Characterization of Proline-Serine-Rich Carboxyl Terminus in Human Sulfotransferase 2B1b: Immunogenicity, Subcellular Localization, Kinetic Properties, and Phosphorylation

Abstract

ABSTRACT:The human sulfotransferase (SULT) 2B1 gene is a member of the SULT2 gene family and encodes two isoforms, SULT2B1a and SULT2B1b. Although messages for both SULT2B1a and SULT2B1b are detectable in human tissues, only SULT2B1b has been identified immunologically. Compared with other human SULTs, SULT2B1b has an extension at the proline-and serine-rich carboxyl (PSC) end of about 53 amino acids. The structure and function of this unique PSC extension were investigated. Constructs of full-length SULT2B1b as well as truncated SULT2B1b without the PSC extension were expressed in Escherichia coli. Removal of the PSC extension significantly decreased the thermostability of the expressed enzyme as well as decreasing the rate of dehydroepiandrosterone sulfation. Rabbit polyclonal antibodies were raised against both the full-length and truncated SULT2B1b proteins. Immunoblot analysis showed that antibodies raised to fulllength SULT2B1b immunoreact only with full-length SULT2B1b, whereas antibodies raised to truncated SULT2B1b react with both full-length and truncated SULT2B1b. Unlike full-length SULT2B1b, truncated SULT2B1b was incapable of translocation to nuclei in transfected human BeWo choriocarcinoma cells. Phosphorylated serines were detected in the PSC extension of full-length SULT2B1b expressed in BeWo cells but not in truncated SULT2B1b. At least one phosphorylated serine was detected in expressed SULT2B1b via two-dimensional gel electrophoresis, immunoblot analysis, and mass spectroscopic analysis. Bacterially expressed full-length SULT2B1b but not truncated SULT2B1b was phosphorylated by casein kinase or Cdc2 protein kinase in vitro. This study suggests that the PSC extension of SULT2B1b is an important site in the immunogenicity, nuclear translocation, kinetic activity, and thermostability of this SULT isoform.

Sulfotransferase 2B1b in human breast: Differences in subcellular localization in African American and Caucasian women

“…These results correlate with those of He et al [10] reporting DHEA sulfation activity in isolated placenta nuclei. SULT2B1b is also the only human SULT isoform that has a proline-rich carboxyl terminus that may be involved in both its nuclear localization and enzyme activity characteristics [15]. The mechanism of nuclear localization is not known, but involves phosphorylation of the unique 3 -tail of SULT2B1b in human BeWo choriocarcinoma cells [15].…”

“…SULT2B1b is also the only human SULT isoform that has a proline-rich carboxyl terminus that may be involved in both its nuclear localization and enzyme activity characteristics [15]. The mechanism of nuclear localization is not known, but involves phosphorylation of the unique 3 -tail of SULT2B1b in human BeWo choriocarcinoma cells [15]. Sulfation activity was evaluated as described in Section 2 in lysate of T-47D breast cancer cells and in cytosol and intact nuclei isolated from fresh breast adenocarcinoma specimen.…”

“…The immunoblot protocol involved using 5% non-fat milk for blocking, primary antibody at a 1:1000 dilution and the appropriate secondary antibody, either goat anti-rabbit or goat anti-mouse IgG HRP conjugate (1:60,000). The following primary antibodies were used: polyclonal rabbit anti-SULT2B1b antiserum generated in our laboratory [15], mouse monoclonal anti-␤-tubulin IgG (Sigma), and mouse monoclonal anti-histone IgG (Chemicon International). Bound antibodies were visualized using the SuperSignal West Pico chemiluminescent substrate system (Pierce).…”

“…SULT2B1b has been localized to the nuclei of placental and breast cells but not lung or prostate cells [8,10,11]. In human BeWo choriocarcinoma cells, nuclear localization of SULT2B1b is associated with serine phosphorylation of a unique carboxyl-terminal proline and serine-rich sequence [15]. The functional role for nuclear localization of SULT2B1b has not been determined although its expression in estrogen responsive tissues would be expected to limit the conversion of DHEA to active estrogens.…”

Human cytosolic sulfotransferase 2B1: Isoform expression, tissue specificity and subcellular localization

“…The 3′-terminal extension has several putative phosphorylation sites as predicted by sequence analysis using the Phosite 1 and Netphos 1 programs. Limited proteolysis and MALDI-TOF mass spectroscopy indicate that a single phosphorylation event is occurring on a 25 aa peptide (figure 2) generated by Glu C digestion from the unique 3′-terminal extension (36). The phosphorylation event may be associated with nuclear localization.…”