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 as well as 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, carbohydrate conjugates of the glycoprotein hormones are 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 peptide hormones, catecholamines, iodothyronines, cholesterol and its steroid/sterol metabolites. Steroid/sterol sulfotransferases are involved in specific physiologic systems and their 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 requires the universal sulfonate donor molecule, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), establishing PAPS as a strategic biologic molecule and making its availability of vital importance. Production of PAPS from ATP and inorganic sulfate is regulated by bifunctional PAPS synthase (PAPSS). Research is concentrated in 2 areas: cloning, biochemical and structural characterization, differential expression and transcriptional regulation of PAPSS isozymes; biochemical and structural characterization, differential expression and transcriptional regulation of isozymes that sulfoconjugate steroids/sterols. Proteins encoded by the two PAPSS genes are 76% identical. Additionally, as a result of alternative splicing PAPSS 2 exists as 2 isoforms. While all PAPSS isozymes demonstrate Michaelis/Menten kinetics, distinct functional differences exist, i.e. PAPSS2 subtypes have a greater catalytic efficiency and are 15-20 times more active than PAPSS1. PAPSS1 is ubiquitously expressed and is the predominant form in human tissues with the exception of liver, whereas specific tissues differentially express PAPSS2 isoforms. Initiation sites for RNA synthesis have been identified for both PAPSS genes; the 5-prime-flanking region upstream of capping sites contains neither a TATAA nor a CCAAT box. Multiple GC/GT boxes are present in proximal promoter regions and use of 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. The enzymes that sulfoconjugate steroids/sterols are designated SULT2 and consist of SULT2A1 and SULT2B1 isozymes. Whereas SULT2A1 consists of a single form, SULT2B1as a result of an alternative exon 1 and differential splicing consists of two isoforms, i.e. SULT2B1a and SULT2B1b. All human SULT2 isozymes have been over expressed and purified for biochemical, functional and structural analyses. SULT2A1, commonly referred to as dehydroepiandrosterone (DHEA) sulfotransferase, has a broad substrate predilection, whereas the SULT2B1 isoforms have a much narrower substrate preference. Importantly, the SULT2B1 isoforms are also structurally distinct from SULT2A1 as well as all other known cognate cytosolic sulfotransferases. The SULT2B1 isoforms, which differ only at their amino termini, exhibit a defined substrate selectivity that is dependent on their unique amino-terminal ends. SULT2B1a avidly sulfonates pregnenolone but poorly sulfonates cholesterol, whereas SULT2B1b functions as a cholesterol sulfotransferase. The fact that the SULT2A1 and SULT2B1 isozymes are differentially expressed and display dissimilar substrate propensities strongly indicates that they have explicit biologic roles to play. We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3'-phospoadenosine-5'-phosphate at 2.9 A and 2.4 A, respectively, as well as SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 A. These structures reveal a different catalytic binding orientation for the substrate from the previously determined structure for prototypical SULT2A1 binding DHEA. In addition, the amino terminal helix, comprising residues D19 to K26, that determines the specificity difference between the SULT2B1 isoforms, becomes ordered upon pregnenolone binding covering the substrate-binding pocket. The specificity difference for cholesterol and pregnenolone between SULT2B1a and SULT2B1b can be traced to the unique amino-terminal residues. Alanine scanning mutagenesis of the 19-DISEI-23 region revealed that only the I20A and I23A mutants knocked out cholesterol sulfonating activity, which could be partially restored by replacement with conservative substitutions such as leucine and methionine. The positions of I20 and I23 are such that they lie on the same side of the helix facing on the inside of the hydrophobic pocket, whereas residues S21 and E22 are solvent-exposed. Residues corresponding to 19-DISEI-23 of SULT2B1b are 4-PPPFH-8 in SULT2B1a. With 3 prolines in a row, it is unlikely that these residues are able to form an a-helix and therefore would be unable to cover the opening to the substrate binding pocket as seen in the SULT2B1b structure. Cholesterol sulfate (CS) is a widely distributed sulfolipid, and in human plasma is quantitatively more significant than any other known sterol sulfate. CS is a multifaceted molecule implicated in a number of biologic systems, e.g. skin and platelets. SULT2B1b is the only SULT2 isozyme expressed in human skin and primary cultures of human epidermal keratinocytes during Ca2+-induced differentiation. Furthermore, expression of SULT2B1b in skin is localized to the granular layer of the living epidermis consistent with this layer containing the highest CS concentration; it is also consistent with the involvement of CS in stratum corneum function and desquamation. Almost 90% of the CS formed during Ca2-induction of keratinocyte differentiation is found in the cellular membrane fraction with only 10% in the soluble fraction. CS promotes platelet aggregation and SULT2B1b is the only SULT2 isozyme expressed in these discoid anucleate particles. SULT2B1b mRNA is stable at 4C but is rapidly lost at 37C, a loss that is prevented by HDL but not LDL, specifically its apoA-I component. Interestingly, platelet membranes contain a specific and saturable CS-binding activity. The mouse ortholog of SULT2B1 demonstrates selective expression of SULT2B1a and SULT2B1b in the central nervous system and skin, respectively, in keeping with the importance of pregnenolone sulfate as a neurosteroid involved in learning and memory processes and the role of CS as a regulatory molecule in keratinocyte differentiation and barrier development. The overwhelming expression of SULT2A1 in liver would be in keeping with its role in general metabolism involving xenobiotics as well as endobiotics. Interestingly, mouse SULT2B1 and SULT2A1 genes are differentially expressed during embryonic development with the former expressed at all stages from E8.5-E19, whereas the latter is not expressed until E19 suggesting a more critical role for SULT2B1 during early development.