Human Sult2b1

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The hydroxysteroid SULT subfamily also includes the two isoforms of SULT2B1. These enzymes represent two transcriptional products from the SULT2B1 gene that is localized at the same gene locus as the SULT2A1 gene (Her et al., 1998; Otterness et al., 1995a). The SULT2B1 gene was initially identified by screening a human placental expressed sequence tag database with an oligonucleotide encoding a highly conserved amino acid sequence motif "RKGxxGDWKNxFT" present in the SULTs (Her et al., 1998). This procedure identified two related cDNA sequences that were derived from the same gene but utilized different transcriptional start sites to incorporate different first exons. The cDNAs were termed SULT2B1a and SULT2B1b. SULT2B1b is 365 amino acids in length, whereas SULT2B1a is 350 amino acids in length. The final 344 amino acids of both sequences are identical since they are derived from the same exons. Expression of the SULT2B1 cDNAs in COS-1 cells utilizing the pCR3.1 vector generated both active proteins. Subsequent expression studies of the histidine (His)-tagged and native forms of the SULT2B1 cDNAs in Escherichia coli demonstrated difficulty in the expression of the native form of SULT2B1b (Meloche and Falany, 2001). However, SULT2B1b was readily expressed with an amino terminal His tag; the active native enzyme could then be obtained by cleavage of the His tag. The expressed enzymes demonstrated selectivity for the sulfation of the 3B-OH position of hydroxysteroids such as DHEA and pregnenolone. No activity was observed using 3a-OH steroids or 3-phenolic steroids as substrates (Meloche and Falany, 2001), but both expressed enzymes were capable of sulfating dihydrotestosterone (Geese and Raftogianis, 2001).

SULT2B1a and SULT2B1b message expression occurs in a number of human tissues including prostate, placenta, trachea, and skin (Her et al., 1998; Geese and Raftogianis, 2001; Meloche and Falany, 2001). The levels of SULT2B1b specific message are generally several-fold greater than those for SULT2B1a. Immunoblot analysis of the expressed native forms of SULT2B1a and SULT2B1b has shown that these proteins can be easily distinguished by their different molecular masses (Meloche and Falany, 2001). In contrast to message levels, immunoblot analysis of several human tissues detects the presence of only SULT2B1b protein (Figure 5.2).

FIGURE 5.2 Expression of SULT2B1b in human MCF-7 breast cancer cells and human breast cancer tissue. Cytosol was prepared from MCF-7 cells and from a specimen of human breast cancer (white female, age 56 years). The cytosols were resolved by SDS-polyacryla-mide gel electrophoresis, transferred to nitrocellulose membrane, and incubated with a rabbit antihuman SULT2B1 antibody (Meloche and Falany, 2001). The secondary antibody was goat antirabbit IgG with protein visualization by chemiluminescence using a West Pico kit (Pierce).

FIGURE 5.2 Expression of SULT2B1b in human MCF-7 breast cancer cells and human breast cancer tissue. Cytosol was prepared from MCF-7 cells and from a specimen of human breast cancer (white female, age 56 years). The cytosols were resolved by SDS-polyacryla-mide gel electrophoresis, transferred to nitrocellulose membrane, and incubated with a rabbit antihuman SULT2B1 antibody (Meloche and Falany, 2001). The secondary antibody was goat antirabbit IgG with protein visualization by chemiluminescence using a West Pico kit (Pierce).

These results suggest that SULT2B1a and SULT2B1b expression in human tissues is controlled at both the transcriptional and translational levels and that SULT2B1b is selectively expressed in human tissues.

The analysis of phenol SULT activity and expression in human tissues was significantly advanced by the presence of phenol SULT activity in blood platelets. The ability to analyze platelet phenol SULT activity resulted in the extensive characterization of the biochemical and pharmacogenetic properties of these enzymes. Purification of MPST (SULT1A3) and PPST (SULT1A1) from platelet and liver, respectively, was instrumental in providing the background for the cloning and expression of the phenol SULT gene subfamily. The phenol SULT family consists of seven members encoded by separate genes that are arranged in clusters on several chromosomes (Freimuth et al., 2000; Weinshilboum et al., 1997).

Molecular characterization of the human SULTs began with the cloning and expression of the cDNA for PPST-1 from a liver cDNA library (Wilborn et al., 1993). With the development of our understanding of the heterogeneity of the SULT family, PPST has subsequently been termed SULT1A1 (Table 5.1). It was demonstrated that the biochemical and enzymatic properties of the expressed PPST-1 protein were similar to those of the platelet PPST enzyme (Wilborn et al., 1993). Polyclonal rabbit antibody raised to either MPST (SULT1A3) or PPST-1 (SULT1A1) reacts strongly with both proteins, but differential migration of the proteins during SDS-polyacrylamide gel electrophoresis facilitates identification of the individual enzymes (Falany et al., 1990; Heroux et al., 1989). The translated molecular mass of SULT1A1 is 34,097 Da (Wilborn et al., 1993), which is slightly less than that of SULT1A3 (MPST; Wood et al., 1994); however, SULT1A1 migrates with a mass of approximately 32 kD, whereas

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