| HOME | HELP | CONTACT US | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Nutrition and Metabolic Diseases, Department of Internal Medicine, Center for Human Nutrition, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390 USA
1 Department of Cell Biology and
2 McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA
(Requests for offprints should be addressed to A K Agarwal; Email: anil.agarwal{at}utsouthwestern.edu)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recently, through positional cloning, we discovered that mutations in AGPAT2 cause congenital generalized lipodystrophy (CGL; Agarwal et al. 2002, Agarwal & Garg 2003), a syndrome with almost complete loss of adipose tissue and predisposition to develop diabetes, hepatic steatosis, and hyperlipidemia (Garg 2004). Besides AGPAT2, there are seven other known isoforms, each of which is encoded by independent genes (Leung 2001, Li et al. 2003, Ye et al. 2005, Agarwal et al. 2006). A multiple sequence alignment of these eight known AGPAT isoforms indicates that several are duplicated, suggesting that several AGPATs are still to be discovered. Furthermore, given the availability of numerous structurally distinct lysophosphatidic acids (LPAs) and fatty acids for acylation intracellularly, we hypothesized that there could be additional AGPAT isoforms which still remain to be identified. These new AGPAT isoforms may differ in substrate specificity, i.e. may prefer specific LPAs and specific fatty acids for sn-2 acylation to perform distinct cellular functions. With the sequencing of the human and mouse genomes and the bioinformatics tools available, we cloned and characterized an additional AGPAT isoform encoding human AGPAT9. This isoform retains AGPAT activity very similar to another isoform AGPAT2.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The National Center for Biotechnology Information (NCBI) and Celera databases were used for the sequence analysis. Multiple protein alignment and phylogenetic tree were created using Vector NTi suites (InforMax, Bethesda, MD, USA).
Cloning of human AGPAT9
The search for additional gene encoding for an acyltransferase was performed by homolog comparison with known acyltransferases deposited in the various databases including NCBI, Celera and expressed sequence tags (ESTs). One of the hits, AK090444 [GenBank] , showed significant homology to the known acyltransferases, including the conserved motifs, suggesting that this sequence may have the acyltransferase activity.
Amplification of human AGPAT9 cDNA
To determine if the sequences are expressed in human tissues, we initially designed primers 1 and 2 (Table 1
) within the coding region to amplify the mRNA from the adipose tissue. The amplified PCR product was purified and the sequence was found to be identical to the predicted open reading frame (ORF). To amplify the entire ORF, additional primers were then designed in the 5' and 3' untranslated regions and amplified in two overlapping fragments using primer pairs 3 and 4, and primer pairs 5 and 6 (Table 1) and the human adipose tissue Marathon Ready cDNA (Clontech). The PCR products were gel purified and sequenced directly using the amplification primers and cloned in TA cloning vector (pCR2.1; Invitrogen). These overlapping PCR fragments were then combined and reamplified using only the 5' and 3' flanking primer pairs 3 and 6 (Table 1). The PCR product was gel purified and cloned in pDrive vector (Qiagen) for further sequencing and cloning steps.
|
To construct the AGPAT9 expression plasmid, the pDrive-AGPAT9 was used as the template and primer pair designed to only amplify the ORF (primers 8 and 10; Table 1
). The restriction sites BamHI and XhoI were incorporated into the primers for ease in subsequent cloning into the mammalian expression vector; pcDNA3.1(+)neo. The amplified product and the vector were digested with XhoI and BamHI and ligated to the same sites. The expression plasmid was restricted with appropriate enzymes to ascertain the orientation of the insert in relation to the promoter. This expression plasmid was named as pAGPAT9-SF.
Generation of wild-type AGPAT9 expression vector (long form)
Initially, we amplified and generated mammalian expression construct based on available nucleotide sequences (GenBank entry AK090444
[GenBank]
), however, on further alignment of human AGPAT9 protein with homologous protein sequences from several other species revealed that there are additional amino terminal protein sequences which were missed in the human. Based on this information, we amplified these 5'-sequences such that an overlapping fragment with the short form of AGPAT9 was generated using primer pairs 7 and 9 (Table 1). The generation of the mammalian expression plasmid was the same as for the short form except primer pairs 10 and 11 (Table 1). This expression plasmid was named as pAGPAT9-LF.
Generation of V5- epitope-tagged wild-type AGPAT9 expression vector (long form)
The construction strategy for the V5 epitope tagged AGPAT9 expression vector was the same as described above, except the AGPAT9-LF was amplified with primer containing the V5 epitope sequences with the removal of the first ATG of the AGPAT9 protein (in order to remove the spurious protein translation). (5'-CGGGATCCATGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGAGGCTGCGGGGATGCGGACCCCG-3' the BamHI restriction site is in bold and the V5 epitope is underlined) and the primer 10 (Table 1).
Generation of EGFP-tagged wild-type AGPAT9 expression vector (long form)
The construction of the wild-type fusion protein with GFP was carried out using the AGPAT9 wild-type plasmid as the template and primer pairs 12 and 13 (Table 1). The amplified product and the plasmid pEGFP-N3 (Clontech) were restricted with XhoI and BamHI and cloned in the similar site. The expression plasmid was sequenced to ascertain the orientation and the correctness for the junction sequences. The human AGPAT1-REP was generated by excising the AGPAT1 coding region from the pDrive-hAGPAT1 (unpublished) to the KpnI and XhoI and cloning in the compatible restriction site of expression vector dsRed-T4-N1 (Clontech).
Genomic localization of human AGPAT9 and gene structure
The human cDNA for AGPAT9 was blasted against the genomic databases (NCBI) which identified the gene located on chromosome 5p15.5 (contig: NT_006576 [GenBank] .15, nt 1 526 408–1 426 586 and Hs5_6733, nt 1 589 408–1 489 586).
Northern blot analysis
To determine the tissue expression pattern and transcript size, a multiple tissue total RNA blot was obtained from Clontech and was hybridized with the probe generated with primer pairs 5 and 6 (274 bp fragment). Briefly, the blot was hybridized with 32P-labeled probe generated from the above fragment overnight at 60 °C. The blot was washed with decreasing concentration of sodium salt at 65 °C. The final wash was at 0.1x SSC; 0.1% SDS for 30 min at 65 °C. The blot was exposed either overnight or for 7 days at –80 °C. The blot was stripped and reprobed with the ß-actin probe as a control as suggested by the manufacturer. The scanned images were quantified using LabWorks 4.0 software (UVP Inc., Upland, CA, USA).
Quantitative real-time PCR in human tissue panel
Quantitative PCR was performed using the TaqMan primer and probes, designed using primer express software, ABI PRISM 7700 sequence detection system and primer express analysis software (Livak 1997; ABI user bulletin 2). The human cDNA panel was from Clontech. Briefly, 100 pg cDNA was added to the forward (5'-TCGCTGACTTCCACAGGTTTG-3') and reverse (5'-GGTGAGGTCTCTGCACAGCTTT-3') primers along with fam-labeled probe (AATACCTGTACCCGGATCA) and universal mix containing AmpliTaq and appropriate buffers. The PCR was followed for 40 cycles of 94 °C for 15 s and 60 °C for 30 s. The cDNA was amplified in duplicate with G3PDH as an internal control. The 
Ct value for each tissue was calculated as Ct = (Ct (tissue) – Ct (G3PDH)).
Generation of stably expressing wild-type AGPAT9 in Chinese Hamster Ovary (CHO) cells
The CHO cells stably expressing AGPAT9 were generated as described earlier (Haque et al. 2005). Stable-transfected CHO cell lines were selected in 500 µg/ml G418 (Sigma). The G418 resistant cells in each well were scored as a single pool. Such pools were expanded for further studies. Cells transfected with pcDNA 3.1(+) vector alone were used as controls. The heterologous expression of human AGPAT9 was verified by reverse-transcriptase polymerase chain reaction (RT-PCR) as described before.
AGPAT enzymatic activity of AGPAT9 in the cell lysate
Three to four independent pools of cells were expanded after selection in G418. The cells were plated in 60 mm dishes and grown to confluence. The medium was then removed and the cells were rinsed twice with PBS and harvested by scraping in 200 µl of 20 mM Tris–HCl (pH 7.5) and 5 mM NaCl containing protease inhibitor cocktail (Roche). Cells were subjected to a single freeze thaw step and then sonicated three times with 7 s burst, followed by a 1 min cooling on ice (Vibracell High Intensity Ultrasonic Liquid Processor; Sonics & Materials Inc., Newtown, CT, USA). Cellular debris and nuclei were removed by centrifugation (1000 g for 10 min at 4 °C), and the post nuclear supernatant was used for the enzymatic activity. Protein concentration was determined by a commercially available colorimetric assay (Bio-Rad Laboratories).
The enzyme activity was determined by measuring the conversion of [3H]LPA to [3H]PA as described previously (Haque et al. 2005, Agarwal et al. 2006). Briefly, the enzymatic reaction was assembled in 200 µl of 100 mM Tris–HCl (pH 7.4), containing the following: 10 µmol/l LPA (oleoyl-sn-1-glycerol-3-phosphate, Avanti Polar Lipids, Alabaster, AL, USA), 50 µmol/l oleoyl CoA (Sigma), 1 µl [3H]oleoyl-LPA (specific activity 30–60 Ci/mmol; Perkin–Elmer Life and Analytic Sciences, Boston, MA, USA), and 1 mg/ml fatty acid free bovine serum albumin. The reaction was started by adding 20 µg cell lysate followed by incubation for 10 min at 37 °C. The reaction was terminated by adding 0.5 ml 1-butanol containing 1N-HCl and to extract phospholipids. The butanol extract was dried under vacuum and the LPA and PA were resolved using the solvent system chloroform–methanol–acetic acid–water (25:10:3:1). Radioactive spots were identified by comigration with unlabeled LPA and PA standard and visualized in iodine vapors. The [3H]LPA and [3H]PA spots were scraped and counted for radioactivity (Tri-Carb Liquid Scintillation Counter 3100TR; Perkin–Elmer Life Sciences). The enzyme activity of each cell pool were determined as described before (Haque et al. 2005, Agarwal et al. 2006).
Lysophosphatidylcholine acyltransferase (LPCAT1) enzymatic activity of AGPAT9-LF in the cell lysate and tissue homogenate
LPCAT1 activity in cell lysate was determined as described previously (Chen et al. 2006, Nakanishi et al. 2006). Cell lysates were obtained from the V5-tagged expressing cells and incubated with [14C]palmitoyl lysophosphatidylcholine in the presence of palmitoyl-CoA. The incubation was terminated by adding chloroform: methanol (2:1 v/v) mixture. Organic phase was dried and resolved on thin layer chromatography (TLC) using solvent system chloroform:-methanol:acetic acid:water (50:25:8:4 v/v). Bands corresponding to standard LPC and PC were visualized by iodine, eluted, and quantitated. The tissue homogenate was prepared in the same buffer as for (AGPAT) assay, centrifuged to remove cell debris, and assayed using 20 µg total protein.
Acyl-CoA specificity
specificity of esterification of sn-2 position of [3H]LPA (sn-1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate) was determined by substituting the fatty acids with the following acyl-CoAs. Decanoyl (C10:0)-, lauroyl (C12:0)-, myristoyl (C14:0)-, palmitoyl (C16:0)-, stearoyl (C18:0)-, oleoyl (C18:1)-, linolenoyl (C18:3; n–3)-, 
-linolenoyl (C18:3;n–6)-, arachidoyl (C20:0)-, arachidonoyl (C20:4;n–6)-, behenoyl (C22:0)-, docosahexaenoyl (C22:6;n–3)-, tricosanoyl (C23:0)-, ligneceroyl (C24:0)-, nervonoyl (C24:1;n–9)-, and hexacosanoyl (C26:0)-CoA. The assay conditions remain the same as described for the acyltransferase activity.
sn-1-acyl-lysophosphaditic acid specificity
To determine the specificity of various LPAs for the enzymatic activity, the following LPAs with various fatty acids at sn-1 position were used for the assay. Myristoyl (C14:0)-, palmitoyl (C16:0)-oleoyl (C18:1), arachidonoyl (C20:4)-, arachidoyl (C20:0)-, linoleoyl (C18:2)- and linolenoyl (C18:3). The enzymatic assay was assembled as before except that unlabeled LPAs and [14C]oleoyl-CoA were used as substrates, and activity was determined by formation of [14C]PA.
Western blot analysis
Total cell lysate (40 µg) from the V5-AGPAT9-LF and vector alone were resolved on 10% SDS–PAGE followed by transfer on to nylon membrane (Millipore, Billerica, MA, USA). The membrane blocked with 5% non fat milk containing 0.2% Tween -20 and then incubated with V5 antibody conjugated to horse radish peroxidase (HRP; Invitrogen) for 2 h at room temperature, the membrane was washed and then incubated ECL reagent (GE Healthcare Piscataway, NJ, USA) to visualize the immunoreactive protein.
Immunofluorescence microscopy
Stable expressing AGPAT9-EGFP CHO cells were grown on cover slip 1 day before the experiment. Cells were fixed/permeabilized by incubation with methanol (–20 °C) for 20 min. Cells were washed 3 x 5 min with PBS and then incubated with primary antibody (sec 61 ß; Upstate, Lake Placid, NY, USA diluted in the ratio of 1:200) for 30 min at 37 °C in a humidified chamber. Cells were then washed 3 x 5 min with PBS and incubated with AlexaFluor 555 coupled fluorescent secondary antibody (Invitrogen) for 30 min at 37 °C in a humidified chamber. After incubation, cells were washed 3 x 5 min with PBS and mounted on a glass slide with Mowiol. Cells were observed with a Zeiss Axioplan 2E, and pictures were captured with a Hamamatsu monochrome digital camera (Hamamatsu City, Japan). Images were analyzed with Openlab (Improvision, Lexington, MA, USA).
For live cell imaging, the cells were seeded in dishes and transfected with equal amount of AGPAT1-REP and AGPAT9-GEP expression plasmids. Cells were imaged directly 72 h after transfection using a DeltaVision RT Deconvolution Microscope (Applied Precision, LLC; Issaquah, WA, USA). The obtained images were deconvoluted to remove the reflective fluorescence using softWoRx software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Based on in silico sequence assembly, the human AGPAT 9 was amplified from the omental adipose tissue RNA. The amplified sequences showed that the start of the ORF for human AGPAT9 was in good context for the Kozak rule in initiating protein translation (Kozak 1984) predicting a protein of 432 residues. However, based on protein sequence homology, only a truncated form had been cloned, missing an N-terminal sequence (Fig. 1). Thus, based on human genomic sequences, we then designed additional primers to amplify the 5'-region sequences. The assembled sequences for AGPAT9-long form (AGPAT9-LF) had an in-frame stop codon located 60 nucleotides upstream in the 5'-untranslated region, suggesting that this ATG codon is most likely used as a translational start codon. The predicted protein consists of 534 residues and molecular weight of 59 147 Da. Alignment of AGPAT9-LF from several species showed that this isoform was highly conserved and is also present in invertebrates like fruit fly (Drosophila melanogaster). The putative conserved motifs, NHX4D and EGTR, seen in almost all proteins of the glycerophospholipid acyltransferase family, were modified to PHX4D and EGTC respectively in both the AGPAT-SF and the AGPAT9-LF.
|
A search against the human genomic databases localized the human gene on chromosome 5p15.5. Based on the predicted sequences and the amplified sequences, the exon–intron boundaries were determined. The gene contains 14 exons with the last exon having a long untranslated region (Table 2). All the exon and intron junctions obeyed the AG-GT dinucleotide acceptor–donor rule. Visual inspection of the sequences revealed two regions of repeat sequences located in introns 1 and 2. As shown in Fig. 2, the first repeat consisted of a sequence of 17 nucleotides and was located 130 nucleotides from exon 2. The other repeat sequence located in intron 2 was nearly 3.5 kb from the splice site junction of exon 3. This repeat sequence, although not a perfect dinucleotide repeat, can still form a stretch of the alternating purine-pyrimidine polynucleotides. It is interesting to note that the short form of AGPAT9 starts in the exon 3 and could be the result of these repeat sequences.
|
|
Northern blot analysis of human tissues showed that the AGPAT9 mRNA was close to 4.4 kb and expressed in several tissues, with the lung and spleen expressing high levels, followed by the brain, heart, and placenta (Fig. 3). We did not observe any alternative transcript, even upon long exposure. In the rodents, a single transcript of about 4 kb has been identified in the lung and spleen (Chen et al. 2006). In the rat, AGPAT9 is also expressed in the stomach, but not in the mouse (Chen et al. 2006) indicating species variation. In our human northern blot, the stomach RNA was not represented. As reported earlier, the human AGPAT2 isoform is highly expressed in the liver and pancreas (Eberhardt et al. 1997) and in the adipose tissue (Agarwal et al. 2004) with relatively low expression in the lung and spleen. These RNA analyses indicate differential pattern of RNA expression of AGPAT2 and AGPAT9 isoforms in human tissues. Whether there are functional redundancies among these isoforms remains unclear.
|
are the Ct values for various tissues after normalization to the housekeeping gene, glycerol-3-phosphate dehydrogenase (G3PDH). Quantitative real-time PCR revealed that AGPAT9 had about twofold higher expression in the lung than in the spleen. AGPAT9 was also expressed in the omental adipose tissue and placenta, but about sevenfold less than the lung. AGPAT9 is expressed poorly in the skeletal muscle.
|
Multiple sequence alignment of the known human acyltransferases revealed that AGPAT9 belongs to the family of acyltransferases (Fig. 4A). Previous studies have revealed that AGPATs 1–5 and 8 have the same two conserved motifs, NHX4D and EGTR, as shown in Fig. 4B. As more of these acyltransferases are cloned and sequenced there appears to be flexibility in these conserved motifs. The conserved proline in AGPAT9 from several species indicates that the asparagine residue in the NHX4D motif is not an absolute requirement for the enzymatic activity. Likewise, substitution of arginine to cysteine in EGTR motif is also tolerated (Fig. 1). In Synechocystis sp. PCC6803, an unrelated acyltransferase had the EGAT sequence instead of the motif EGTR (Weier et al. 2005). In a recent study, using in vitro translated mouse acyltransferases, it was shown that Agpat 3, 4, and 5 possessed acyltransferase activity, which was only 41–48% of mouse Agpat2 (Lu et al. 2005).
|
In stably expressing CHO cells, the AGPAT9-LF-GEP protein showed an endoplasmic reticulum (ER) like expression pattern. This was confirmed by colocalizing AGPAT9-LF-GFP with the ER protein Sec 61 ß (Fig. 5A). AGPAT9-LF-GFP also colocalized with the ER proteins disulfide isomerase and the ER-chaperone GRP78/BIP (data not shown). Since previous studies have shown that AGPAT1, 2, 6, and 8 are all localized to the ER, we tried to determine whether, the AGPAT9-LF is localized to the same or different subdomain of ER. We transiently transfected CHO cells with AGPAT9-LF-GFP and AGPAT1-REP and captured images for both the proteins using live cells imaging. Shown in Fig. 5B are the fluorescence for AGPAT9-LF and AGPAT1 indicating that the two proteins appear to colocalize to similar ER domains. However, further colocalization studies with higher resolution will further confirm this observation.
|
The human AGPAT9-SF has moderate acyltransferase activity, only 22.6% when compared with the other isoform, human AGPAT2, using oleoyl-CoA as the acyl donor and 1-oleoyl glycerol-3-phosphate as the acceptor (data not shown). We then created a V5- epitope tagged AGPAT9-LF and determined the acyltransferase activity as above. This long form has much higher activity (72.8% when compared with AGPAT2) than the AGPAT9-SF, indicating that the amino terminus residues increase its activity over threefold (Fig. 6A and B).
|
To assess the substrate specificity of AGPAT9-LF, LPAs with various fatty acids at the sn-1 position were incubated with lysates of CHO cells over expressing wild-type AGPAT9 and [14C]oleoyl-CoA. Wild-type AGPAT9 accommodated myristoyl, linoleoyl, and palmitoyl fatty acids at the sn-1 position of the LPA, synthesizing PAs at about the same rate as the preferred substrate, sn-1-oleoyl-LPA (Table 4). Other LPAs, such as sn-1-linolenoyl, and sn-1-arachidonoyl were esterified at 50–70% the rate of sn-1-oleoyl-LPA. However, sn-1-arachidoyl form of LPA was not esterified.
|
). All other acyl-CoAs were poor substrates when compared with oleoyl-CoA (Table 5).
|
|
While this study was in progress, two independent studies showed that the mouse homolog has acyl-CoA:LPCAT1 activity and that the enzyme is highly expressed in the lung (Chen et al. 2006, Nakanishi et al. 2006). Although, the mouse clone is 88% identical to our human AGPAT9, in our hands stable CHO lines expressing AGPAT9 lacked LPCAT activity, whether determined using whole cell lysate (Fig. 7A) or in cell fractions (data not shown). Such LPCAT activity, even though small, could be observed in the mouse lung and spleen tissue homogenates (Fig. 7B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The AGPAT9 protein is highly conserved among species, and homologs are found in rodents, vertebrate fishes and invertebrate fruit fly (GenBank entry as a hypothetical protein). Our search did not show this protein to be present in plants, but this may be due to incomplete genome annotation in many of these species. The high degree of homology (Fig. 1) and conservation among species suggests that the AGPAT9 gene evolved very early during evolution suggesting that this enzyme serves an important function in cellular physiology in tissues other than the lung as indicated by previous investigators (Chen et al. 2006, Nakanishi et al. 2006). However, unlike mouse LPCAT1 (Chen et al. 2006, Nakanishi et al. 2006), AGPAT9 does not appear to use LPC as substrate in our in vitro studies, which would argue against its sole role in lung physiology. Our data show that the enzyme is present in the spleen as well, suggesting its role in the immune system. Secondly, fruit flies lack tissue equivalent to the lung. Finally, presence of the LPCAT activity in the lung and spleen tissues represents the total LPCAT activity, which may or may not be due to the LPCAT1 activity. Lack of LPCAT activity in the recombinant protein will require further studies with more robust expression system.
The gene structure has revealed some interesting features. The presence of intergenic 17 nucleotide repeat sequence in intron 1 and alternating purine pyrimidine (APP) sequences in the intron 2 could influence the alternate splicing with shorter transcript originating in exon 3 of the AGPAT9 gene (Hui et al. 2005). However, not directly demonstrated, these nucleotide repeat sequences, have been shown to influence the expression of a number of genes including 11ß-hydroxysteroid dehydrogenase 2 (Agarwal 2001). The fact that the short form of the AGPAT9 is also enzymatically active and the APP regulates the expression of several genes, might suggest why we initially cloned the short form for AGPAT9. The study of the enzymatic activities of the long and short forms of AGPAT9 also reveal that the first 102 residues at the amino terminus of the protein increase the enzymatic activity, suggesting a physiological role of alternative splicing in this gene.
This study corroborates the previous study that AGPAT9 localizes to the ER using different set of ER-resident proteins, Sec 61 ß. This study also shows that AGPAT1, another ER-resident acyltransferase, and AGPAT9 partially colocalize, i.e. they are present in the similar ER domain.
The two signature motifs are highly conserved among the acyltransferase superfamily, NHX4D, and EGTR (Agarwal & Garg 2003, Leung 2001), but these motifs have amino acid substitutions in AGPAT9. The NHX4D motif has asparagine substituted with proline while in the EGTR motif arginine is replaced with cysteine, EGTC. Thus, asparagine in NHX4D and arginine in EGTR may be conserved amongst acyltransferases, but are not required for absolute enzymatic activity, at least in AGPAT9 (this study) and in AGPAT8, as reported previously (Agarwal et al. 2006). Thus, the conserved NHX4D motif can now be represented as (N/P) HX4D where asparagine is not critical for the enzymatic activity.
We have previously reported the role of hydrophobic residues downstream to the NHX4D motif to be important for the enzymatic activity (Agarwal et al. 2006). Similar, strongly hydrophobic region is obvious in the AGPAT9 as well (Fig. 8); suggesting that these hydrophobic residues might similarly fold to form a hydrophobic tunnel to accommodate the acyl chain, as observed with the tertiary structure of squash (Cucurbita moschata) chloroplast gpat (Turnbull et al. 2001a,b, Slabas et al. 2002, Tamada et al. 2004). Analysis of the predicted secondary structure of gpat and AGPATs show that the positioning of the HX4D domain is in the loop region between a beta strand (ßc) and alpha helix (
6). The positioning of the other secondary structure elements also fall in a similar way for AGPAT2 and AGPAT9, which is comparable to that of the gpat. It is important to note that even though AGPATs share ~12–15% sequence homology with gpat, these proteins are similar in the secondary structure prediction and hydrophobicity distribution, which would be the key features in determining the fold of the protein and also the active site hydrophobic tunnel. This indicates that the entire hydrophobic region is critical for the enzymatic activity and not the EGT(R/D/C) motif as observed in acyltransferases cloned till now. As shown in Fig. 8, the secondary structure prediction also indicates that the active site of the proteins, AGPAT2 and AGPAT9, should fold similar to the gpat as determined by X-ray crystal structure (Turnbull et al. 2001a,b, Slabas et al. 2002, Tamada et al. 2004). This would strongly suggest that the cloned enzyme should have sn-2 acyltransferase activity similar to AGPAT2 whose activity is now established (Fig. 9).
|
It is interesting to observe that so many AGPATs are now cloned. Isoforms 1–5, 8, and 9 all appear to have varying degree of acyltransferase activity. Most, isoforms 1, 2, and 6–9 are all localized to ER. This raises an important question: why would cells express all of these AGPATs localized to the ER? Are the activities redundant? Answers to these questions will emerge when detailed investigation of these AGPATs are carried out, in terms of hormonal and nutritional regulation, both at the transcriptional and translational level, tissue expression, substrate specificities and subcellular localization. To understand the physiological function of each of these AGPATs either by knocking down the activities in cultured cells or by developing the knockout mouse model shall reveal the function for each of these in specific cell types or in an integrated animal system. It is quite likely that each of these AGPATs perform a unique function within the cells, which maintain cellular membrane structure and/or produce specific phospholipids signaling molecule. Identification and characterization of AGPAT9 is a step towards understanding the role of these acyltransferases in human physiology.
|
Acknowledgements |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Agarwal AK & Garg A 2003 Congenital generalized lipodystrophy: significance of triglyceride biosynthetic pathways. Trends in Endocrinology and Metabolism 14 214–221.[CrossRef][Web of Science][Medline]
Agarwal AK, Arioglu E, de Almeida S, Akkoc N, Taylor SI, Bowcock AM, Barnes RI & Garg A 2002 AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome 9q34. Nature Genetics 31 21–23.[CrossRef][Web of Science][Medline]
Agarwal AK, Barnes RI & Garg A 2004 Genetic basis of congenital generalized lipodystrophy. International Journal of Obesity and Related Metabolic Disorders 28 336–339.
Agarwal AK, Barnes RI & Garg A 2006 Functional characterization of human 1-acylglycerol-3-phosphate acyltransferase isoform 8: cloning, tissue distribution, gene structure and enzymatic activity. Archives of Biochemistry and Biophysics 449 64–76.[CrossRef][Web of Science][Medline]
Beigneux AP, Vergnes L, Qiao X, Quatela S, Davis R, Watkins SM, Coleman RA, Walzem RL, Philips M, Reue K et al. 2006 Agpat6-a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium. Journal of Lipid Research 47 734–744.
Chen X, Hyatt BA, Mucenski ML, Mason RJ & Shannon JM 2006 Identification and characterization of a lysophosphatidylcholine acyltransferase in alveolar type II cells. PNAS 103 11724–11729.
Coleman RA & Lee DP 2004 Enzymes of triacylglycerol synthesis and their regulation. Progress in Lipid Research 43 134–176.[CrossRef][Web of Science][Medline]
Eberhardt C, Gray PW & Tjoelker LW 1997 Human lysophosphatidic acid acyltransferase. cDNA cloning, expression, and localization to chromosome 9q34.3. Journal of Biological Chemistry 272 20299–20305.
Garg A 2004 Acquired and genetic lipodystrophies. New England Journal of Medicine 350 1220–1234.
Haque W, Garg A & Agarwal AK 2005 Enzymatic activity of naturally occurring 1-acylglycerol-3-phosphate-O-acyltransferase 2 mutants associated with congenital generalized lipodystrophy. Biochemical and Biophysical Research Communications 327 446–453.[CrossRef][Web of Science][Medline]
Hui J, Hung LH, Heiner M, Schreiner S, Neumuller N, Reither G, Haas SA & Bindereif A 2005 Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing. EMBO Journal 24 1988–1998.[CrossRef][Web of Science][Medline]
Kozak M 1984 Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature 308 241–246.[CrossRef][Medline]
Leung DW 2001 The structure and functions of human lysophosphatidic acid acyltransferases. Frontiers in Bioscience 6 d944–d953.[CrossRef][Web of Science][Medline]
Li D, Yu L, Wu H, Shan Y, Guo J, Dang Y, Wei Y & Zhao S 2003 Cloning and identification of the human LPAAT-zeta gene, a novel member of the lysophosphatidic acid acyltransferase family. Journal of Human Genetics 48 438–442.[CrossRef][Web of Science][Medline]
Lu B, Jiang YJ, Zhou Y, Xu FY, Hatch GM & Choy PC 2005 Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart. Biochemical Journal 385 469–477.[CrossRef][Web of Science][Medline]
McMaster CR & Jackson TR 2004 Phospholipid synthesis in mammalian cells. In Lipid Metabolism and Membrane Biogenesis, pp 5–30. Ed. G Daum. Berlin Heidelberg: Springer-Verlag.
Nakanishi H, Shindou H, Hishikawa D, Harayama T, Ogasawara R, Suwabe A, Taguchi R & Shimizu T 2006 Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells and possible involvement in surfactant production. Journal of Biological Chemistry 281 20140–20147.
Rost B, Yachdav G & Liu J 2004 The Predict Protein server. Nucleic Acids Research 32 W321–W326.
Slabas AR, Kroon JT, Scheirer TP, Gilroy JS, Hayman M, Rice DW, Turnbull AP, Rafferty JB, Fawcett T & Simon WJ 2002 Squash glycerol-3-phosphate (1)-acyltransferase. Alteration of substrate selectivity and identification of arginine and lysine residues important in catalytic activity. Journal of Biological Chemistry 277 43918–43923.
Tamada T, Feese MD, Ferri SR, Kato Y, Yajima R, Toguri T & Kuroki R 2004 Substrate recognition and selectivity of plant glycerol-3-phosphate acyltransferases (GPATs) from Cucurbita moscata and Spinacea oleracea. Acta Crystallographica. Section D. Biological Crystallography 60 13–21.[CrossRef][Medline]
Turnbull AP, Rafferty JB, Sedelnikova SE, Slabas AR, Schierer TP, Kroon JT, Nishida I, Murata N, Simon JW & Rice DW 2001a Crystallization and preliminary X-ray analysis of the glycerol-3-phosphate 1-acyltransferase from squash (Cucurbita moschata). Acta Crystallographica. Section D. Biological Crystallography 57 451–453.[CrossRef][Medline]
Turnbull AP, Rafferty JB, Sedelnikova SE, Slabas AR, Schierer TP, Kroon JT, Simon JW, Fawcett T, Nishida I, Murata N et al. 2001b Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase. Structure 9 347–353.[Medline]
Vergnes L, Beigneux AP, Davis R, Watkins SM, Young SG & Reue K 2006 Agpat6 deficiency causes subdermal lipodystrophy and resistance to obesity. Journal of Lipid Research 47 745–754.
Webber KO & Hajra AK 1993 Purification of dihydroxyacetone phosphate acyltransferase from guinea pig liver peroxisomes. Archives of Biochemistry and Biophysics 300 88–97.[CrossRef][Web of Science][Medline]
Weier D, Muller C, Gaspers C & Frentzen M 2005 Characterisation of acyltransferases from Synechocystis sp. PCC6803. Biochemical and Biophysical Research Communications 334 1127–1134.[CrossRef][Web of Science][Medline]
Ye GM, Chen C, Huang S, Han DD, Guo JH, Wan B & Yu L 2005 Cloning and characterization a novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase gene AGPAT7. DNA Sequence 16 386–390.[Web of Science][Medline]
Received in final form 15 March 2007
Accepted 28 March 2007
Made available online as an Accepted Preprint 30 March 2007
This article has been cited by other articles:
|
|
 |
|
  S. Sukumaran, R. I Barnes, A. Garg, and A. K Agarwal Functional characterization of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 10/glycerol-3-phosphate acyltransferase isoform 3 J. Mol. Endocrinol., June 1, 2009; 42(6): 469 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Takeuchi and K. Reue Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis Am J Physiol Endocrinol Metab, June 1, 2009; 296(6): E1195 - E1209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Gimeno and J. Cao Thematic Review Series: Glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity J. Lipid Res., October 1, 2008; 49(10): 2079 - 2088. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Q. Chen, M.-S. Kuo, S. Li, H. H. Bui, D. A. Peake, P. E. Sanders, S. J. Thibodeaux, S. Chu, Y.-W. Qian, Y. Zhao, et al. AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase J. Biol. Chem., April 11, 2008; 283(15): 10048 - 10057. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhao, Y.-Q. Chen, T. M. Bonacci, D. S. Bredt, S. Li, W. R. Bensch, D. E. Moller, M. Kowala, R. J. Konrad, and G. Cao Identification and Characterization of a Major Liver Lysophosphatidylcholine Acyltransferase J. Biol. Chem., March 28, 2008; 283(13): 8258 - 8265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jain, N. Stanford, N. Bhagwat, B. Seiler, M. Costanzo, C. Boone, and P. Oelkers Identification of a Novel Lysophospholipid Acyltransferase in Saccharomyces cerevisiae J. Biol. Chem., October 19, 2007; 282(42): 30562 - 30569. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | CONTACT US | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
. GeneBank accession numbers: Human AGPAT9; this study and NM_024830, NP_0791061; mouse: NM_145376, NP_663351; rat: XM_341747, XP_341748; opossum: AAFR02008846; stickleback; from trace database and zebrafish: XM_701606, XP_706698; fly: NP_001036265.
