1 cells (13Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Google Scholar). Recently, of the of in has The Biol. Chem. 2008; 283: Scholar). to the as the of the ER are from the of a CE by an ER protein with its catalytic the R.G. K. Anderson Rudel L.L. and membrane a serine for activity to of the endoplasmic reticulum Biol. 2000; Scholar, S. D. Liu M.S. J. in the endoplasmic reticulum Biol. Chem. 1999; Scholar), in in macrophages the N. Y. Cholesterol and Cell Biol. 2006; Scholar). to the and ER may be more has been is on and its overexpression the of with the ER S. J. K. N. H. Fujimoto T. to and to the endoplasmic Cell Sci. 2005; Scholar, S. K. S.J. M. R.G. localization of to effects of and of Biol. Chem. 2005; Scholar). Therefore, it may not to a role for NCEH in the hydrolysis of CE in the CE in the is a of transport of the to the ER for and transport to the NCEH may CE in the by this The hydrolysis of neutral in by ER enzymes is also in and and and which are to the ER hydrolyzes in of R. M. K. is a major adipocyte Biol. Chem. 2004; Scholar). was reported that nCEH activity is by in or macrophages D. Neutral cholesterol esterase activity in macrophages and its by protein Biol. Chem. Scholar, of a neutral cholesteryl ester hydrolase by in Biophys. Scholar, C.A. J.A. Yeaman S.J. and of hormone-sensitive lipase in 1991; Scholar). In this the nCEH activity of from MPMs was by but the difference was not we reported that NCEH a major role in the nCEH activity of MPMs (13Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Google Scholar). the it is that HSL a role NCEH in the nCEH activity of macrophage as and on the of expression of NCEH and HSL the nCEH activity of macrophage is significantly by In NCEH is a single-membrane-spanning ER protein that to the ER membrane with an N-terminal catalytic and lipid-binding are located in the ER lumen. The N-terminal serves as a signal sequence that NCEH to the ER lumen. N-linked glycosylation at is for its enzymatic activity. of the mechanisms by which NCEH cholesterol from macrophage foam cells the for the of novel for the of has been as an of or in of this we reported the and of mice cholesterol ester cholesterol ester hydrolase endoplasmic reticulum protein hormone-sensitive lipase murine peritoneal macrophage neutral cholesterol ester hydrolase neutral cholesterol ester hydrolase protein protein A p-nitrophenyl butyrate triacylglycerol hydrolase