Human Sulfotransferase SULT1C1: cDNA Cloning, Tissue-Specific Expression, and Chromosomal Localization1

SHORT COMMUNICATION Human Sulfotransferase SULT1C1: cDNA Cloning, Tissue-Specific Expression, and Chromosomal Localization1 Chengtao Her,* G. Pal Kaur,† Raghbir S. Athwal,† and Richard M. Weinshilboum*,2 *Department of Pharmacology, Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, Minnesota 55905; and †The Fels Institute for Cancer Research and Molecular Biology, Temple University Medical School, Philadelphia, Pennsylvania 19140 Received October 7, 1996; accepted February 6, 1997

We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as ‘‘ST1C1’’—although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated ‘‘SULT.’’ The new human cDNA was cloned from a fetal liver–spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5*-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2. q 1997 Academic Press

Sulfate conjugation is an important pathway in the biotransformation of many drugs, xenobiotic compounds, hormones, and neurotransmitters (13). The cytosolic sulfotransferase (ST) enzymes that catalyze these reactions are members of an emerging gene superfamily, five members of which are currently known to be expressed in human tissues (14). In vertebrates, these enzymes can be divided into two large families, the phenol STs (PSTs) and the hydroxysteroids STs Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. U66036 and U66037. 1 This work was supported in part by NIH Grants RO1 GM 28157 and RO1 GM 35720 as well as a supplement to RO1 GM 35720 supported by the Office of Research on Women’s Health. 2 To whom correspondence and reprint requests should be addressed. Telephone: (507) 284-2246. Fax: (507) 284-9111.

(HSSTs), members of which share at least 45% amino acid sequence identity. The PST family may be further subdivided into at least two subfamilies, the PSTs and estrogen STs (ESTs), members of which share at least 60% amino acid sequence identity (13, 14). Five human ST enzymes, three PSTs, one EST, and one HSST, have been described (14). cDNAs and genes for all five of these enzymes have been cloned, and the chromosomal localizations of their genes have been determined (14). The three human PST genes have been localized to the short arm of chromosome 16 within a human ‘‘PST gene complex’’ located between bands 16p11.2 and 16p12.1 (1, 3, 7, 12). The human EST gene, STE, was mapped to 4q13.1 (6), and the human DHEA ST gene, STD, was localized to 19q13.3 (11). However, the question of the total number of cytosolic ST enzymes expressed in human tissues remains unresolved. In the present study, we report the isolation of a cDNA for a novel human ST enzyme, its tissue-specific expression, and the chromosomal localization of its gene. Cytosolic ST enzymes contain at least four areas of highly conserved amino acid sequence (9, 14). Site-directed mutagenesis experiments have indicated that one of the sequence motifs located near the carboxyl terminus of these enzymes, an area that has been referred to as ‘‘region IV,’’ might be involved in the binding of the cosubstrate for the reaction, 3*-phosphoadenosine-5*-phosphosulfate (4, 9). We took advantage of those observations to seek additional human cytosolic STs in the expressed sequence tag database (2) by searching for the highly conserved region IV signature sequence, RKGxxGDWKNxFT, in which ‘‘x’’ represents any amino acid (14). That approach resulted in the identification of IMAGE CloneID 296479 (8), GenBank Accession No. N74646, a clone that had been isolated from a human fetal liver–spleen cDNA library. The clone was obtained from the American Type Culture Collection (Rockville, MD) and was sequenced completely on both strands (GenBank Accession No. U66036). This new human cDNA contained a 176-bp 5*-untranslated region (UTR), an 888-bp open reading frame (ORF), and a 251-bp 3*-UTR, followed by a poly(A) tract. A polyadenylation signal, AATAAA, was located GENOMICS

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19 nucleotides 5* upstream from the poly(A) tract. The 296-amino-acid protein encoded by the ORF included all four highly conserved sequence motifs that have been described in cytosolic ST enzymes (9, 14). The sequence of this new hypothetical human ST enzyme was 52, 53, 52, 48, and 36% identical to the amino acid sequences of the five known human cytosolic ST enzymes, TS PST1, TS PST2, TL PST, EST, and DHEA ST, respectively (14). Therefore, even though this new protein appeared to be a member of the PST ‘‘family’’ of enzymes on the basis of sequence, it did not fit within either of the currently well-defined subfamilies within this family; i.e., its sequence was less than 60% identical to those of EST or the three PSTs. When amino acid sequence comparisons were performed with those of all reported cytosolic STs in any species, the protein encoded by the new human cDNA displayed highest amino acid sequence identity, 62%, with that encoded by a rat liver cDNA that was designated ST1C1 (10). The protein encoded by this rat cDNA not only catalyzed the sulfation of 4-nitrophenol, a prototypic substrate for PSTs, it also catalyzed the sulfate conjugation of the carcinogen N-hydroxyl-2-acetylaminofluorene, an essential step in the ‘‘metabolic activation’’ of this compound (10). Our observation of high sequence homology with the rat ST1C1 cDNA suggested that our new cDNA might encode a human orthologue of the rat enzyme—or that it might be a member of an orthologous subfamily separate and distinct from other PST subfamilies (14). We next performed mRNA dot blot and Northern blot analyses to determine tissues in which this new human ST enzyme might be expressed. Dot blot analysis was performed with poly(A)/ RNA isolated from 50 human tissues (Fig. 1). Positive signals were detected with mRNA isolated from adult human stomach, kidney, and thyroid as well as fetal liver and kidney (Fig. 1). The strongest signals were obtained with adult stomach and kidney (Fig. 1). Human Multiple Tissue Northern Blots (Clontech Laboratories, Inc.) were then used to perform Northern blot analyses (data not shown). The predominant mRNA transcript was approximately 1.4 kb in length and was easily detectable in preparations from adult human stomach and thyroid, as well as fetal human kidney. Fainter signals were detected with mRNA from adult kidney and fetal liver. Therefore, results of the Northern blot analyses were consistent with those obtained by performing mRNA dot blot analysis (Fig. 1). We next attempted to determine the chromosomal localization of the gene that encoded this new human ST enzyme to find whether it differed from the locations on chromosomes 4, 16, and 19 of currently known and characterized genes for cytosolic ST enzymes in humans (1, 3, 6, 7, 11, 12). As a first step, we determined the partial sequence of the 5*-terminus of the first intron within the cDNA ORF by performing rapid amplification of genomic DNA ends, RAGE, with human genomic DNA RAGE panels as template (6). PCR primers

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FIG. 1. Human SULT1C1 mRNA dot blot analysis. (A) ‘‘Map’’ of the loading pattern for a Human mRNA Master Blot (Clontech Laboratories, Inc., Palo Alto, CA). (B) Dot blot analysis of the Human mRNA Master blot performed with human SULT1C1 cDNA as a probe. The cDNA probe had been radioactively labeled with [a-32P]dCTP by random priming (Oligolabeling Kit, Pharmacia, Piscataway, NJ). Each dot contained 93–448 ng of poly(A)/ RNA. The positive signal from the E. coli DNA is probably artifactual.

were designed based on the assumption that the locations of splice junctions within the gene encoding the new cDNA would be similar to those within the genes of other cytosolic ST enzymes (14). Two human cDNAspecific forward primers were used, FLSF2 (5*-TGGCCCTGACCTCAGACCTGGGGAAACA-3*) for the initial amplification and FLSF103 (5*-TTCGAGGCCAAACCAGATGATCTC-3*) for the second, ‘‘nested’’ reaction. The reverse primers used for both rounds of amplification were designed on the basis of the nucleo-

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tide sequence of the ‘‘anchor,’’ pBluescript DNA. Amplification conditions were those described previously (6). A 248-bp amplification product (GenBank Accession No. U66037) was obtained with SstI RAGE panel DNA as template. This product overlapped the cDNA sequence by 49 bp and contained 126 bp of partial intron sequence located between nucleotides 151 and 152 within the cDNA ORF, with the ‘‘A’’ in the ATG translation initiation codon designated /1. Comparison of the location of this presumed splice junction with those of other known human ST genes indicated that its location was highly conserved (14). Knowledge of this intron sequence then made it possible to use a PCR-based approach to determine the chromosomal localization of the gene. Chromosomal localization was determined by performing the PCR with template DNA from NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panels 1 and 2 (Coriell Institute for Medical Research, Camden, NJ). The primers used to perform those amplifications were exon-based forward primer FLSF2 and intronbased reverse primer IN1R1 (5*-AGCCTGTGCTGGCTCAGCAGGTCTT-3*). When the PCR was performed under conditions identical to those used to perform the RAGE experiments with NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel 2, an amplification product of the predicted size was obtained only with template DNA from the parental human cell line and from hybrid cell line GM/NA10826B, a line that retains only human chromosome 2. To confirm the chromosome 2 localization, NIGMS Human/ Rodent Somatic Cell Hybrid Mapping Panel 1 DNA was also used as template to perform the PCR with the same primers, and an amplification product of the predicted length was obtained with template DNA from hybrid cell lines GM/NA09925, GM/NA09926, GM/ NA09927, GM/NA09928, GM/NA09930A, and GM/ NA09934. In this case, concordance and discordance percentages for a chromosome 2 localization were 100 and 0%, respectively. We then used the same primers to perform PCR amplifications with template DNA from a radiation reduced human chromosome 2/rodent hybrid panel (5). Results of that analysis indicated that the gene was located between bands 2q11.1 and 2q11.2 (Fig. 2). In summary, we have isolated and sequenced a cDNA that encodes a novel human cytosolic ST that may be a human orthologue of the rat enzyme ST1C1 (10). Both mRNA dot blot and Northern blot analyses indicated that this cDNA was expressed in the adult human stomach, kidney, and thyroid, as well as in fetal kidney and liver. The gene for this enzyme was mapped to human chromosome 2, between bands 2q11.1 and 2q11.2. Future functional studies of the protein encoded by the cDNA, a protein that is almost certainly an ST enzyme, will be required to determine its role in the biotransformation of exogenous and/or endogenous substrates in humans. A recent Sulfotransferase Nomenclature Workshop held in Drymen, Scotland, sug-

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FIG. 2. Human SULT1C1 gene chromosomal localization. The ideogram depicts schematically the PCR-based sublocalization of the human SULT1C1 gene between bands 2q11.1 and 2q11.2 on the basis of data obtained with human chromosome 2/rodent hybrid cell DNA as template for the PCR. Each cell line studied is listed, as well as whether that line was positive (/) or negative (0) for the presence of SULT1C1 by PCR analysis. The portion(s) of human chromosome 2 present in each cell line is (are) also depicted.

gested that the symbol SULT be used to designate all cytosolic ST cDNAs and genes, a suggestion that has been accepted by the HUGO Nomenclature Committee. Therefore, we suggest that this cDNA be referred to as human SULT1C1 and that its gene be designated SULT1C1. ACKNOWLEDGMENT We thank Luanne Wussow for her assistance with the preparation of the manuscript.

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