Properties of the Mouse Intestinal Acyl-CoA:Monoacylglycerol Acyltransferase, MGAT2
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m302835200
ISSN1083-351X
AutoresJingsong Cao, Paul Burn, Yuguang Shi,
Tópico(s)Alcohol Consumption and Health Effects
ResumoAcyl-CoA:monoacylglycerol acyltransferase (MGAT) plays an important role in dietary fat absorption by catalyzing a rate-limiting step in the re-synthesis of diacylglycerols in enterocytes. The present study reports further characterization of MGAT2, a newly identified intestinal MGAT (Cao, J., Lockwood, J., Burn, P., and Shi, Y. (2003) J. Biol. Chem. 278, 13860–13866) for its substrate specificity, requirement for lipid cofactors, optimum pH and Mg2+, and other intrinsic properties. MGAT2 enzyme expressed in COS-7 cells displayed a broad range of substrate specificity toward fatty acyl-CoA derivatives and monoacylglycerols, among which the highest activities were observed with oleoyl-CoA and rac-1-monolauroylglycerol, respectively. MGAT2 appeared to acylate monoacylglycerols containing unsaturated fatty acyls in preference to saturated ones. Lipid cofactors that play roles in signal transduction were shown to modulate MGAT2 activities. In contrast to oleic acid and sphingosine that exhibited inhibitory effects, phosphatidylcholine, phosphatidylserine, and phosphatidic acid stimulated MGAT2 activities. Using recombinant murine MGAT2 expressed in Escherichia coli, we demonstrated conclusively that MGAT2 also possessed an intrinsic acyl-CoA:diacylglycerol acyltransferase (DGAT) activity, which could provide an alternative pathway for triacylglycerol synthesis in the absence of DGAT. In contrast to the inhibitory effect on MGAT2 activities, nonionic and zwitterionic detergents led to a striking activation of DGAT activity of the human DGAT1 expressed in mammalian cells, which further distinguished the behaviors of the two enzymes. The elucidation of properties of MGAT2 will facilitate future development of compounds that inhibit dietary fat absorption as a means to treat obesity. Acyl-CoA:monoacylglycerol acyltransferase (MGAT) plays an important role in dietary fat absorption by catalyzing a rate-limiting step in the re-synthesis of diacylglycerols in enterocytes. The present study reports further characterization of MGAT2, a newly identified intestinal MGAT (Cao, J., Lockwood, J., Burn, P., and Shi, Y. (2003) J. Biol. Chem. 278, 13860–13866) for its substrate specificity, requirement for lipid cofactors, optimum pH and Mg2+, and other intrinsic properties. MGAT2 enzyme expressed in COS-7 cells displayed a broad range of substrate specificity toward fatty acyl-CoA derivatives and monoacylglycerols, among which the highest activities were observed with oleoyl-CoA and rac-1-monolauroylglycerol, respectively. MGAT2 appeared to acylate monoacylglycerols containing unsaturated fatty acyls in preference to saturated ones. Lipid cofactors that play roles in signal transduction were shown to modulate MGAT2 activities. In contrast to oleic acid and sphingosine that exhibited inhibitory effects, phosphatidylcholine, phosphatidylserine, and phosphatidic acid stimulated MGAT2 activities. Using recombinant murine MGAT2 expressed in Escherichia coli, we demonstrated conclusively that MGAT2 also possessed an intrinsic acyl-CoA:diacylglycerol acyltransferase (DGAT) activity, which could provide an alternative pathway for triacylglycerol synthesis in the absence of DGAT. In contrast to the inhibitory effect on MGAT2 activities, nonionic and zwitterionic detergents led to a striking activation of DGAT activity of the human DGAT1 expressed in mammalian cells, which further distinguished the behaviors of the two enzymes. The elucidation of properties of MGAT2 will facilitate future development of compounds that inhibit dietary fat absorption as a means to treat obesity. Acyl-CoA:monoacylglycerol acyltransferase (MGAT) 1The abbreviations used are: MGAT, acyl-CoA:monoacylglycerol acyltransferase; DGAT, acyl-CoA:diacylglycerol acyltransferase; mMGAT, mouse MGAT; Rac, racemic; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid.1The abbreviations used are: MGAT, acyl-CoA:monoacylglycerol acyltransferase; DGAT, acyl-CoA:diacylglycerol acyltransferase; mMGAT, mouse MGAT; Rac, racemic; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid. catalyzes the synthesis of diacylglycerols from monoacylglycerols and long chain fatty acyl-CoAs, a first step in the monoacylglycerol pathway contributing >80% of glycerolipid synthesis in the intestinal mucosa (1Johnston J.M. Snyder F. Lipid Metabolism in Mammals. Plenum Press, New York1977: 151-188Crossref Google Scholar, 2Lehner R. Kuksis A. Prog. Lipid Res. 1996; 35: 169-201Crossref PubMed Scopus (254) Google Scholar). The dietary fat (mainly triacylglycerols) is digested into fatty acids and sn-2-monacylglycerols and absorbed into enterocytes where fatty acids and sn-2-monoacylglycerols are utilized to sequentially re-synthesize diacylglycerols and triacylglycerols by MGAT and acyl-CoA: diacylglycerol acyltransferase (DGAT), allowing for the lipid to be transported into the circulation system by chylomicron. Therefore, MGAT plays a critical role in intestinal dietary fat absorption. MGAT may also play a role in signaling since its enzymatic product diacylglycerol is an activator of protein kinase C as well as an intermediate in the synthesis of phospholipids (3Ron D. Kazanietz M.G. FASEB J. 1999; 13: 1658-1676Crossref PubMed Scopus (551) Google Scholar, 4Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4215) Google Scholar, 5Nakamura S. Asaoka Y. Yoshida K. Sasaki Y. Nishizuka Y. Adv. Second Messenger Phosphoprotein Res. 1993; 28: 171-178PubMed Google Scholar, 6Hjelmstad R.H. Bell R.M. Biochemistry. 1991; 30: 1731-1740Crossref PubMed Scopus (52) Google Scholar, 7Lehner R. Kuksis A. Biochim. Biophys. Acta. 1992; 1125: 171-179Crossref PubMed Scopus (28) Google Scholar). Hepatic MGAT activity was found at high levels in suckling rats, diabetic and hibernating animals (8Coleman R.A. Haynes E.B. J. Biol. Chem. 1984; 259: 8934-8938Abstract Full Text PDF PubMed Google Scholar, 9Mostafa N. Everett D.C. Chou S.C. Kong P.A. Florant G.L. Coleman R.A. J. Comp. Physiol. B Biochem. Syst. Environ. 1993; 163: 463-469PubMed Google Scholar, 10Mostafa N. Bhat B.G. Coleman R.A. Biochim. Biophys. Acta. 1993; 1169: 189-195Crossref PubMed Scopus (24) Google Scholar), suggesting that it could be regulated by the high influx of fatty acids. MGAT may also play an important role in systemic regulation of glycerolipid biosynthesis since its sn-2-monoacylglycerol is a competitive inhibitor of glycerol-3-phosphate acyltransferase (11Polheim D. David J.S. Schultz F.M. Wylie M.B. Johnston J.M. J. Lipid Res. 1973; 14: 415-421Abstract Full Text PDF PubMed Google Scholar, 12Coleman R.A. Biochim. Biophys. Acta. 1988; 963: 367-374Crossref PubMed Scopus (12) Google Scholar). The contribution of MGAT to adipose glycerolipid synthesis is also demonstrated (13Jamdar S.C. Cao W.F. Arch. Biochem. Biophys. 1992; 296: 419-425Crossref PubMed Scopus (20) Google Scholar).The biochemical properties of MGAT has been extensively investigated in intestine of various animal species (14Johnston J.M. Paultauf F. Schiller C.M. Schultz L.D. Biochim. Biophys. Acta. 1970; 218: 124-133Crossref PubMed Scopus (46) Google Scholar, 15Paltauf F. Johnston J.M. Biochim. Biophys. Acta. 1971; 239: 47-56Crossref PubMed Scopus (26) Google Scholar, 16Lehner R. Kuksis A. Itabashi Y. Lipids. 1993; 28: 29-34Crossref PubMed Scopus (27) Google Scholar, 17Manganaro F. Kuksis A. Can. J. Biochem. Cell Biol. 1985; 63: 341-347Crossref PubMed Scopus (28) Google Scholar) as well as in the intestine and liver of suckling and adult rats (8Coleman R.A. Haynes E.B. J. Biol. Chem. 1984; 259: 8934-8938Abstract Full Text PDF PubMed Google Scholar, 18Bhat B.G. Wang P. Coleman R.A. J. Biol. Chem. 1994; 269: 13172-13178Abstract Full Text PDF PubMed Google Scholar, 19Coleman R.A. Methods Enzymol. 1992; 209: 98-104Crossref PubMed Scopus (80) Google Scholar, 20Coleman R.A. Haynes E.B. J. Biol. Chem. 1986; 261: 224-228Abstract Full Text PDF PubMed Google Scholar, 21Coleman R.A. Walsh J.P. Millington D.S. Maltby D.A. J. Lipid Res. 1986; 27: 158-165Abstract Full Text PDF PubMed Google Scholar, 22Bhat B.G. Wang P. Coleman R.A. Biochemistry. 1995; 34: 11237-11244Crossref PubMed Scopus (17) Google Scholar), kidney (23Barac-Nieto M. Cohen J.J. Nephron. 1971; 8: 488-499Crossref PubMed Google Scholar, 24Trimble M.E. Life Sci. 1978; 22: 883-890Crossref PubMed Scopus (3) Google Scholar, 25Trimble M.E. Harrington Jr., W.W. Bowman R.H. Curr. Probl. Clin. Biochem. 1977; 8: 362-370PubMed Google Scholar), and rat adipocytes (13Jamdar S.C. Cao W.F. Arch. Biochem. Biophys. 1992; 296: 419-425Crossref PubMed Scopus (20) Google Scholar). Because of its association with the microsomal membranes and its alleged involvement in an enzyme complex, MGAT has been difficult to purify to homogeneity (26Lehner R. Kuksis A. J. Biol. Chem. 1995; 270: 13630-13636Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Several partial purifications of MGAT from rat intestinal membranes and neonatal liver have been reported previously (17Manganaro F. Kuksis A. Can. J. Biochem. Cell Biol. 1985; 63: 341-347Crossref PubMed Scopus (28) Google Scholar, 27Bhat B.G. Bardes E.S. Coleman R.A. Arch. Biochem. Biophys. 1993; 300: 663-669Crossref PubMed Scopus (23) Google Scholar). However, properties on a pure MGAT have never been extensively studied because of the lack of a cloned gene encoding the enzyme.The recent cloning and identification of an intestinal MGAT enzyme, MGAT2 (28Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), allows us to evaluate the intrinsic characteristics of the enzyme. The mouse MGAT2 is most abundantly expressed in the small intestine (28Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) where the highest MGAT activity was detected. MGAT2 can catalyze the acylation of each of sn-1-monoacylglycerol, sn-2-monoacylglycerol, and sn-3-monoacylglycerol (28Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). MGAT2-transfected cells also displayed DGAT activity. However, many biochemical characteristics such as acyl donor and acceptor preference and specificity, pH and magnesium optimum, potential activators and inhibitors, and other intrinsic properties await further investigation. By expression of MGAT2 in mammalian cells as well as in bacterial cells, this study examined these properties of the enzyme, mMGAT2.EXPERIMENTAL PROCEDURESMaterials—Rac-1-octanoylglycerol,rac-1-lauroylglycerol,rac-1-palmitoylglycerol, rac-1-stearoylglycerol, rac-1-oleoylglycerol, rac-1-linoleoylglycerol, rac-1-linolenoylglycerol, 1-O-palmityl-rac-glycerol, phosphatidylcholine, phosphatidic acid, phosphatidylserine, lysophosphatidic acid, Triton X-100, SDS, CHAPS, rac-glycerol 3-phosphate, sn-glycerol 2-phosphate, 1-O-hexadecyl-rac-glycerol, and fatty acyl-CoAs (malonyl-CoA, n-octanoyl-CoA, lauroyl-CoA, palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA, arachidoyl-CoA, linoleoyl-CoA, and arachidonoyl-CoA) were purchased from Sigma. All of the other monoacylglycerols (sn-2-oleoylglycerol), diacylglycerols (rac-1,2-dioctanoylglycerol, sn-1,3-dioctanoylglycerol, rac-1,2-dilauroylglycerol, sn-1,3-dilauroylglycerol, rac-1,2-dipalmitoylglycerol, sn-1,3-dipalmitoylglycerol, rac-1,2-distearoylglycerol, sn-1,3-distearoylglycerol sn-1,2-dioleoylglycerol, rac-1,2-dioleoylglycerol, sn-1,3-dioleoylglycerol, sn-1,3-dilinoleoylglycerol, and sn-1,3-linolenoylglycerol) and triacylglycerols (1,2,3-triocatanoylglycerol, 1,2,3-trilauroylglycerol, 1,2,3-tripalmitoylglycerol, 1,2,3-trioleoylglycerol, 1,2,3-tristearoylglycerol, 1,2,3-trilinoleoylglycerol, 1,2,3-trilinolenoylglycerol, and 1,2,3-triarachinoylglycerol) used as substrates or standards were obtained from Doosan Serdary Research Laboratories (Toronto, Ontario, Canada). [14C]Oleoyl-CoA (50 mCi/mmol) and sn-2-[3H]monooleoylglycerol (60 Ci/mmol) were from American Radiolabeled Chemicals Inc. (St. Louis, MO).Expression of MGAT2 and DGAT1 in Mammalian Cells—A mammalian expression plasmid coding full-length mouse MGAT2 was engineered as described previously (28Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Similarly, the human DGAT1 coding sequence was amplified from human small intestine cDNA (BD Biosciences and Clontech, Palo Alto, CA) and cloned into NotI and EcoRV sites of the pcDNA3.1/Hygro(–) mammalian expression vector (Invitrogen). COS-7 cells were maintained under the conditions recommended by American Tissue Culture Collection (Manassas, VA) and transiently transfected with vectors with or without mouse MGAT2 or human DGAT1 cDNA as described previously (28Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Forty-eight hours after transfection, cells were harvested in ice-cold phosphate-buffered saline, pelleted by centrifugation, homogenized, and assayed immediately or frozen in liquid N2 for later use.In Vitro Assays for MGAT and DGAT Activity—Cell pellets were homogenized in 20 mm NaCl with three short 10-s pulses from a Brinkmann Polytron. The resultant homogenates were used to assess the activity of MGAT and DGAT in transfected mammalian cells. The protein concentration in homogenates was determined by a BCA Protein Assay Kit (Pierce) according to manufacturer's instructions. MGAT and DGAT activity was determined at room temperature in a final volume of 100 or 200 μl as previously described (28Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). MGAT activity was determined by measuring the incorporation of [14C]oleoyl moiety into diacylglycerol with [14C]oleoyl-CoA (acyl donor) and various monoacylglycerols (acyl acceptors) or by measuring the incorporation of acyl moiety into diacylglycerol with various acyl-CoAs and sn-2-[3H]monooleoylglycerol. The incorporation of [14C]oleoyl moiety into trioleoylglycerol with [14C]oleoyl-CoA and sn-1,2-dioleoylglycerol was measured to obtain DGAT activity. The acyl acceptors were introduced into the reaction mixture by liposomes prepared with phosphatidylcholine/phosphatidylserine (molar ratio ≈1:5). Unless indicated otherwise, the reaction mixture contained 100 mm Tris/HCl, pH 7.0, 5 mm MgCl2, 1 mg/ml bovine serum albumin free fatty acids (Sigma), 200 mm sucrose, 20 μm of various cold acyl-CoAs or [14C]oleoyl-CoA (50 mCi/mmol), 2 μm sn-2-[3H]monooleoylglycerol (60 Ci/mmol) or 200 μm of various cold acyl acceptors, and 50–100 μg of cell homogenate protein or 0.5 μg of partially purified protein from Escherichia coli. The indicated concentration of specific phospholipids, detergents, or other inhibitors and activators was delivered into reactions together with substrates. After a 10–20-min incubation at room temperature, lipids were extracted with chloroform/methanol (2:1, v/v). After centrifugation to remove debris, aliquots of the organic phase-containing lipids were dried under a speed vacuum and separated by the Linear-K Preadsorbent TLC plate (Waterman Inc., Clifton, NJ) with hexane:ethyl ether:acetic acid (80: 20:1, v/v/v). The separation was always performed under the conditions where sn-1,2-(2,3)-diacylglycerol and sn-1,3-diacylglycerols were clearly resolved. Individual lipid moieties were identified by standards with exposure to I2 vapor. The TLC plates were exposed to a PhosphorScreen to assess the formation of 14C- or 3H-labeled lipid products. Phosphor-imaging signals were visualized using a Storm 860 (Amersham Biosciences) and quantitated using ImageQuant software.Expression and Purification of mMGAT2 from Escherichia coli— mMGAT2 cDNA was subcloned into an E. coli expression vector for bacterial expression and purification of mouse MGAT2 enzyme. E. coli cell line DH5α was transformed with either empty vector (mock-transformed) or vector containing mMGAT2. Two liters of cultures in LB medium were incubated at 37 °C until A 600 reached 0.7. Expression of recombinant mMGAT2 was then induced by adding 1 mm isopropyl-β-d-thiogalactopyranoside for 16 h at 18 °C. Cells were homogenized by sonication in an extraction buffer containing 50 mm Tris/HCl, pH 7.5, 150 mm NaCl, 5 mm β-mercaptoethanol, and protease inhibitors. The clarified extract was loaded onto a heparin column (Amersham Biosciences) followed by washing the column sequentially with 40 ml of Tris buffer A (50 mm Tris/HCl, pH 7.4, 150 mm NaCl, 10% glycerol, 5 mm mercaptoethanol, and protease inhibitors) and 50 ml of Tris buffer A containing 2 m NaCl. Bound proteins were eluted with a linear gradient of 150 mm to 1.5 m NaCl. The protein was identified with 12.5% SDS-PAGE gel by Coomassie Blue staining and Western blot analysis.RESULTSSubstrate Specificity of mMGAT2 toward Fatty Acyl-CoA Derivatives and Monoacylglycerols—First, the preference of mMGAT2 toward fatty acyl-CoA derivatives was determined in assays using sn-2-[3H]monooleoylglycerol as acyl acceptor. The experiments were conducted under the conditions where 2 μm sn-2-[3H]monooleoylglycerol was incubated with 20 μm of various fatty acyl-CoA derivatives for 20 min. As shown in Fig. 1, A and B (quantitative analysis of signals in panel A), MGAT2 displayed a broad substrate pattern toward various fatty acyl-CoA derivatives including n-octanoyl-CoA (C8:0), lauroyl-CoA (C12:0), palmitoyl-CoA (C16:0), stearoyl-CoA (C18:0), arachidoyl-CoA (C20:0), oleoyl-CoA (18:1), linoleoyl-CoA (18:2), and arachidonoyl-CoA (20:4). MGAT activity displayed a preference of long chain acyl-CoA derivatives in the range of C8:0–C18:0 but began to decline when arachidoyl-CoA (C20:0) was used as acyl donor. The greatest activity was observed with palmitoyl-CoA (C16:0), stearoyl-CoA (C18:0), and oleoyl-CoA (C18:1). MGAT2 showed dichotomy activities toward unsaturated fatty acids, exhibiting a decreased activity with linoleoyl-CoA (C18:2) compared with C18:0 and C18:1 but a significant enhanced activity with arachidonoyl-CoA (C20:4) compared with C20:0 (Fig. 1, A and B). In accordance with increased MGAT activity, a significant portion of diacylglycerols was converted to triacylglycerols, which are presumably catalyzed by DGAT activity of MGAT2 or the endogenous DGAT activity of COS-7 cells (Fig. 1, A and B). The observed difference in the ratio of triacylglycerol/diacylglycerol between panels A and C of Fig. 1 is probably because of the different ratio of acyl-CoAs and monoacylglycerols in the reaction. More triacylglycerol was formed under the conditions with a high ratio of acyl-CoA, which may be facilitated by excess of acyl-CoA.Second, the substrate specificity of mMGAT2 toward monoacylglycerols containing various fatty acyl chains was determined in assays using [14C]oleoyl-CoA. Because the specific activities of MGAT2 toward either rac-1-monoacylglycerols or sn-2-monoacylglycerols are similar (28Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), the rac-1-monoacylglycerols were used in the assay. The experiments were performed under the conditions where 50 μm of each monoacylglycerol was incubated with 20 μm [14C]oleoyl-CoA for 20 min. The experiment compared the MGAT activities toward monoacylglycerols containing saturated fatty acids (rac-1-octanoylglycerol (C8:0), rac-1-lauroylglycerol (C12:0), rac-1-palmitoylglycerol (C16:0), and rac-1-stearoylglycerol (C18:0)), unsaturated fatty acids (rac-1-oleoylglycerol (C18:1), rac-1-linoleoylglycerol (C18:2), and rac-1-linolenoylglycerol (C18:3)), and an ether analogue (rac-1-O-palmitylglycerol). In contrast to the preference to acyl-CoAs with longer carbon chain, MGAT2 catalyzed more efficiently the acylation of rac-1-monoacylglycerols containing shorter fatty acyl chains as shown in Fig. 1C and quantitative analysis in Fig. 1D. A much weaker activity was observed with rac-1-monostearoylglycerol, inconsistent with previous observations (28Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 29Bierbach H. Digestion. 1983; 28: 138-147Crossref PubMed Scopus (24) Google Scholar, 30Yen C.L. Farese Jr., R.V. J. Biol. Chem. 2003; 278: 18532-18537Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Furthermore, MGAT2 displayed striking preference toward monoacylglycerols containing unsaturated fatty acids in an order of C18:3>C18:2>C18:1>C18:0. A glycerol ether derivative, rac-1-O-palmitylglycerol can be also utilized as substrates for MGAT2 (Fig. 1C, arrow) although less efficiently than its acyl analogue, rac-1-palmitoylglycerol. In contrast, glycerol 3-phosphate and glycerol 2-phosphate are not substrates for MGAT2 (data not shown). The utilization of any of the acyl substrates in the assays did not exceed 20% of the original amount.pH and Mg2+ Profiles of MGAT2—The pH profile of the MGAT2 was determined using 100 mm Tris buffers between pH 6.0 and 9.0 with an interval of 0.5. As shown in Fig. 2, A and B (quantitative analysis), the optimal activity of MGAT2 was found at pH 7.0 with a broad profile between pH 7 and 9 with more 1,3-diacylglycerol formed at higher pH values. The effect of MgCl2 on the activity of MGAT was also examined in this study because it was reported that the addition of magnesium into the reaction affected MGAT and DGAT activities from various tissues or isolated microsomes (13Jamdar S.C. Cao W.F. Arch. Biochem. Biophys. 1992; 296: 419-425Crossref PubMed Scopus (20) Google Scholar, 29Bierbach H. Digestion. 1983; 28: 138-147Crossref PubMed Scopus (24) Google Scholar, 31Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). The presence of lower concentrations of MgCl2 ( 100 mm) of magnesium produced marked inhibition of the reaction (Fig. 2, C and D).Fig. 2Effects of pH values (A and B) and Mg2 + concentrations (C and D) on MGAT activity of MGAT2 expressed in mammalian cells. The reaction was conducted by incubating 200 μm sn-2-monooleoylglycerol and 25 μm [14C]oleoyl-CoA for 10 min at room temperature in the presence of 50 μg of cell homogenate from mouse MGAT2-transfected COS-7 cells followed by lipids extraction and TLC assay as described under "Experimental Procedures." Radioactivity of diacylglycerols (DAG) in A and C were quantitated in B and D, respectively. Data are representative of two independent experiments. FA, fatty acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of Detergents on MGAT and DGAT Activities—It has been suggested that some of the detergents may affect the MGAT activity by serving as competitive substrate because of their structural similarities to fatty acids (26Lehner R. Kuksis A. J. Biol. Chem. 1995; 270: 13630-13636Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The effects of various detergents on MGAT and DGAT activity from primary tissues such as small intestine (26Lehner R. Kuksis A. J. Biol. Chem. 1995; 270: 13630-13636Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 29Bierbach H. Digestion. 1983; 28: 138-147Crossref PubMed Scopus (24) Google Scholar), neonatal liver (27Bhat B.G. Bardes E.S. Coleman R.A. Arch. Biochem. Biophys. 1993; 300: 663-669Crossref PubMed Scopus (23) Google Scholar), and adipocytes (29Bierbach H. Digestion. 1983; 28: 138-147Crossref PubMed Scopus (24) Google Scholar) have been extensively investigated because the presence of detergents is usually necessary to solubilize the enzymes from membranes. However, these experiments were conducted using tissues that may contain several isoforms of each enzyme, making the results difficult to interpret. In this study, we examined the effects of nonionic (Triton X-100), ionic (SDS), or zwitterionic (CHAPS) detergents on MGAT and DGAT activity from MGAT2- and DGAT1-transfected mammalian cells. At a low concentration, none of the detergents posed any measurable effect on DGAT activities. However, Triton X-100, SDS, and CHAPS exhibited severe inhibitory effects on MGAT activity of MGAT2 when the concentration of detergents reached 1.0, 0.1, and 1.0%, respectively (Fig. 3, A and B). The observed DGAT activity from MGAT2-transfected cell homogenate was more liable and sensitive to inactivation by detergents as determined by the formation of triacylglycerol, which started diminishing in the presence of 0.01% Triton X-100, 0.01% SDS, and 0.1% CHAPS, respectively (Fig. 3, A and B). At 0.1% detergent concentration, SDS abolished both MGAT2 and DGAT1 activities and Triton X-100 only diminished the innate DGAT activity of MGAT2 as shown in Fig. 3, A and C, with quantitative analysis shown in Fig. 3, B and D. Surprisingly, at 1.0% concentration, both Triton X-100 and CHAPS significantly enhanced the DGAT activity from DGAT1-transfected cell homogenate. Thus, both Triton X-100 and CHAPS can be used at relatively high concentration (1.0%) to distinguish the MGAT2 activity from that of DGAT1.Fig. 3Effect of detergents on MGAT activity of mouse MGAT2 (A and B) and DGAT activity of human DGAT1 (C and D). The reaction was conducted by incubating 200 μm sn-2-monooleoylglycerol (for MGAT activity of mouse MGAT2) or sn-1,2-dioleoylglycerol (for DGAT activity of human DGAT1) and 25 μm [14C]oleoyl-CoA for 20 min at room temperature with 50 μg of cell homogenate from mouse MGAT2-transfected COS-7 cells in the absence or presence of indicated concentrations of various detergents followed by lipids extraction and TLC assay as described under "Experimental Procedures." Radioactivity of diacylglycerols (DAG) or triacylglycerols (TAG) in A and C were quantitated in B and D, respectively. Data are representative of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Activators and Inhibitors—Bhat et al. (18Bhat B.G. Wang P. Coleman R.A. J. Biol. Chem. 1994; 269: 13172-13178Abstract Full Text PDF PubMed Google Scholar) reported that anionic phospholipids and anionic lysophospholipids stimulated MGAT activity, whereas fatty acids and sphingosine inhibited enzyme activity derived from neonatal liver microsomes. The addition of phospholipids greatly increased DGAT activity in the lipid body fraction of an oleaginous fungus (32Kamisaka Y. Nakahara T. J. Biochem. (Tokyo). 1996; 119: 520-523Crossref PubMed Scopus (10) Google Scholar). In contrast, phosphatidylcholine was shown to inhibit triacylglycerol synthetase activity derived from intestinal mucosa (26Lehner R. Kuksis A. J. Biol. Chem. 1995; 270: 13630-13636Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Such a discrepancy may be attributed to the presence of different isoforms of MGAT in these studies as reported in small intestine and neonatal liver (20Coleman R.A. Haynes E.B. J. Biol. Chem. 1986; 261: 224-228Abstract Full Text PDF PubMed Google Scholar). In order to clarify the issue with the MGAT2 enzyme, we characterize the properties of the intestinal MGAT2 using a variety of known inhibitors and activators of MGAT including phospholipids, oleic acid, and sphingosine. Acyl acceptor, sn-2-monooleoylglycerol, in ethanol was delivered into reaction mixture together with the indicated amounts of activators or inhibitors. The amount of ethanol used in the assay was <1%, which did not affect the enzyme activity. phosphatidylcholine, phosphatidylserine, and phosphatidic acid activated MGAT activity in a dose-dependent manner as shown in Fig. 4A. The three phospholipids showed similar potency of activation. Lysophosphatidic acid displayed a biphasic effect. Whereas lysophosphatidic acid activated the enzyme activity at relatively lower concentrations, a high concentration of lysophosphatidic acid exhibited a marked inhibitory effect (Fig. 4A). In contrast, oleic acid and sphingosine were potent inhibitors for MGAT2 activity (Fig. 4B).Fig. 4Effect of phospholipids and lysophospholipids (A) and neutral lipids (B) on MGAT activity of mouse MGAT2 expressed in mammalian cells. MGAT activity was measured by incubating 200 μm sn-2-monooleoylglycerol and 25 μm [14C]oleoyl-CoA in the absence or presence of indicated concentrations of lipids, which were delivered to the reaction together with sn-2-monooleoylglycerol. MGAT activity was expressed as percent of control activity measured in the absence of activators or inhibitors. Data represent the mean of two independent experiments. PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine; LPA, lysophosphatidic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characteristics of MGAT and DGAT Activity of Partially Purified MGAT2 from Escherichia coli—To further investigate the intrinsic properties of MGAT2 enzyme, we expressed and purified recombinant MGAT2 in E. coli. Bacterial expression offers a unique advantage over the mammalian and insect expression systems since E. coli does not express en
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