ACCELERATED PUBLICATION
Identification of Acyl Coenzyme A:Monoacylglycerol Acyltransferase 3, an Intestinal Specific Enzyme Implicated in Dietary Fat Absorption*

Dong ChengDagger, Thomas C. Nelson, Jian Chen, Stephen G. Walker, Judith Wardwell-Swanson, Rupalie Meegalla, Rebecca Taub, Jeffrey T. Billheimer, Michael Ramaker, and John N. Feder§

From the Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, New Jersey 08543

Received for publication, January 29, 2003, and in revised form, February 25, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol using 2-monoacylglycerol and fatty acyl coenzyme A. This enzymatic reaction is believed to be an essential and rate-limiting step for the absorption of fat in the small intestine. Although the first MGAT-encoding cDNA, designated MGAT1, has been recently isolated, it is not expressed in the small intestine and hence cannot account for the high intestinal MGAT enzyme activity that is important for the physiology of fat absorption. In the current study, we report the identification of a novel MGAT, designated MGAT3, and present evidence that it fulfills the criteria to be the elusive intestinal MGAT. MGAT3 encodes a ~36-kDa transmembrane protein that is highly homologous to MGAT1 and -2. In humans, expression of MGAT3 is restricted to gastrointestinal tract with the highest level found in the ileum. At the cellular level, recombinant MGAT3 is localized to the endoplasmic reticulum. Recombinant MGAT3 enzyme activity produced in insect Sf9 cells selectively acylates 2-monoacylglycerol with higher efficiency than other stereoisomers. The molecular identification of MGAT3 will facilitate the evaluation of using intestinal MGAT as a potential point of intervention for antiobesity therapies.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Triacylglycerol (TAG),1 an important molecule for eukaryotic fuel storage, is synthesized by two major pathways, the glycerol 3-phosphate pathway and the monoacylglycerol pathway (1). The glycerol 3-phosphate pathway is present in all tissues, whereas the monoacylglycerol pathway is restricted to the enterocytes of the small intestine (1). The monoacylglycerol pathway is believed to be critical for the packaging of dietary fat into chylomicron lipoprotein particles (2).

Acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) (2-acylglycerol O-acyltransferase, EC 2.3.1.22) is the enzyme that initiates the monoacylglycerol pathway (3). For insoluble dietary fat such as TAG to be absorbed by the small intestine, dietary fat molecules must first be digested by pancreatic lipase into soluble free fatty acids and 2-monoacylglycerol (4). These products are quickly absorbed into enterocytes. Within minutes of their appearance in the lumen of the small intestine, MGAT uses these molecules as substrates to form diacylglycerol (DAG). DAG is further acylated by acyl coenzyme A:diacylglycerol acyltransferase (DGAT) to re-form TAG. The newly formed TAG molecules are then packaged with other complex lipids such as cholesterol ester, phospholipids, and small amounts of protein to form round lipoprotein particles called chylomicrons. Chylomicrons, 90% of which are comprised of TAG, are quickly secreted into the lymph where they serve as a source of energy (3, 5).

Similar to other neutral lipid synthesis proteins, MGAT is an intrinsic membrane protein that to date has not been purified to homogeneity from any source. The molecular identity of the gene encoding the intestinal MGAT has been elusive. The first cDNA clone shown to encode MGAT enzyme activity, designated MGAT1, was identified by Yen et al. (6). However, MGAT1 is expressed in stomach, kidney, white adipose, and brown adipose tissues but not in the small intestine. Hence MGAT1 cannot account for the high intestinal MGAT enzyme activity that is important for the physiology of fat absorption (6).

In the current study, we have identified a novel cDNA, designated MGAT3, whose gene product fulfills the criteria as the MGAT responsible for the absorption of dietary fat. The RNA expression profile of human MGAT3 is highly restricted to gastrointestinal tract. Specifically MGAT3 transcripts are found at steady state in the ileum at levels ~45,000 times greater than those observed in the majority of the other tissues analyzed. When recombinant MGAT3 is expressed in baculovirus it produces robust MGAT enzyme activity. Importantly recombinant MGAT3 appears to possess superior substrate specificity for the acylation of 2-monoacylglycerol over other stereoisomers, a requirement that must be fulfilled by the authentic intestinal MGAT.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification and Cloning of Human MGAT3-- A comprehensive BLAST search was conducted using Saccharomyces cerevisiae ScDGAT2 sequence (accession number YOR245C) (7) against the human genome sequence. The resultant sequences were used to capture the existing expressed sequence tag. The expressed sequence tag information and Genscan program were then used to predict the coding sequence of the candidate genes. The candidate genes were profiled using TaqManTM quantitative PCR. One of them, which was expressed highly in the intestine (see Fig. 3), was designated MGAT3 and chosen for further study. To clone the MGAT3 cDNA, an antisense oligo, designated Oligo A, with biotin on the 5'-end was designed with the following sequence: 5'-biotin-GCCCACTGCTTCTAGATGCTGCTTCTGCAAGGTTTTGGAAGTGGTTGGGGGCTGCAGGGTTGTGGCAACTCCCATTGCAG-3'. To enrich the single strand circular cDNAs that hybridize with this sequence, an aliquot of 0.2 ng of Oligo A was mixed with 6 µg of several single-stranded circular cDNA libraries (Invitrogen) in 50% formamide. The mixture was heated to 95 °C for 2 min and hybridized in 50% formamide, 0.75 M NaCl, 0.02 M NaPO4, pH 7.2, 2.5 mM EDTA, 0.1% SDS at 42 °C for 26 h. The Oligo A/cDNA hybrids were incubated with streptavidin magnetic beads in high ionic salt buffer containing 10 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mM EDTA, pH 8.0 at 42 °C for 60 min with agitation every 5 min. The beads were captured with a magnet and were washed three times in 200 µl of 0.1× saline/sodium phosphate/EDTA, 0.1% SDS at 45 °C. The enriched single-stranded cDNAs were released from Oligo A-streptavidin magnetic bead complex by incubation with 0.1 N NaOH for 10 min. The released single strand cDNAs were ethanol-precipitated and converted into double strands by primer extension with the following oligo: 5'-GAGCTTCTGCAATGGGAGTT-3'. The double-stranded cDNAs were then transformed into Escherichia coli DH12S cells, and clones with the correct predicted insert size were sequenced. The sequence has been deposited in GenBankTM under accession number AY229854.

TaqMan Quantitative PCR Analysis of Human MGAT3-- Total RNA was isolated using the TriZol protocol (Invitrogen). For real time PCR, all primer and probe sequences were searched against GenBankTM data bases to ensure the target specificity. The oligo sequences (Forward Primer, ACTCTGGCCCTTCTCTGTTTTTT; Reverse Primer, AACGCCTTCCACCTTGGTT; Probe, TCCCAGTCCACATAGAGCCACACCAAG) were obtained from Applied Biosystems (ABI, Foster City, CA). Quantitative sequence detection was carried out on an ABI PRISM 7700 by adding to the reverse-transcribed reaction mixture (derived from 10 ng of DNase-treated total RNA) with 2.5 µM Forward and Reverse Primers, 2.0 µM Probe, 500 µM dNTPs, and 5 units of AmpliTaq GoldTM. The PCR was then held at 94 °C for 12 min followed by 40 cycles of 94 °C for 15 s and 60 °C for 30 s. The threshold cycle (Ct) of the lowest expressing tissue was used as the base line of expression. Expression levels of all other tissues were expressed as the relative abundance to that tissue by calculating the difference in Ct value. The -fold differences between the base line and the other tissues were calculated as 2Delta Ct.

Expression of Recombinant Human MGAT3 in Insect Cells-- The predicted coding sequence of human MGAT3 was fused with an NH2-terminal FLAG epitope (MGDYKDDDDG) and expressed in Spodoptera frugiperda (Sf9) insect cells using the Bac-to-Bac® system (Invitrogen) according to the manufacturer's instructions. Recombinant FLAG-human DGAT2 was expressed in the same manner as a control. Generally cells were infected with viruses (multiplicity of infection >3) for 2 days. After harvest, cells were washed with PBS and then homogenized with a probe sonicator in Homogenization Buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 1× protease inhibitor mixture (Roche Molecular Biochemicals). Membrane fractions (100,000 × g pellets) were stored at -80 °C until use for enzymatic assays. For immunoblot analysis, aliquots of 2 µg of membrane proteins were loaded on SDS-polyacrylamide gels and probed with an anti-FLAG M2 IgG (Sigma).

MGAT Enzymatic Assays-- MGAT activities were assayed for 5-10 min at 37 °C in a final volume of 200 µl according to a protocol modified from Coleman (8). Each reaction contained 10 µg of membrane proteins in Assay Buffer (100 mM Tris-HCl, pH7.5, 5 mM MgCl2, 1.25 mg/ml bovine serum albumin, 250 mM sucrose, 800 µM phosphatidylcholine liposomes). Generally 50 µM acyl coenzyme A and 200 µM sn-2-monoacylglycerol (delivered in acetone; final acetone concentration <2%) were used. Reactions were started by adding protein and terminated by adding 4 ml of chloroform/methanol (2/1, v/v). The extracted lipids were dried and separated by thin layer chromatography (TLC) with hexane/ethyl ether/acetic acid (85/15/0.5, v/v/v). Identities of TAG, DAG, monoacylglycerol (MAG), free fatty acid (FFA), and other lipids were verified with lipid standards (Sigma) after staining with iodine vapor. The specific enzyme activities were determined by the incorporation of [14C]oleoyl-CoA (20,000 dpm/nmol) or sn-2-[3H]monooleoylglycerol (20,000 dpm/nmol). The resultant chromatograms were analyzed with STORM PhosphorImager.

MGAT3 Expression and Subcellular Localization in COS-7 Cells-- COS-7 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected with either the pcDNA3-FLAG-MGAT3 construct or the pcDNA3 vector alone. Twenty-four hours post-transfection cells were switched to the medium containing 500 µg/ml G418. After 10 days in selective medium, the cells were reseeded onto poly-D-lysine-coated coverslips for immunocytochemistry. Following overnight cell attachment the coverslips were fixed with 3.0% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and then blocked for 1 h at 4 °C with PBS containing 4% nonfat dried milk, 0.2% Triton X-100. The samples were then incubated with 4 µg/ml anti-FLAG IgG (Sigma) followed by incubation with TRITC-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Lab) for 1 h at 4 °C. Additional samples were also stained with anti-calnexin antibody (Stressgen), a marker for the endoplasmic reticulum (ER), and were used as a reference for ER staining. The coverslips were examined on a Zeiss 510 LSM microscope using the appropriate bandpass filters for TRITC.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fig. 1A shows the comparison of human MGAT3 with its homologues. It bears 49% identity, 60% similarity to DGAT2; 44% identity, 51% similarity to MGAT1; and 46% identity, 55% similarity to MGAT2, respectively. Hence MGAT3 is a novel member of the DGAT2 gene family. The cDNA of MGAT3 encodes a 341-amino acid protein. Hydrophobicity analysis suggests that the predicted MGAT3 protein contains up to five potential transmembrane domains but does not contain a classic signal sequence at its NH2 terminus (Fig. 1B).


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Fig. 1.   A, alignment of the predicted human MGAT3 amino acid sequence with human MGAT1, human MGAT2, and human DGAT2 (GenBankTM accession numbers AF384163, AP000649, and AF384161, respectively) (6, 16). The multiple sequence alignment was performed with the ClustalW algorithm in VectorNTI program. Amino acids that are identical in all family members are indicated by a star (*), conserved residues are indicated by a colon (:), and similar residues are indicated by a period (·). B, hydrophobicity analysis of the predicted human MGAT3. The analysis was performed using the Kyte and Doolittle algorithm (17). The positive score is proportional to the degree of hydrophobicity.

To facilitate the detection of the recombinant human MGAT3 protein, a copy of FLAG epitope was fused in-frame at its NH2 terminus. Upon the infection by recombinant MGAT3 virus, Sf9 cell membrane extracts produced a band of ~36 kDa on SDS-polyacrylamide gels as detected by anti-FLAG IgG immunoblots (Fig. 2A, lane 2). The size of the protein is in agreement with the predicted mass. This ~36-kDa band is missing in wild type virus and is different from that obtained with recombinant FLAG-tagged human DGAT2 (lanes 1 and 3). To assess MGAT3 subcellular localization, FLAG epitope-tagged MGAT3 was expressed in COS-7 cells. Immunofluorescence microscopy using anti-FLAG IgG demonstrates a reticular staining pattern in the transfected cells (Fig. 2B). This staining pattern was indistinguishable from that of calnexin, a known ER protein (data not shown). Hence we conclude that recombinant MGAT3 is localized to the ER.


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Fig. 2.   Expression and characterization of recombinant human MGAT3. A, immunoblot with anti-FLAG IgG. Sf9 cells were infected with either wild type baculovirus or MGAT3 or DGAT2 recombinant viruses, respectively, and aliquots of membrane extracts were subjected to immunoblot analysis as described under "Experimental Procedures." B, immunofluorescence microscopy of FLAG-MGAT3 expressed in COS-7 cells. C, TLC MGAT enzyme assay. Aliquots of 10 µg of virus-infected Sf9 cell membrane proteins were subjected to MGAT enzyme assays at 37 °C for 6 min. For exogenous substrates, 200 µM sn-2-monooleoylglycerol and 50 µM [14C]oleoyl coenzyme A (20,000 dpm/nmol) were incubated with the membrane extracts. Lipid extracts were separated with TLC, and the chromatogram was exposed to STORM PhosphorImager for 12 h. MAG, denoted as nonspecific band **; Unknown, the other nonspecific band with unknown chemical nature denoted as band *. D, MAG stereoisomer specificity. Fifty micromolar [14C]oleoyl coenzyme A (20,000 dpm/nmol) and 200 µM 1-MAG, 2-MAG, or 3-MAG were assayed as in C. E, preference of fatty acyl-CoAs. Two hundred micromolar sn-2-[3H]monooleoylglycerol (20,000 dpm/nmol) and different acyl-CoAs at 50 µM were assayed. C2, acetyl-CoA; M, malonyl-CoA; C4:0, butyryl-CoA; C12:0, lauroyl-CoA; C16:0, palmitoyl-CoA; C18:0, stearoyl-CoA; C20:0, arachidoyl-CoA; C18:1, oleoyl-CoA; C18:2, linoleoyl-CoA; C20:4, arachidonoyl-CoA; WT, wild type.

Aliquots of Sf9 cell membrane extracts were used to conduct MGAT assays using radioactive [14C]oleoyl-CoA as the tracer. The TLC chromatogram shows that upon the addition of exogenous 2-monooleoylglycerol as a substrate, recombinant MGAT3 membranes produced two bands corresponding to 1,2- and 1,3-DAGs, respectively (Fig. 2C, lane 5). These DAG products were missing from membrane extracts of wild type or DGAT2 virus-infected cells (lanes 4 and 6). Interestingly both MGAT3 and DGAT2 membranes produced a TAG band (lanes 5 and 6), although wild type virus failed to produce detectable TAG (lane 4), indicating that the bands are due to the expression of recombinant protein. A possible explanation is that insect cell membrane possess endogenous MGAT activity, and its DAG product can be used by recombinant DGAT2 as a substrate to form TAG. This rationale argues that human MGAT3 also possesses DGAT activity. To test this possibility, exogenous DAG was added directly as a substrate. Indeed, similar to DGAT2, MGAT3 membranes were able to acylate DAG to form TAG (lanes 8 and 9). To calculate the enzymatic activity quantitatively, labeled DAG and TAG bands were cut from the TLC chromatogram and subjected to liquid scintillation counting. The specific activities of MGAT3 were ~22.8 nmol/min/mg for DAG and ~9.3 nmol/min/mg for TAG synthesis. The specific activities for the wild type virus were ~0.5 nmol/min/mg for DAG and ~0.2 nmol/min/mg for TAG, respectively. These results establish that the MGAT3 cDNA encodes a membrane protein that contains dual MGAT and DGAT activities.

To assess MAG stereoisomer specificity, sn-1-monooleoylglycerol (1-MAG), sn-2-monooleoylglycerol (2-MAG), and sn-3-monopalmitoylglycerol (3-MAG) were assayed (Fig. 2D). The results indicate that MGAT3 prefers 2-MAG as the substrate (compare lane 5 with lanes 2 and 8) to synthesize DAG. Interestingly, regardless of which type of MAGs were used, the amount of TAG synthesized did not change. It is possible that the background MGAT activity of endogenous insect cell membrane does not possess stereospecificity for MAG. When the background level of DAG is produced, it is used by DGAT activity encoded by MGAT3 to form TAG. To test this possibility directly, DGAT2 membrane was assayed in parallel (lanes 3, 6, and 9). Indeed recombinant DGAT2 membranes were able to synthesize TAG using three different MAG stereoisomers.

To assess the substrate optimum for acyl-CoAs, different acyl-CoAs were assayed for their ability to acylate radioactive sn-2-[3H]monooleoylglycerol. As shown in Fig. 2E, palmitoyl-CoA (C16:0) and oleoyl-CoA (C18:1) are the best substrates for MGAT3.

Quantitative TaqMan PCR was used to demonstrate that MGAT3 is selectively expressed in the digestive system (Fig. 3). The tissue with the highest expression steady state RNA level is ileum whose relative expression level was found to be ~45,000 times greater than that observed in the majority of the other tissues analyzed. The tissues with the next highest expression levels make up the remaining portions of the lower gastrointestinal tract, i.e. jejunum, duodenum, colon, cecum, and the rectum. Transcripts are notably missing from the stomach and the esophagus and trachea. The only other non-gastrointestinal related tissue to show appreciable expression is the liver. These data provide further evidence that MGAT3 is the elusive MGAT gene whose expression accounts for the high level of intestinal MGAT enzyme activity.


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Fig. 3.   TaqMan quantitative PCR analysis of MGAT3 in various normal human tissues. The steady state levels of MGAT3 are highest in the small intestine, most notably the ileum.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the current study, we report the cloning and characterization of a new human MGAT cDNA, a novel member of the DGAT2 gene family. During the revision of this manuscript, another MGAT cDNA, designated MGAT2, was reported by Cao et al. (18). We compared sequences of the current cDNA with human MGAT2 and found that they are distinct (Fig. 1A). Hence we designate our cDNA as MGAT3.

Similar to MGAT1 and -2, the recombinant human MGAT3 is capable of catalyzing MGAT enzyme reaction. Importantly MGAT3 is distinct from the other two MGAT enzymes in the following three aspects. 1) Enzymatically MGAT3 has a superior stereoselectivity in using MAG that contains an acyl-moiety at the sn-2 position; neither MGAT1 nor MGAT2 possess such stereoselectivity (6, 18). 2) The best acyl donors for MGAT3 are palmitoyl- and oleoyl-, the two most abundant species of fatty acids found in the small intestine; the best acyl donor for MGAT1 is arachidonoyl-, which is low in abundance in the intestine (6). 3) The expression of human MGAT3 is restricted to gastrointestinal tract with its highest expression in the ileum; the expression of MGAT1 is not found in the small intestine but in the stomach (6), and the MGAT2 expression is not restricted to the small intestine but is also found in kidney and stomach. In the intestine, the major digestive products of TAG produced by pancreatic lipase are 2-MAG (4) and the long chain fatty acids palmitate and oleate (5, 9). Hence the superior MGAT3 substrate selectivity for these types of fatty acids and its high level of expression in the small intestine would allow it to fulfill the physiological function of synthesizing TAG from the large quantities of free fatty acids and 2-monoacylglycerol absorbed in the intestine. These properties of MGAT3 allow us to propose that it is indeed another elusive intestinal MGAT that is essential for intestinal fat absorption.

From detergent-solubilized rat intestinal microsomes, Lehrner and Kuksis (10) reported the purification of MGAT and DGAT activities in a protein complex. In retrospect, the co-purification of these two enzyme activities could be explained by the fact that both MGAT2 and MGAT3 appear to encode dual MGAT/DGAT activities. The expression of MGAT2 and MGAT3 in the intestine and their MGAT/DGAT dual function also explain why DGAT1 knockout mice have an alternative TAG synthesis mechanism to support the fat absorption and chylomicron formation (11, 12).

The successful introduction of Orlistat to the market has demonstrated that drugs that inhibit the absorption of dietary fat can be efficacious for obesity treatment (13, 14). However, the mechanism for Orlistat, which is to inhibit pancreatic lipase, leads to the undesirable side effect of fecal leakage (15). An alternative approach may be to allow for the digestion of fat to fatty acids in the gut but block the uptake or the packaging of fatty acids into chylomicron particles. Inasmuch as intestinal MGAT is a critical enzyme for this pathway, MGAT2 and MGAT3 appear to be attractive therapeutic targets for the obesity treatment. The conceivable liability of inhibiting intestinal MGAT is the potential side effects associated with the accumulation of FFA and 2-MAG in the enterocytes. The molecular identification of MGAT2 and MGAT3 will facilitate the evaluation of this potential therapeutic approach.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY229854.

Dagger To whom correspondence may be addressed: Pharmaceutical Research Inst., Bristol-Myers Squibb Co., P. O. Box 5400, Princeton, NJ 08543-5400. Tel.: 609-818-5480; Fax: 609-818-3600; E-mail: dong.cheng@bms.com.

§ To whom correspondence may be addressed: Pharmaceutical Research Inst., Bristol-Myers Squibb Co., P. O. Box 5400, Princeton, NJ 08543-5400. Tel.: 609-818-4772; Fax: 609-818-6935; E-mail: John.Feder@bms.com.

Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.C300042200

    ABBREVIATIONS

The abbreviations used are: TAG, triacylglycerol; MAG, monoacylglycerol; DAG, diacylglycerol; FFA, free fatty acid; MGAT, acyl coenzyme A:monoacylglycerol acyltransferase; DGAT, acyl coenzyme A:diacylglycerol acyltransferase; TLC, thin layer chromatography; Ct, threshold cycle; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; ER, endoplasmic reticulum; 1-MAG, sn-1-monooleoylglycerol; 2-MAG, sn-2-monooleoylglycerol; 3-MAG, sn- 3-monopalmitoylglycerol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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