Highly conserved mouse and human brain sulfotransferases: molecular cloning, expression, and functional characterization

Abstract

By employing reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5′-rapid amplification of cDNA ends technique, we have cloned a novel mouse sulfotransferase cDNA. Database search led to the identification of a human gene encoding the homologue of this newly discovered mouse sulfotransferase. RT-PCR technique was employed to clone the cDNA encoding the human enzyme. Sequence analysis revealed that the novel mouse and human sulfotransferases display nearly 98% identity in their amino acid sequences. Their amino acid sequence identity to other known cytosolic sulfotransferases, however, was found to be below 36%. These two highly conserved sulfotransferases therefore appear to belong to a family different from the two major mammalian cytosolic sulfotransferase gene families. Northern blot analysis revealed the neuronal tissue-specific expression of these two novel sulfotransferases. Recombinant mouse and human brain sulfotransferases, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as 33 kD proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified mouse and human brain sulfotransferases displayed enzymatic activities toward endogenous and xenobiotic compounds, including l-triiodothyronine, thyroxine, estrone, p-nitrophenol, 2-naphthylamine, and 2-naphthol. Using mouse brain filtrate as substrate, both brain sulfotransferases were shown to catalyze specifically the sulfation of only a few compounds.

Introduction

In mammals, sulfation represents a major pathway for the biotransformation/excretion of drugs and xenobiotics, as well as endogenous compounds such as catecholamines, cholesterol, steroid and thyroid hormones, and bile acids (Mulder and Jakoby, 1990, Falany and Roth, 1993, Weinshilboum and Otterness, 1994). The responsible enzymes, collectively called the ‘cytosolic sulfotransferases’, constitute a superfamily of enzymes that catalyze the transfer of a sulfonate group from the active sulfate, 3′-phosphoadenosine 5′-phosphosulfate, to a substrate compound containing a hydroxyl or an amino group (Lipmann, 1958). Based on the amino acid sequences of known mammalian cytosolic sulfotransferases, two major gene families, the phenol sulfotransferase (PST) family (designated SULT1) and hydroxysteroid sulfotransferase (HSST) family (designated SULT2), have been categorized (Yamazoe et al., 1994, Weinshilboum et al., 1997). The PST family presently consists of four sub-families, PSTs (SULT1A), Dopa/tyrosine (or thyroid hormone) sulfotransferases (SULT1B), hydroxyarylamine (or acetylaminofluorene) sulfotransferases (SULT1C), and estrogen sulfotransferases (SULT1E). Taking into account the two recently discovered human hydroxysteroid sulfotransferases (Her et al., 1998), the HSST family now comprises two sub-families, SULT2A and SULT2B.

Most of the cytosolic sulfotransferases identified to date are capable of utilizing somewhat broad spectra of compounds as substrates, and many of them display overlapping substrate specificities (Mulder and Jakoby, 1990, Falany and Roth, 1993, Weinshilboum and Otterness, 1994). Most of them are also expressed in a rather wide range of tissues. For example, the two major human PSTs, the monoamine (M)-form PST and simple phenol (P)-form PST, have been detected in human tissues including brain, liver, intestine, lung, adrenal gland, and blood platelets (Weinshilboum and Otterness, 1994). Expression in such a variety of tissues seems compatible with their proposed involvement in the detoxification process. By searching the GenBank database, we have recently identified two partial mouse sulfotransferase cDNA clones (Accession # H31054 and W59156) which, based on sequence analysis, fall into none of the sulfotransferase sub-families mentioned above. Intriguingly, these two partial cDNAs, which presumably encode different parts of the same mouse sulfotransferase, displayed greater than 97% identity at the amino acid sequence level with corresponding coding regions of an apparently homologous human sulfotransferase gene (GenBank Accession # HS388M5). Such a high degree of sequence homology strongly implies that these homologous sulfotransferase enzymes may play a fundamental role in mammals. We were therefore interested in examining in greater detail the structural and functional properties of these two novel sulfotransferases, as well as their tissue distribution. The latter information may provide further insight into their functional involvement in vivo.

We report in this communication the molecular cloning, expression, purification, and characterization of the mouse and human brain sulfotransferases. By employing Northern and Western blotting techniques, the tissue-specific distribution of these novel sulfotransferases among mouse and human organs/tissues was investigated.