The Biotransformation Of Endobiotics By Sulfonation

Child Health And Human Development

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Abstract

Sulfonation is a fundamental process in the biotransformation of endobiotics as well as drugs and xenobiotics. Sulfonation is essential for normal growth and development and maintenance of the internal milieu. Sulfonated macromolecules such as glycosaminoglycans and proteoglycans are involved in cell surface and connective tissue structures and bone formation. Sulfonation of tyrosine residues is a widespread post-translational modification of many secretory and membrane proteins as well as neuroendocrine peptides. In addition to the tyrosine sulfonation, carbohydrate conjugates of the glycoprotein hormones are also subject to modification whereby the addition of a sulfonate group onto a saccharide moiety creates a unique structural motif with important functional consequences. Sulfolipids such as sphingolipids and galactoglycerolipids are concentrated in the brain, peripheral nerves and reproductive tissues. Sulfonation is also of major importance in the biotransformation of low molecular weight compounds such as catecholamines, iodothyronines, cholesterol, oxysterols, steroids and vitamins. Steroid/sterol sulfotransferases play a fundamental role in specific physiological systems and associated disorders, e.g. the menstrual cycle, sperm capacitation and fertility; hormone-dependent tumors of the prostate and breast; obesity and diabetes; lung maturation and the respiratory distress syndrome; anxiety, stress and seizure disorders. Sulfonation has a marked effect on the biological activity of steroids/sterols, regardless of whether they are acting via a genomic or nongenomic mechanism, by modulating availability of the biologically active form. Sulfonation cannot occur in the absence of the universal sulfonate donor molecule, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which establishes PAPS as a strategic biological molecule and making its availability of vital importance. The production of PAPS from ATP and inorganic sulfate is regulated by the bifunctional enzyme, PAPS synthase (PAPSS). We are currently concentrating our research activity in two areas: 1) cloning, biochemical and structural characterization, differential expression and transcriptional regulation of human PAPSS isozymes; 2) biochemical and structural characterization, differential expression and transcriptional regulation of the human hydroxysteroid sulfotransferase that sulfoconjugates cholesterol and oxysterols. The genes for human PAPSS 1 and 2 are located on chromosomes 4 and 10, respectively. The proteins encoded by these two genes are 76% identical. Additionally, PAPSS 2 exists in two forms, i.e. 2a and 2b. While all three isoforms demonstrate Michaelis/Menten kinetics, there are distinct functional differences, i.e. the PAPSS2 subtypes have a greater catalytic efficiency and are 15-20 times more active than PAPSS1. Multiplex PCR revealed that PAPSS1 is ubiquitously expressed and is the predominant form in all human tissues except the liver, whereas specific tissues differentially express the PAPSS2 subtypes. Interestingly, human adult cartilage expresses PAPSS1 to a greater degree than PAPSS2, whereas in the growth plate of developing bones, PAPSS2 is clearly the predominant isoform. In studies of transcriptional regulation, the sites for initiation of RNA synthesis have been identified for both genes, and the 5?-flanking region upstream of the capping sites contains neither a TATAA nor a CCAAT box. Multiple GC/GT boxes are present in the proximal promoter regions, and both genes are under the influence of the Sp1 family of transcription factors, particularly Sp1 and Sp2. In addition, the transcription factor AP2 (alpha and beta) is involved in the regulation of the gene for PAPSS2. These in vitro studies have utilized human brain, liver, cartilage and adrenocortical cell lines. Quantitative RTPCR using a light cycler system is being used to further investigate differential tissue expression of the PAPSS isoforms during development using the guinea pig, rabbit and mouse as animal models. In collaboration with the Max-Plank Institute in Dortmund, Germany, crystallization of the PAPSS synthase 1 and resolution of its three dimensional structure is by x-ray crystallography in progress. Sulfonation of cholesterol and hydroxylated metabolites (oxysterols) has far-reaching physiological significance. For instance, sulfonation of cholesterol is an important metabolic step during normal skin development and creation of the barrier. Cholesterol sulfonate functions as an essential signal transducer (e.g. stimulates protein kinase C isoforms, especially the eta isoform). Epidermal cornification involves the cross-linking of precursor proteins, a process dependent on the activity of transglutaminase-1, which in turn is dependent on the accumulation of cholesterol sulfonate, an activator of the transglutaminase-1 gene. Sulfonated oxysterols are involved in the regulation of an important class of orphan nuclear receptors. It is notable that until recently, a specific cholesterol sulfotransferase had not been identified and characterized. We have now identified and begun to characterize the sulfotransferase that sulfonates both cholesterol and specific oxysterols. Structurally, the cholesterol/sterol sulfotransferase protein is distinct from all previously cloned human steroid sulfotransferases in that is has extended amino- and carboxyterminal regions, with the carboxyterminal end being especially rich in proline residues. In contrast to the two termini, however, the core region of the cholesterol sulfotransferase protein is highly homologous to other members of the steroid sulfotransferase family. Mutational analysis revealed that removing the carboxy-terminus from cholesterol sulfotransferase has no effect on catalytic activity; on the other hand, removing the extended amino-terminus resulted in almost complete loss of enzymatic activity. This finding strongly suggests that the amino-terminus is responsible for the unique substrate selectivity demonstrated by this steroid sulfotransferase isoform. Further mutational studies are underway to explore this discovery in more detail. Additionally, studies regarding the transcriptional regulation of human cholesterol sulfotransferase are in progress. Interestingly, an ortholog of human cholesterol sulfotransferase has now been cloned in mice, which currently is also under investigation with the aim of developing a gene knockout model. In collaboration with the Laboratory of Reproductive and Developmental Toxicology, NIEHS, x-ray crystallization to solve the three dimensional structure of human cholesterol sulfotransferase is in progress. Furthermore, crystallization of specific mutant constructs of human cholesterol sulfotransferase is also planned.