Characterization of proline-serine-rich carboxyl terminus in human sulfotransferase 2B1b: immunogenicity, subcellular localization, kinetic properties, and phosphorylation.
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 full-length 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.
He
Dongning
D
Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1670 University Blvd., Volker Hall, Room G133M, Birmingham, AL 35294, USA.
Falany
Charles N
CN
D045744:Cell Line, Tumor; D003687:Dehydroepiandrosterone / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004795:Enzyme Stability; D006801:Humans; D015151:Immunoblotting; D042541:Intracellular Space / Q000201:enzymology; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D010766:Phosphorylation; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013696:Temperature; D014162:Transfection / Q000379:methods
D007527:Isoenzymes; D013431:Sulfates; D012694:Serine; D003687:Dehydroepiandrosterone; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D015238:Sulfotransferases; C433248:SULT2B1 protein, human