A novel mouse liver-sulfotransferase like (mL-STL) gene was firstly identified in our laboratory with unknown physiological function. Computer assisted analysis revealed that the mL-STL gene encodes a complete cytosolic sulfotransferase (SULT) domain. SULT is a superfamily of enzymes that catalyze the transfer of a sulfate group from a cofactor, 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to the hydroxyl group or amino group of substrates in a process called sulfonation. Different SULT family members recognize specific subsets of substrates. Dendrogram analysis indicated that the mL-STL protein shares a high amino acid sequence similarity with the SULT2A subfamily, which is known to sulfonate hydroxysteroids. This result suggests that the mL-STL protein can catalyze the SULT2A substrates. However, further study is required to confirm the substrate specificity of the mL-STL protein.
The first objective of my study is to characterize whether the mL-STL protein is a functional cytosolic SULT enzyme which can catalyze the SULT2A substrates. To study the sulfotransferase activity of the mL-STL protein, the recombinant mL-STL protein was produced and subjected to sulfotransferase assay. The mL-STL cDNA was subcloned into a prokaryotic mini-pRSETA expression vector and the recombinant mL-STL protein was successfully expressed in BL21(DE3)pLysS E. coli strain. The pRSETA-mL-STL protein was then column purified, confirmed by mass spectrometry and used for sulfotransferase assays with substrates from different SULT families. Our result indicated that the purified pRSETA-mL-STL protein showed the highest sulfonation catalytic efficiency to the SULT2A-catalyzed bile acids including Na chenodeoxycholate (Na CDC) followed by Na cholate, ϖ-muricholic acid (ϖ-MC) and Na taurochenodeoxycholate (Na tauroCDC). The pRSETA-mL-STL protein only showed a relatively low sulfonation activity to other bile acids including lithocholic acid (LCA), Na deoxycholate (Na DC), Na taurolithocholic acid (Na tauroLC), β-muricholic acid (ϖ-MC) as well as ϖ-muricholic acid (ϖ-MC). No sulfotransferase activity was detected toward other hydroxylsteroids including DHEA, pregnenolone, corticosterone, cholesterol, 22(H)-hydroxycholesterol [22(H)-HC] and 22(R)-hydroxycholesterol [22(R)-HC]. Also, the pRSETA-mL-STL protein showed no detectable activity to the putative substrates of other SULT members including phenols, naphthols, dopamine and estrones. To confirm this sulfonation activity in eukaryotic system, the mL-STL DNA was cloned into an eukaryotic vector pcDNA4/HisMaxC and the recombinant HisMax-mL-STL protein was stably-expressed in AML-12 and HEK293 cell lines for sulfotransferase assay. The result showed that the stably-expressed mammalian HisMax-mL-STL protein has the same substrate specificity toward Na CDC and followed by Na cholate, α-MC and Na tauroCDC. Taken together, the present results confirmed that the mL-STL protein is a new mouse cytosolic sulfotransferase which can only catalyze unique bile acid substrates of SULT2A family in vitro.
My second objective is to investigate the regulation mechanism of mL-STL protein expression by peroxisome proliferator-activated receptor α (PPARα). In a previous study in our laboratory, the mL-STL mRNA expression was found to be dramatically up-regulated by PPARα during energy deprivation. PPARα is a ligand-binding transcription factor and it can regulate its target gene expression by heterodimerizing with retinoid X receptor (RXR) and binding to the specific regions of target genes called the peroxisome proliferator response element (PPRE). Since the mL-STL gene expression is also regulated by PPARα, we would like to study whether the mL-STL gene is a PPARα target gene which contains a functional PPRE. To characterize the putative PPRE of mL-STL gene, the sequence of mL-STL 5’ flanking region and intron 1 was in-silico analyzed and several putative functional PPREs were predicted. The genomic DNA sequences containing the putative PPREs of the mL-STL gene were subcloned into a pGL4.17 dual-luciferase reporter system. The recombinant PPRE reporter system was co-transfected with PPARα and RXR expression vectors together with their specific ligands into four kinds of mammalian cell lines to study its transcriptional activity. The luciferase assay results showed that the mL-STL intron 1 region (-10736/-10224) possessed transcriptional response activity in AML-12 cell line only, which indicated a possible functional PPRE in intron 1 of mL-STL gene in a tissue-specific manner. However, the putative PPRE in this region was not confirmed by site-directed mutagenesis and whether mL-STL i s a d irect PPARα-target gene was still unknown. Further characterization of the functional PPRE of the mL-STL gene might increase our understandings on how PPARá modulate the mL-STL protein expression during energy deprivation.
|Advisor:||Fung, Wing Ping|
|School:||The Chinese University of Hong Kong (Hong Kong)|
|School Location:||Hong Kong|
|Source:||DAI-B 76/08(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||Bile acid, Energy metabolism, Sulfotransferase|
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