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 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 small peptide hormones, catecholamines, iodothyronines, cholesterol and its metabolites steroids, oxysterols and vitamin D. Steroid/sterol sulfotransferases play a fundamental role in specific physiologic 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 biologic activity of steroids/sterols, regardless of whether they act 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 biologic 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 focused on two research areas: 1) cloning, biochemical and structural characterization, differential expression and transcriptional regulation of human, guinea and rabbit PAPSS isozymes. 2) Biochemical and structural characterization, differential expression and transcriptional regulation of the human hydroxysteroid sulfotransferase that sulfoconjugates cholesterol, pregnenolone 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. The biologic significance of having two forms of an enzyme carrying out an identical function, i.e. synthesis of PAPS is not presently appreciated. Multiplex PCR reveals that PAPSS1 is ubiquitously expressed and is the predominant form in human tissues with the exception of the liver, whereas specific tissues differentially express the PAPSS2 subtypes. Interestingly, adult cartilage expresses PAPSS1 to a greater degree than PAPSS2, whereas in the growth plate of developing bones, PAPSS2 is the predominant isoform. This finding, in part, explains why mutations involving the PAPSS2 gene result in a dwarfing disorder. In studies of transcriptional regulation, the sites for initiation of RNA synthesis have been identified for both genes, and the 5-prime-flanking region upstream of the capping sites contains neither a TATAA nor a CCAAT box. Multiple GC/GT boxes are, however, present in the proximal promoter regions and utilizing human brain, liver, cartilage and adrenocortical cell lines, revealed that both genes are under the influence of the Sp1 family of transcription factors, particularly Sp1 and Sp2. Real-time PCR is being used to further investigate differential tissue expression of PAPSS isoforms during development using guinea pig and rabbit animal models. In collaboration with the Max-Plank Institute in Dortmund, Germany, crystallization of PAPSS synthase 1 with resolution of its three dimensional structure by x-ray crystallography is 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 sulfate 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 sulfate, an essential activator of the transglutaminase-1 gene. Sulfonated oxysterols are involved in the regulation of an important class of orphan nuclear receptors. We have now identified the hydroxysteroid sulfotransferase that sulfonates cholesterol (SULT2B1). As a result of an alternative exon 1, the gene for human 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 hydroxysteroid sulfotransferase SULT2A1 isozyme is the presence of extended amino- and carboxy-terminal ends in the former. Removal of 53 amino acids from the relatively long carboxy-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. In the gene for SULT2B1, exon IB encodes for only the unique amino-terminal region of SULT2B1b; however, exon IA encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene for SULT2B1 employs exon IB, cholesterol sulfotransferase is synthesized, whereas if exon IA is used, pregnenolone sulfotransferase is produced. Real-time PCR reveals that the SULT2B1a isoform is exclusively expressed in the central nervous system, whereas SULT2B1b is the predominant SULT2 isozyme expressed in skin. The significance of this is that pregnenolone sulfate acts as an essential neurosteroid, whereas cholesterol sulfate is fundamentally involved is keratinocyte differentiation. Studies regarding the transcriptional regulation of the human cholesterol sulfotransferase gene are in progress. Interestingly, an ortholog of the human SULT2B1 gene has now been cloned in mice and is under investigation with the aim of developing a gene knockout model. In collaboration with the Laboratory of Reproductive and Developmental Toxicology, NIEHS, efforts are under way to solve the three dimensional structure of the human SULT2B1 isoforms by x-ray crystallography.