From the Endocrine Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285
Received for publication, March 19, 2003 , and in revised form, April 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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The biochemical properties of MGAT has been extensively investigated in intestine of various animal species (1417) as well as in the intestine and liver of suckling and adult rats (8, 1822), kidney (2325), and rat adipocytes (13). Because of its association with the microsomal membranes and its alleged involvement in an enzyme complex, MGAT has been difficult to purify to homogeneity (26). Several partial purifications of MGAT from rat intestinal membranes and neonatal liver have been reported previously (17, 27). 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 (28), allows us to evaluate the intrinsic characteristics of the enzyme. The mouse MGAT2 is most abundantly expressed in the small intestine (28) 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 (28). 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.
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EXPERIMENTAL PROCEDURES |
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Expression of MGAT2 and DGAT1 in Mammalian CellsA mammalian expression plasmid coding full-length mouse MGAT2 was engineered as described previously (28). 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 (28). 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 ActivityCell 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
(28). 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 50100 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 1020-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 DH5 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
A600 reached 0.7. Expression of recombinant mMGAT2 was
then induced by adding 1 mM
isopropyl-
-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
-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.
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RESULTS |
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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 (28), 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 (2830). 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 MGAT2The 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 (13, 29, 31). The presence of lower concentrations of MgCl2 (<30 mM) showed no significant effects on MGAT2 activity, whereas higher concentrations (>100 mM) of magnesium produced marked inhibition of the reaction (Fig. 2, C and D).
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Effects of Detergents on MGAT and DGAT ActivitiesIt 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 (26). The effects of various detergents on MGAT and DGAT activity from primary tissues such as small intestine (26, 29), neonatal liver (27), and adipocytes (29) 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.
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Activators and InhibitorsBhat et al. (18) 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 (32). In contrast, phosphatidylcholine was shown to inhibit triacylglycerol synthetase activity derived from intestinal mucosa (26). 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 (20). 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).
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Characteristics of MGAT and DGAT Activity of Partially Purified MGAT2 from Escherichia coliTo 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 endogenous MGAT, DGAT, and other synthetic enzymes of triacylglycerols. The cell extract was loaded on a peptide-tagged affinity column and eluted with a linear gradient of 150 mM to 1.5 M NaCl. The eluted fractions were subjected to SDS-PAGE or enzyme assays. The Coomassie Blue-stained SDS-PAGE profile from MGAT2-transformed cells revealed a major polypeptide band migrating at an apparent molecular mass of 38 kDa (Fig. 5A, fraction 2729) that was absent in mock-transformed cells. The 38-kDa peptide is consistent with the molecular mass of 38.6 kDa predicted from the open reading frame of the mouse MGAT2 gene and human MGAT2 expressed in mammalian cells with an apparent molecular mass of 39 kDa on SDS-PAGE (data not shown). We next tested the fractions for both MGAT and DGAT activities. As shown in Fig. 5B, MGAT activity was only detected from fractions that contains the partially purified MGAT2 enzyme but not from similar fractions purified from the mock-transformed E. coli cells. Although the detected MGAT activity is much weaker than that from the MGAT2 enzyme expressed in the mammalian cells, the results suggest that the bacterial expressed enzyme is active. To address the issue of whether the MGAT2 enzyme possesses intrinsic DGAT activity as detected in previous experiments using MGAT expressed in mammalian cells (28), we also tested the purified fractions for DGAT activity. As demonstrated in Fig. 5C, fractions that showed MGAT activity also catalyzed the synthesis of triacylglycerol, thus confirming that MGAT2 enzyme possesses intrinsic DGAT activity. Furthermore, treatment with Triton X-100 abolished the DGAT activity of recombinant enzyme from the bacterial source (Fig. 5D), which is consistent with the property of the MGAT2 enzyme expressed in the mammalian cells shown in Fig. 3A.
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DISCUSSION |
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MGAT2 expressed in COS-7 cells displayed a broad range of substrates toward either fatty acyl-CoA derivatives or monoacylglycerols containing various fatty acyl chains. Consistent with the substrate specificity of MGAT1 expressed in insect cells (34) and MGAT activity in human intestinal mucosa (29), MGAT2 preferred fatty acyl-CoAs with longer carbon chains in the range below C18 while the activity began to decline when the length of carbon chains exceeded C20. With regards to monoacylglycerols, MGAT2 appeared to prefer acyls with short carbon chain, contrasting with a reported preference of longer ones (29). MGAT2 acylated monoacylglycerols containing unsaturated fatty acyls, such as essential linoleic acid, in preference to saturated ones. These findings coincide with the concept that the essential fatty acids play important roles in normal development of the retina and brain and thus could be selectively preserved by specific tissues (36, 37). Consistent with our findings, human MGAT2 and human MGAT3 were reported to possess the similar substrate specificity pattern (30, 35). Variations of substrate specificity among different investigators may be attributed to different resources of enzymes, substrate concentrations, and assay systems employed (29, 34, 37, 38).
The properties of MGAT2 and MGAT1 were similar in several respects but differed from the MGAT activity observed in neonatal rat liver. For example, both MGAT1 and MGAT2 expressed in mammalian cells utilized sn-1-monoacylglycerol and sn-2-monoacylglycerol (28, 34) while partially purified MGAT from suckling rat liver acylated 1-monoacylglycerol at only 4% of activity observed with sn-2-monoacylglycerol (27). In addition, MGAT2 expressed in mammalian cells acylated 1-O-hexadecyl-rac-glycerol efficiently (Fig. 1B), whereas the ether analog of monoacylglycerol was catalyzed very poorly by MGAT from rat liver (27). These findings together with previously reported different responses to various treatments including temperature, proteolysis, detergents, protein modification reagents, and divalent cations between liver MGAT and intestine MGAT (20) strongly indicate that liver might express isoforms different from MGAT1 and MGAT2. However, this is not conclusive as the MGAT enzyme may be differently anchored in liver, intestine, and mammalian cell lines or tissue-specific cofactors may exist to modulate the function of MGAT. It was reported that the hepatic isoform could be developmentally up-regulated to meet the need to use fatty acids for energy or to preserve essential fatty acids (20). Further in vivo studies detecting hepatic expression of MGAT1, MGAT2, and MGAT3 during these specific physiological conditions will be very informative in clarifying this issue.
Current reports also investigated possible roles of phospholipids and lipid cofactors in regulating MGAT2 activities. MGAT activities in microsomes has previously been shown to be affected by lipid cofactors that modulate the activity of most membrane-bound enzymes (18, 22). Some of the lipid cofactors may serve as coordinators between the monoacylglycerol and glycerol 3-phosphate pathways of triacylglycerol biosynthesis. It remains to be elucidated on the degree of interaction between the two pathways since both routes presumably compete for the same pool of acyl-CoA and both produce diacylglycerol intermediates. Indirectly, the monoacylglycerol route may also affect the rate of fatty acid oxidation by modulating the level of acyl-CoA. Our data show that several metabolic intermediates and their derivatives from the glycerol 3-phosphate pathway impose mildly positive effects on the MGAT activities. In contrast, sphingosine and oleic acid demonstrated a potent inhibitory effect on MGAT2 enzyme activity, which is supported by previous reports (18, 22).
It has been proposed that the biosynthesis of triacylglycerols in the enterocyte was catalyzed by a synthetase complex consisting of MGAT, DGAT, and fatty acyl-CoA synthetase (39). This study reports for the first time the properties of an individual recombinant MGAT expressed in E. coli. This E. coli expression system offers a unique advantage to the mammalian expression system since the bacterial system does not express endogenous MGAT and DGAT. Our finding that E. coli expressed MGAT2 is enzymatically active suggests that MGAT2 can function without forming a complex with other biosynthetic enzymes. It should be noted that the activity of MGAT2 expressed in E. coli appears much weaker than in mammalian cells, suggesting the existence of other cofactors for full activation of MGAT2. In addition to the formation of a synthase complex, the requirement of a small cytoplasmic protein for the activation of rat hepatic and intestinal synthesis of triacylglycerol, which has been purified from plasma, was reported previously (4043).
We previously reported that MGAT2 expressed in mammalian cell lines also possess weak but significant DGAT activity (28). The same phenomenon was observed in MGAT1 and MGAT3 (30, 35). We postulated that this DGAT activity could be due to either the intrinsic properties of MGAT2 or enhanced endogenous DGAT activity by the presence of MGAT2 in mammalian cell lines. To clarify this issue, we analyzed MGAT2 for both MGAT and DGAT activities using purified MGAT2 enzyme expressed in E. coli where MGAT and DGAT background should be negligible. Consistent with what was observed in mammalian systems, the purified MGAT2 from the bacterial resource also displayed weak DGAT activity in addition to the predicted MGAT activity (Fig. 5, B and C). This finding provides convincing evidence that MGAT2 possesses intrinsic DGAT, which is further supported by the observation that treatment with a low dose of nonionic detergent, Triton X-100, abolished the DGAT activity of MGAT2 without affecting the MGAT activity itself. The intrinsic DGAT activity of mouse MGAT2 is not surprising since mouse MGAT2 shares 47.5% sequence homology with DGAT2. Likewise, DGAT2 also possesses a MGAT activity (34). Additionally, the human MGAT2 is closely adjacent to the DGAT2 gene located at 11q13.5, suggesting that the two genes originated from duplication. Hence, it will be interesting to investigate whether DGAT enzymes expressed in the E. coli system also possess intrinsic MGAT activities, and if so, whether DGAT2 enzymes have more MGAT activity than DGAT1, which shares much less homologies with MGAT genes. Such a dual function of intestinal MGAT2 and MGAT3 offers an alternative pathway for triacylglycerol synthesis when DGAT enzymes are deficient. It is possible that overexpression of MGAT2 could have a synergistic effect on the endogenous DGAT activity since they are reported to form a complex with other triacylglycerol biosynthetic enzymes. However, our preliminary experiments demonstrated that the addition of partially purified MGAT2 from E. coli cells did not increase the activity of DGAT overexpressed in COS-7 cell.2 The interaction among MGAT, DGAT, and other triacylglycerol biosynthetic enzymes such as fatty acyl-CoA synthetase could be addressed in future studies by cotransfection experiments in mammalian cells or other systems.
Detergents have been widely used in solubilizing membrane-associated proteins and protein complexes including MGAT, DGAT, and acyl-CoA acyltransferase from various tissues, although they mostly led to a substantial loss of enzyme activities (17, 26, 27, 29). In view of our results, MGAT activity of MGAT2 was more stable and resistant against treatment with detergents than its intrinsic DGAT activity since 0.1% Triton X-100 or CHAPS did not result in the loss of MGAT activity, whereas they did so with DGAT activity from the same preparation. However, high concentration of detergents (1.0%) led to a substantial loss of activities of MGAT2. It is possible that the nonionic detergents served as competitive inhibitors for the substrate-binding site of MGAT because of their structural similarities with fatty acids or that the lipid-protein interaction is needed for optimal activity of MGAT2. One of the most striking effects of detergent treatment is that both Triton X-100 and CHAPS at high concentration (1%) significantly enhanced the DGAT activity of DGAT1 expressed in COS-7 cells. Although the underlining mechanism remains elusive, it is possible that these detergents could serve as dispersing agents for the hydrophobic diacylglycerols or lead to the exposure of more active sites of DGAT1 to its substrates, therefore increasing the opportunity of interaction between the enzyme and substrates. The finding that MGAT2 also has intrinsic DGAT activity and that nonionic and zwitterionic detergents demonstrated an activating effect on DGAT1 activities may have repercussions on the interpretations of previously published work on MGAT using microsomes that contain both MGAT and DGAT enzyme solubilized with detergents.
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FOOTNOTES |
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To whom correspondence should be addressed: Endocrine Research, DC 0545, Lilly
Research Laboratories, Lilly Corporate Center, Eli Lilly and Company,
Indianapolis, IN 46285. Tel.: 317-276-6753; Fax: 317-276-9574; E-mail:
shi_yuguang{at}lilly.com.
1 The 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.
2 J. Cao, P. Burn, and Y. Shi, unpublished data.
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REFERENCES |
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