Mutational Analysis of Human Hydroxysteroid Sulfotransferase SULT2B1 Isoforms Reveals That Exon 1B of the SULT2B1 Gene Produces Cholesterol Sulfotransferase, whereas Exon 1A Yields Pregnenolone Sulfotransferase

Abstract

As a result of an alternative exon 1, the gene for human hydroxysteroid sulfotransferase (SULTB1) encodes for two peptides differing only at their amino termini. The SULT2B1b isoform preferentially sulfonates cholesterol. Conversely, the SULT2B1a isoform avidly sulfonates pregnenolone but not cholesterol. The outstanding structural feature that distinguishes the SULT2B1 isoforms from the prototypical SULT2A1 isozyme is the presence of extended amino- and carboxyl-terminal ends in the former. Investigating the functional significance of this unique characteristic reveals that removal of 53 amino acids from the relatively long carboxyl-terminal end that is common to both SULT2B1 isoforms has no effect on the catalytic activity of either isoform. On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to SULT2B1b results in loss of cholesterol sulfotransferase activity, whereas removal of 8 amino acids from the amino-terminal end that is unique to SULT2B1a has no effect on pregnenolone sulfotransferase activity. Deletion analysis along with site-directed mutagenesis of SULT2B1b reveal that the amino acid segment 19–23 residues from the amino terminus and particularly isoleucines at positions 21 and 23 are crucial for cholesterol catalysis. In the gene for SULT2B1, exon 1B encodes for only the unique amino-terminal region of SULT2B1b; however, exon 1A encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene forSULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas if exon 1A is used, pregnenolone sulfotransferase is produced. As a result of an alternative exon 1, the gene for human hydroxysteroid sulfotransferase (SULTB1) encodes for two peptides differing only at their amino termini. The SULT2B1b isoform preferentially sulfonates cholesterol. Conversely, the SULT2B1a isoform avidly sulfonates pregnenolone but not cholesterol. The outstanding structural feature that distinguishes the SULT2B1 isoforms from the prototypical SULT2A1 isozyme is the presence of extended amino- and carboxyl-terminal ends in the former. Investigating the functional significance of this unique characteristic reveals that removal of 53 amino acids from the relatively long carboxyl-terminal end that is common to both SULT2B1 isoforms has no effect on the catalytic activity of either isoform. On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to SULT2B1b results in loss of cholesterol sulfotransferase activity, whereas removal of 8 amino acids from the amino-terminal end that is unique to SULT2B1a has no effect on pregnenolone sulfotransferase activity. Deletion analysis along with site-directed mutagenesis of SULT2B1b reveal that the amino acid segment 19–23 residues from the amino terminus and particularly isoleucines at positions 21 and 23 are crucial for cholesterol catalysis. In the gene for SULT2B1, exon 1B encodes for only the unique amino-terminal region of SULT2B1b; however, exon 1A encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene forSULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas if exon 1A is used, pregnenolone sulfotransferase is produced. sulfotransferase 3′-phosphoadenosine 5′-phosphosulfate reverse transcription glutathione S-transferase The cloning of a novel hydroxysteroid sulfotransferase (SULT)1 subfamily in human (1Her C. Wood T.C. Eichler E.E. Mohrenweiser H.W. Ramagli L.S. Siciliano M.J. Weinshilboum R.M. Genomics. 1998; 53: 284-295Crossref PubMed Scopus (135) Google Scholar) and mouse (2Sakakibara Y. Yanagisawa K. Takami Y. Nakayama T. Suiko M. Liu M.-C. Biochem. Biophys. Res. Commun. 1998; 247: 681-686Crossref PubMed Scopus (39) Google Scholar) species has been a significant development in the field of cytosolic sulfotransferases (3Weinshilboum R.M. Otterness D.M. Aksoy I.A. Wood T.C. Her C. Raftogianis R.B. FASEB J. 1997; 11: 3-14Crossref PubMed Scopus (392) Google Scholar, 4Nagata K. Yamazoe Y. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 159-176Crossref PubMed Scopus (177) Google Scholar). The gene for the novel human hydroxysteroid sulfotransferase (SULT2B1) maps to chromosome 19q13.4, which is ∼500 kb telomeric to the location of the prototypical hydroxysteroid sulfotransferase gene SULT2A1(5Her C. Aksoy I.A. Kimura S. Brandriff B.F. Wasmuth J.J. Weinshilboum R.M. Genomics. 1995; 29: 16-23Crossref PubMed Scopus (45) Google Scholar). It should be noted, however, that the gene for SULT2A1encodes for a single polypeptide, whereas the SULT2B1 gene, as a result of an alternative exon 1, encodes for two subtypes differing only at their amino-terminal ends. Thus, the twoSULT genes produce three functionally related polypeptides, the physiologic significance of which is not entirely understood. SULT2A1, which is commonly referred to as dehydroepiandrosterone sulfotransferase, has a broad substrate predilection involving, in addition to dehydroepiandrosterone, a variety of neutral steroids including androgenic steroids. It also sulfonates estrogenic steroids as well as bile acids (6Radominska A. Comer K.A. Zimniak P. Falany J. Iscan M. Falany C.N. Biochem. J. 1990; 272: 597-604Crossref PubMed Scopus (112) Google Scholar, 7Aksoy A. Otterness D.M. Weinshilboum R.M. Drug Metab. Dispos. 1993; 21: 268-275PubMed Google Scholar, 8Falany C.N. Wheeler J., Oh, T.S. Falany J.L. J. Steroid Biochem. Mol. Biol. 1994; 48: 369-375Crossref PubMed Scopus (164) Google Scholar, 9Falany C.N. FASEB J. 1997; 11: 206-216Crossref PubMed Scopus (520) Google Scholar); it does not, however, sulfonate cholesterol (10Javitt N.B. Lee Y.C. Shimizu C. Fuda H. Strott C.A. Endocrinology. 2001; 142: 2978-2984Crossref PubMed Scopus (75) Google Scholar). The SULT2B1 isoforms, in contrast to the SULT2A1 isozyme, have a more selective substrate preference and will not, to all intents and purposes, sulfonate testosterone, estradiol, or bile acids (10Javitt N.B. Lee Y.C. Shimizu C. Fuda H. Strott C.A. Endocrinology. 2001; 142: 2978-2984Crossref PubMed Scopus (75) Google Scholar, 11Meloche C.A. Falany C.N. J. Steroid Biochem. Mol. Biol. 2001; 77: 261-269Crossref PubMed Scopus (87) Google Scholar, 12Geese W.J. Raftogianis R.B. Biochem. Biophys. Res. Commun. 2001; 288: 280-289Crossref PubMed Scopus (79) Google Scholar). Notably, the SULT2B1 isoforms, particularly SULT2B1b, sulfonate cholesterol (10Javitt N.B. Lee Y.C. Shimizu C. Fuda H. Strott C.A. Endocrinology. 2001; 142: 2978-2984Crossref PubMed Scopus (75) Google Scholar), and although a clear functional difference between the SULT2B1 isoforms is now emerging, a physiologic distinction between them is not well understood. From a structural point of view, the outstanding feature of the SULT2B1 isoforms, as compared with the SULT2A1 isozyme as well as other cloned steroid and cognate cytosolic sulfotransferases, is their extended amino- and carboxyl-terminal ends. This characteristic intrigued us as to whether there might be functional effects ascribable to either the carboxyl-terminal ends, which are structurally common to the two proteins, and/or the amino-terminal ends, which are structurally unique. The question was, therefore, posed: do either the amino- or the carboxyl-terminal ends of the human SULT2B1 isoforms have an influence on substrate specificity and/or catalysis? Interestingly, the results of these experiments reveal the selective importance of the unique amino-terminal end of the SULT2B1b isoform for both substrate specificity and catalysis. This is in contrast to the unique amino-terminal end of the SULT2B1a isoform, which neither influences substrate specificity nor is required for catalysis. All steroids, sterols, 2-hydroxypropyl-β-cyclodextrin, and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) were obtained from Sigma. [3H]cholesterol (60 Ci/mmol) and [3H]pregnenolone (17.5 Ci/mmol) were purchased from PerkinElmer Life Sciences. Silica gel TLC plates were obtained from Analtech (Newark, MA). Organic solvents were purchased from J. T. Baker Inc. and Mallinckrodt. Prokaryotic expression vectors for human SULT2B1a (GenBankTM accession no. U92314) and SULT2B1b (GenBankTM accession no. U92315) were obtained using PCR atSalI and NotI restriction sites of pGEX-6P-3 fromAmersham Biosciences. Recombinant SULT2B1 subtypes were purified using the GST gene fusion system (Amersham Biosciences) according to a previously reported procedure (13Fuda H. Shimizu C. Lee Y.C., H., A. Strott C.A. Biochem. J. 2002; 364: 497-504Crossref PubMed Google Scholar). Eluates were collected and analyzed by SDS-polyacrylamide gel electrophoresis, and cleavage proteins were visualized with the GelCode Blue Stain reagent from Pierce. Protein concentrations were determined using the BCA protein assay kit (Pierce) and bovine serum albumin as a standard. Amino- and carboxyl-terminal truncations were generated using PCR.PfuTurbo Hotstart DNA polymerase (Stratagene, La Jolla, CA) and primers as described in TableI were used for PCR under the following conditions: predenaturing at 95 °C for 2 min followed by 18 cycles of denaturing at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 6 min. PCR products were digested with SalI and NotI and ligated intoSalI/NotI-digested pGEX-6P-3 vector directly. Sequencing identified appropriate clones.Table IOligonucleotide primers used in mutagenesis of human SULT2B1 isoformsConstructSense/AntisensePrimersSULT2B1a1. COOHS3′-GTCGACATGGCGTCTCCCCCACCTTTCCAC-3′AS5′-AGCGGCCGCTTAGTCTTCATCCCAGGGGAAGGTCGGCATC-3′2. NH3S5′-ACGCGTCGACAGCCAGAAGTTGCCAGGTGAATAC-3′AS5′-GCGGCCGCTTATTATGAGGGTCGTGGGTGCGG-3′SULT2B1b1. COOHS5′-GTCGACATGGACGGGCCCGCCGAGCCCCAGATC-3′AS5′-AGCGGCCGCTTAGTCTTCATCCCAGGGGAAGGTCGGCATC-3′2. NH3(8)S5′-ACGCGTCGACATCCCGGGCTTGTGGGACACC-3′AS5′-GCGGCCGCTTATTATGAGGGTCGTGGGTGCGG-3′3. NH3(18)S5′-ACGCGTCGACATCTCGGAAATCAGCCAGAAG-3′AS5′-GCGGCCGCTTATTATGAGGGTCGTGGGTGCGG-3′4. NH3(23)S5′-ACGCGTCGACAGCCAGAAGTTGCCAGGTGAATAC-3′AS5′-GCGGCCGCTTATTATGAGGGTCGTGGGTGCGG-3′5. D19AS5′-GGACACCTATGAAGATGCCATCTCGGAAATCAGC-3′AS5′-GCTGATTTCCGAGATGGCATCTTCATAGGTGTCC-3′6. I20AS5′-GACACCTATGAAGATGACGCCTCGGAAATCAGCCAG-3′AS5′-CTGGCTGATTTCCGAGGCGTCATCTTCATAGGTGTC-3′7. I20ES5′-GACACCTATGAAGATGACGAATCGGAAATCAGCCAG-3′S5′-CTGGCTGATTTCCGATTCGTCATCTTCATAGGTGTC-3′8. I20QS5′-GACACCTATGAAGATGACCAATCGGAAATCAGCCAG-3′AS5′-CTGGCTGATTTCCGATTGGTCATCTTCATAGGTGTC-3′9. I20LS5′-GACACCTATGAAGATGACCTCTCGGAAATCAGCCAG-3′AS5′-CTGGCTGATTTCCGAGAGGTCATCTTCATAGGTGTC-3′10. I20MS5′-GACACCTATGAAGATGACATGTCGGAAATCAGCCAG-3′AS5′-CTGGCTGATTTCCGACATGTCATCTTCATAGGTGTC-3′11. I20KS5′-GACACCTATGAAGATGACAAATCGGAAATCAGCCAG-3′AS5′-CTGGCTGATTTCCGATTTGTCATCTTCATAGGTGTC-3′12. S21AS5′-CACCTATGAAGATGACATCGCGGAAATCAGCCAG-3′AS5′-CTGGCTGATTTCCGCGATGTCATCTTCATAGGTG-3′13. E22AS5′-GAAGATGACATCTCGGCAATCAGCCAGAAGTTG-3′AS5′-CAACTTCTGGCTGATTGCCGAGATGTCATCTTC-3′14. I23AS5′-GATGACATCTCGGAAGCCAGCCAGAAGTTGCCAGG-3′AS5′-CCTGGCAACTTCTGGCTGGCTTCCGAGATGTCATC-3′15. I23ES5′-GATGACATCTCGGAAGAAAGCCAGAAGTTGCCAGG-3′AS5′-CCTGGCAACTTCTGGCTTTCTTCCGAGATGTCATC-3′16. I23QS5′-GATGACATCTCGGAACAAAGCCAGAAGTTGCCAGG-3′AS5′-CCTGGCAACTTCTGGCTTTGTTCCGAGATGTCATC-3′17. I23LS5′-GATGACATCTCGGAACTCAGCCAGAAGTTGCCAGG-3′AS5′-CCTGGCAACTTCTGGCTGAGTTCCGAGATGTCATC-3′18. I23MS5′-GATGACATCTCGGAAATGAGCCAGAAGTTGCCAGG-3′AS5′-CCTGGCAACTTCTGGCTCATTTCCGAGATGTCATC-3′19. I23KS5′-GATGACATCTCGGAAAAAAGCCAGAAGTTGCCAGG-3′AS5′-CCTGGCAACTTCTGGCTTTTTTCCGAGATGTCATC-3′Human SULT2B1a had 52 amino acids removed from the carboxyterminus (COOH) and 8 amino acids removed from the amino terminus (NH3): Human SULT2B1b also had 52 amino acids removed from the carboxyl terminus as well as 8, 18 and 23 amino acids removed from the aminoterminus. In addition, point mutations of a DISEI amino acid sequence (residues 19–23) in the aminoterminal region of SULT2B1b were carried out (compare Fig. 2). Underlined nucleotides indicateSalI and NotI restriction sites. Open table in a new tab Human SULT2B1a had 52 amino acids removed from the carboxyterminus (COOH) and 8 amino acids removed from the amino terminus (NH3): Human SULT2B1b also had 52 amino acids removed from the carboxyl terminus as well as 8, 18 and 23 amino acids removed from the aminoterminus. In addition, point mutations of a DISEI amino acid sequence (residues 19–23) in the aminoterminal region of SULT2B1b were carried out (compare Fig. 2). Underlined nucleotides indicateSalI and NotI restriction sites. Mutations involving specific amino acid residues of the amino-terminal region of SULT2B1b were generated using QuikChange XL site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene). Briefly, 25 ng of pGEX-SULT2B1b were used as a template and mutated nucleotide primers as described in Table I. PCR conditions were: predenaturing at 95 °C for 2 min, followed by 18 cycles of denaturing at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 6 min. After digestion with DpnI, 2 μl of PCR product were used to transform XL10-Gold competent cells provided with the kit. Sequencing identified appropriate clones. Sulfotransferase activity was determined using radiolabeled cholesterol and pregnenolone. Twenty-μl reaction volumes contained the same concentration of either an overexpressed and purified wild type protein or a mutant construct (4.0 μg/tube, SULT2B1a, and 0.4 μg/tube, SULT2B1b for cholesterol (5 μm); 0.4 μg/tube, SULT2B1a, and 0.4 μg/tube, SULT2B1b for pregnenolone (20 μm)) in 0.1 mm Tris-HCl buffer (pH 7.5) containing 5 mmMgCl2, 0.2 mm 2-hydroxypropyl-β-cyclodextrin, and 4% ethanol (v/v). Reactions were carried out at 37 °C for 5 min and stopped by placing tubes in boiling water for 5 min. To each reaction tube, 10 μl of 5 mg/ml of either cholesterol sulfate or pregnenolone sulfate in 10% 2-hydroxypropyl-β-cyclodextrin were added as a carrier, and 5 μl were applied to a TLC plate, which was developed using a solvent system consisting of chloroform/methanol/acetone/acetic acid/water (8:2:4:2:1). After drying, the TLC plate was exposed to I2 vapor to visualize the steroid/sterol sulfate spots, which were excised, and the radioactivity was determined by liquid scintillation spectrometry. For the kinetic analyses, assays were similarly carried out using the following purified protein preparation: SULT2B1a, 4.0 μg, and SULT2B1b, 0.4 μg in the cholesterol assay, and SULT2B1a, 0.4 μg, SULT2B1b, 0.4 μg in the pregnenolone assay. The wild type protein preparations used throughout this investigation as well as all mutated protein preparations were judged to be >90% pure based on analysis by SDS-PAGE and protein staining (data not presented). A steady state analysis of the SULT2B1 isoforms using cholesterol and pregnenolone as substrates is depicted in Fig.1. The SULT2B1a isoform avidly sulfonates pregnenolone, whereas sulfonation of cholesterol is minimal. In contrast, the SULT2B1b isoform preferentially sulfonates cholesterol, reaching a maximum at a substrate concentration of 5 μm. SULT2B1b, however, also sulfonates pregnenolone, although a steady state is not reached until the substrate concentration reaches ∼20 μm, at which point the sulfonation of pregnenolone essentially equals that of cholesterol. Nevertheless, the greater efficiency of the SULT2B1b isoform for cholesterol is revealed by a kcat/K m ratio of 11.1 ± 1.2m−1S−1 × 103 for cholesterol versus 1.6 ± 0.4m−1S−1 × 103 for pregnenolone. Notably, neither SULT2B1 isoform sulfonates dehydroepiandrosterone efficiently (data not presented). Removal of 53 amino acids from the carboxyl-terminal end that is common to both SULT2B1a and SULT2B1b (compare Fig. 2) does not significantly reduce the ability of either SULT2B1a to sulfonate pregnenolone or SULT2B1b to sulfonate cholesterol (Fig.3). Likewise, removal of 8 amino acids from the amino-terminal end that is unique to the SULT2B1a isoform (compare Fig. 2) does not significantly alter pregnenolone sulfotransferase activity (Fig. 3). On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to the SULT2B1b isoform (compare Fig. 2) results in an almost complete loss in cholesterol sulfotransferase activity (Fig. 3).Figure 3Sulfotransferase activity after truncation of the amino- and carboxyl-terminal ends of the SULT2B1 isoforms.Wild type (WT) of SULT2B1a and SULT2B1b and constructs of each isoform lacking either the unique amino-terminal end ((−)NH 3) or 52 amino acids from the common carboxyl-terminal end ((−)COOH) (compare Fig. 2) were prepared as GST fusion proteins, cleaved, affinity-purified, and assayed for either pregnenolone (SULT2B1a) or cholesterol (SULT2B1b) sulfotransferase activity as described under “Experimental Procedures” using 20 and 5 μm, respectively, of each substrate. Negative control (Neg. Ctr.) indicates the result when a wild type preparation is assayed in the absence of the PAPS cofactor. The top of each column represents the mean of five replicates, and error barsindicate the standard deviation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast to the removal of the first 23 amino acids from the amino-terminal end of SULT2B1b, which leads to a loss in catalysis, cholesterol sulfotransferase activity is essentially unaffected after removal of the first 8 amino acids (Fig. 4). Although removal of the first 18 amino acids results in an apparent increase in catalytic activity (Fig. 4), the of for the amino-terminal protein is not significantly from the of for wild The loss in catalytic activity that between the and amino-terminal truncations of SULT2B1b indicates that the sequence between residues 18 and (compare Fig. 2) is crucial for this sequence was analyzed by (Fig. and result in of cholesterol sulfotransferase activity that are that of wild although m for these respectively, and μm, are not significantly from the m of wild In contrast to the both the and result in a complete loss of cholesterol sulfotransferase activity (Fig. Interestingly, the have no effect on the ability of this isoform to sulfonate pregnenolone (compare Fig. (data not This was by the effect of a variety of amino acids other for the isoleucines at positions 20 and were carried out for each with results that are essentially the same (Fig. involving a and a amino as well as a involving a but amino acid with a in to result in loss of catalytic activity to the (Fig. On the other hand, of a results in and of catalytic activity (Fig. of a for but with a in results in a at 20 and at in cholesterol sulfotransferase activity (Fig. m of for wild type and for for for and for are not significantly the SULT2A1 and SULT2B1 are at the amino acid however, the extended amino- and carboxyl-terminal ends of the SULT2B1 isoforms are increase to Interestingly, all previously cloned of the cytosolic sulfotransferase and sulfotransferases as well as hydroxysteroid sulfotransferases, have that from to amino whereas SULT2B1a and SULT2B1b of and amino the extended amino- and carboxyl-terminal ends of the SULT2B1 isoforms there a significant structural between the SULT2A1 and SULT2B1 in their a at sites of and specific amino acid residues in of cytosolic sulfotransferases K. M. J. Biochem. Toxicol. 2001; PubMed Scopus Google Scholar, Y. M. Biol. 1997; PubMed Scopus Google Scholar) are with but (compare Fig. 2). with the and of PAPS are structural is a at in SULT2B1a and in SULT2B1b along with a at in SULT2B1a and in The significance of this is that in the of human the of a is to with the of a that the an in of the catalytic with the between the and sulfate of PAPS S. M. J. Biol. 2002; PubMed Scopus Google Scholar). The of the SULT2B1 isoforms residues that are in all cytosolic sulfotransferases, residues now to be in with the PAPS cofactor. This the extended amino- and carboxyl-terminal ends as the outstanding feature of the SULT2B1 isoforms and the structural distinction between them and the prototypical SULT2A1 isozyme as well as other of the cytosolic sulfotransferase The functional significance of the extended carboxyl-terminal end of the SULT2B1 isoforms is not is that this which is in might a in (10Javitt N.B. Lee Y.C. Shimizu C. Fuda H. Strott C.A. Endocrinology. 2001; 142: 2978-2984Crossref PubMed Scopus (75) Google Scholar). it is that the relatively long carboxyl-terminal extension amino which is structurally common to both SULT2B1 isoforms, be removed a significant in the catalytic of either isoform. On the other hand, removal of the unique amino-terminal ends, which the SULT2B1 isoforms, removal of the unique amino-terminal end of SULT2B1a consisting of 8 amino acids does not significantly alter catalytic activity, whereas removal of the unique amino-terminal end of consisting of 23 amino acids has a influence on cholesterol catalysis. The however, the first 18 amino acids of the SULT2B1b amino-terminal region are not for catalytic activity, which leads to the the acid segment between residues 18 and from the amino terminus of SULT2B1b is the crucial structural feature of this isoform that is for cholesterol sulfotransferase activity. the isoleucines this acid segment of SULT2B1b are in catalytic based on the of a variety of amino acid it that only a amino acid with a of at these does not whereas essentially as well as although is also to significant activity. of the produce m to the wild type the of these Conversely, and amino acid at either of the positions are to activity. Interestingly, in the SULT2B1a isoform, the amino acid residues at positions to the isoleucines at positions 20 and 23 in SULT2B1b, are and (compare Fig. 2). The unique amino-terminal end of SULT2B1b is for the ability of this isoform to sulfonate cholesterol. Conversely, the unique amino-terminal end of SULT2B1a is not required for this isoform to sulfonate pregnenolone, and functional significance is not The importance of the amino-terminal end of SULT2B1b for cholesterol specificity as well as is by the in of the truncations and amino acid carried out on this isoform, which influence cholesterol there is no effect of of these on the ability of SULT2B1b to sulfonate pregnenolone. In with the unique amino-terminal end of SULT2B1b it essentially It that the unique amino terminus of SULT2B1 isoform is for whereas a related structural characteristic in the other isoform is not required for it to this or should the have been The gene for human SULT2B1 of an exon 1B, an exon and the SULT2B1a isoform is by 1A and and the SULT2B1b isoform is by exon 1B, the nucleotides of exon plus (1Her C. Wood T.C. Eichler E.E. Mohrenweiser H.W. Ramagli L.S. Siciliano M.J. Weinshilboum R.M. Genomics. 1998; 53: 284-295Crossref PubMed Scopus (135) Google Scholar). 1B is of a region consisting of nucleotides and the region for the first 23 amino acids of SULT2B1b, which represents the amino-terminal region that is unique to this isoform (1Her C. Wood T.C. Eichler E.E. Mohrenweiser H.W. Ramagli L.S. Siciliano M.J. Weinshilboum R.M. Genomics. 1998; 53: 284-295Crossref PubMed Scopus (135) Google Scholar). on the other hand, in addition to a encodes for the first amino acids of SULT2B1a, of which only the first 8 amino acids are unique to this isoform (1Her C. Wood T.C. Eichler E.E. Mohrenweiser H.W. Ramagli L.S. Siciliano M.J. Weinshilboum R.M. Genomics. 1998; 53: 284-295Crossref PubMed Scopus (135) Google Scholar). Thus, when the gene for human SULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas when the gene exon pregnenolone sulfotransferase is produced. This an question the significance of this In of the expression of the gene for human SULT2B1, two as particularly and It is now that cholesterol sulfate an in development and of the T. T. M. S. M. K. S. K. Res. 2000; PubMed Scopus Google Scholar, C. J. PubMed Google Scholar, S. T. M. K. Y. K. T. J. 1998; PubMed Scopus (45) Google Scholar, K. Wood P. A. J. Res. 2001; PubMed Google Scholar). as determined by expression of the human SULT2B1b isoform, which now as a cholesterol sulfotransferase, is in in other with the of the and the Y. C. C. and C. A. Although expression of the human SULT2B1 isoforms in the system to be the human as determined by reverse to only the SULT2B1a isoform W.J. Raftogianis R.B. Biochem. Biophys. Res. Commun. 2001; 288: 280-289Crossref PubMed Scopus (79) Google Scholar). Interestingly, the mouse SULT2B1a to be almost in H. H. and C. A. The significance of these is that pregnenolone, which is efficiently by the of the SULT2B1a isoform, is now as an E.E. P. M. Rev. 2001; PubMed Scopus Google Scholar, Rev. 2001; PubMed Google Scholar, K.A. Rev. 2001; PubMed Google Scholar, E.E. Res. Rev. 2001; PubMed Scopus Google Scholar).