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THE BIOTRANSFORMATION OF ENDOBIOTICS BY SULFONATION
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Charles A. Strott, MD, Head, Section on Steroid Regulation Young C. Lee, PhD, Staff Scientist |
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We aim to gain insight into the molecular mechanisms and biologic implications of modifying substances by sulfonation, a fundamental process in the biotransformation of endobiotics as well as of drugs and xenobiotics. Sulfonation, the transfer of an SO3 group from the universal donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an acceptor molecule, is essential for normal growth and development and for maintenance of the internal milieu. Sulfonated macromolecules such as glycosaminoglycans and proteoglycans are involved in cell surface and connective tissue structures and bone formation. Sulfolipids are concentrated in the brain, peripheral nerves, and reproductive tissues. Sulfonation of tyrosine residues is a widespread posttranslational modication of many secretory and membrane proteins. Furthermore, glycoprotein hormones are modified by the sulfonation of specific saccharide moieties, creating unique structural motifs, with important functional consequences. Sulfonation is also of major importance in the biotransformation of low molecular weight compounds such as catecholamines, iodothyronines, neuroendocrine peptides, cholesterol, and the cholesterol metabolites oxysterols, bile acids, vitamin D, and steroid hormones. By modulating availability of the biologically active steroid/sterol form, sulfotransferases can influence biologic activity and thus play an essential role in specific physiologic systems and associated disorders. Human hydroxysteroid sulfotransferases SULT2A1, SULT2B1a, and SULT2B1b Fuda, Lee, Higashi, Yanai, Javitt, Strott Often referred to as hydroxysteroid sulfotransferases, the enzymes that sulfoconjugate neutral steroids constitute the SULT2 family, which consists of two subfamilies, SULT2A1 and SULT2B1. Whereas the SULT2A1 subfamily consists of a single form, the SULT2B1 subfamily consists of the two isoforms SULT2B1a and SULT2B1b, which derive from an alternative exon 1. We have cloned, overexpressed, and purified all human SULT2 subfamily members for biochemical, functional, and structural analyses. SULT2A1 is commonly referred to as dehydroepiandrosterone (DHEA) sulfotransferase because DHEA is considered the preferred substrate, although the isozyme has a broad substrate specificity. It would seem that such labeling is arbitrary as SULT2A1, depending on the species, can have an equal or greater affinity for other neutral steroids, e.g., pregnenolone. In contrast to SULT2A1, the SULT2B1 isoforms sulfonate DHEA less efficiently and have a much narrower substrate preference. Importantly, the SULT2B1 isoforms are structurally distinct from SULT2A1 as well as from all other known cognate cytosolic sulfotransferases. Furthermore, the SULT2B1 isoforms, which differ only at their amino-terminal ends, exhibit a more defined substrate selectivity, e.g., SULT2B1a avidly sulfonates pregnenolone but poorly sulfonates cholesterol, whereas SULT2B1b functions as a cholesterol sulfotransferase. It is notable that the substrate specificity demonstrated by the SULT2B1 isoforms is in part dependent on the uniqueness of their amino-terminal ends. The fact that the SULT2A1 and SULT2B1 isozymes are differentially expressed and display dissimilar substrate specificities strongly suggests that they play distinct biologic roles. The cloning of the human SULT2B1 gene, a significant advance in the area of steroid/sterol sulfotransferases, has led to the finding that the SULT2B1b subtype acts as a specific cholesterol sulfotransferase. It is noteworthy that cholesterol sulfate is a widely distributed sulfolipid and, in human plasma, is quantitatively more significant than any other known sterol sulfate. Although knowledge of the physiologic role(s) of cholesterol sulfate is still evolving, the steroid has been implicated in a number of biologic systems. To date, the clearest and most investigated physiologic role for cholesterol sulfate is in keratinocyte differentiation and development of the barrier. It has also been implicated in adrenal and gonadal steroid synthesis, in the regulation of cholesterol and fatty acid synthesis, in controlling sperm capacitation, in the regulation of serine proteases, and in the induction of platelet aggregation. The fact that cholesterol sulfate circulates in blood and its emerging role as a regulatory molecule suggest that it might be categorized as a hormone. Fuda H, Lee YC, Shimizu C, Javitt NB, Strott CA. Mutational analysis of human hydroxysteroid sulfotransferase SUTLT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cho-lesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase. J Biol Chem 2002;277:36161-36166. Strott CA. Hydroxysteroid sulfotransferases SULT2A1, SULT2B1a and SULT2B1b. In: Coughtrie MWH, Pacici GM, eds. Human Cytosolic Sulfotransferases. London: Taylor & Francis, 2003; in press. Crystal structure of human cholesterol sulfotransferase (SULT2B1b) Fuda, Lee, Strott; in collaboration with Pedersen We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3'-phospoadenosine-5'-phosphate (PAP) at 2.9 Å and 2.4 Å, respectively, as well as that of SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 Å. These structures reveal a different catalytic binding orientation for the substrate from the previously determined structure for prototypical hydroxysteroid sulfotransferase (SULT2A1) bound to DHEA. In addition, the amino-terminal helix, comprising residues D19 to K26, which determines the specificity difference between the SULT2B1 isoforms, becomes ordered as pregnenolone binding covers the substrate-binding pocket. We previously suggested that the specificity difference for cholesterol and pregnenolone between the SULT2B1a and SULT2B1b isoforms could be traced to the unique amino-terminal residues 19-DISEI-23. Results from alanine scanning mutagenesis of the 19-DISEI-23 region revealed that only the I20A and I23A mutants knocked out cholesterol sulfonating activity. We then showed that this activity could be partially restored by conservative substitutions such as leucine and methionine. I20 and I23 lie on the same side of the helix facing on the inside of the hydrophobic pocket, whereas residues S21 and E22 are exposed to solvent. Interestingly, the residues corresponding to 19-DISEI-23 of SULT2B1b are 4-PPPFH-8 in SULT2B1a. With three prolines in a row, it is unlikely that the residues are able to form an alpha-helix and therefore would be unable to cover the opening to the substrate binding pocket as seen in the SULT2B1b structure. Although we do not yet have a crystal structure of SULT2B1b with cholesterol bound, the position of pregnenolone is such that the O20 ketone is only 3.4 Å from atom CD1 of I20 and the C21 atom is 6.0 Å from atom CD1 of I23. Thus, the residues may be in positions to form positive van der Waal's interactions with the longer side chain of the C17 atom of cholesterol. Slight conformational changes of the protein, however, may be necessary to accommodate the longer side chain of cholesterol. Lee KA, Fuda H, Lee YC, Negishi M, Strott CA, Pedersen LC. Crystal structure of human cholesterol sulfotransferase (SULT2B1b) in the presence of pregnenolone and PAP: rationale for specificity differences between prototypical SULT2A1 and the SULT2B1 isoforms. J Biol Chem 2003;278:44593-44599. Expression of cholesterol sulfotransferase (SULT2B1b) in human skin and primary cultures of human keratinocytes Higashi, Fuda, Yanai, Lee, Strott; in collaboration with Kanzaki Although its physiologic significance remains unclear, cholesterol sulfate is present in relatively high concentrations in the epidermis of human skin, particularly in the granular layer. We examined expression of the gene encoding cholesterol sulfotransferase (SULT2B1b) and found, consistent with the involvement of cholesterol sulfate in keratinocyte development, progressive expression of SULT2B1b mRNA in primary cultures of normal human epidermal keratinocytes (NHEK) during Ca2+-induced differentiation. The rise in SULT2B1b mRNA expression was associated with parallel increases in the expression of protein and enzymatic activity. Of the three known steroid/sterol sulfotransferase isozymes, only SULT2B1b was expressed in normal human skin and cultured NHEK, as would be expected if the sole purpose of SULT2 expression in skin were the production of cholesterol sulfate, given that SULT2B1b functions as a selective cholesterol sulfotransferase. The expression of SULT2B1b in skin was localized to the granular layer of the living epidermis, in contrast to the barrier protein involucrin, an early marker of keratinocyte differentiation strongly expressed in the immediate suprabasal spinous layer. The intense expression of SULT2B1b in the granular layer of the epidermis is consistent with the layer containing the highest concentration of epidermal cholesterol sulfate. A variety of regulatory effects have been ascribed to cholesterol sulfate in the epidermis, such as regulation of transcription, protein kinase C, and lipid metabolism, suggesting multifunctional actions for this molecule. To further our understanding of the role of cholesterol sulfate, we conducted experiments to determine when and where during keratinocyte development the sulfotransferase producing cholesterol sulfate is expressed. Immunocytochemical analysis revealed that SULT2B1b was localized to the granular layer of the epidermis, suggesting that it is a late marker of keratinocyte differentiation. If SULT2B1b were an early marker of keratinocyte differentiation, it would be expected to have an expression pattern similar to that of involucrin, which was clearly expressed by cells in the immediate suprabasal region or spinous layer, i.e., the earliest cells in the pathway toward keratinocyte differentiation. However, SULT2B1b had an expression pattern that was the same as that of another barrier protein laggrin, an acknowledged late marker of differentiation. The epidermis is a perpetually renewing tissue whereby keratinocytes arise from stem cells in the basal layer and move through a series of cellular differentiation events until, as dead squames, they are finally sloughed off from the outer stratum corneum. Cholesterol sulfate might play a significant role in the proper functioning of the stratum corneum and the normal sloughing of dead cells, a process termed desquamation. For instance, cholesterol sulfate is able to retard desquamation by inhibiting serine proteases in the stratum corneum that are responsible for the degradation of cell adhesion structures known as desmosomes. In addition, cholesterol sulfate can influence cell cohesiveness by affecting the stability of corneocyte lipid bilayers. The inhibitory influence of cholesterol sulfate on normal desquamation, which presumably occurs maximally in the lower stratum corneum would subsequently be removed by the presence of a cholesterol sulfate sulfohydrolase in the outer stratum corneum, thus allowing normal desquamation to occur. This hypothesis is supported by the finding of markedly elevated cholesterol sulfate in the stratum corneum of patients with recessive X-linked ichthyosis, a scaling disorder that is a form of abnormal desquamation resulting from a deficiency in stratum corneum steroid sulfatase activity. Almost 90 percent of the cholesterol sulfate formed during Ca2+-induction of keratinocyte differentiation was found in the cellular membrane fraction, with only 10 percent in the soluble cell fraction. This finding, along with previous reports of cholesterol sulfate localization to membranes of erythrocytes and spermatozoa, suggests that cellular membrane localization of cholesterol sulfate might be a general phenomenon. It has been suggested that cholesterol sulfate is involved in membrane structure and stability, as indicated by its ability to protect red blood cells against osmotic lysis or fusion with animal viruses. We are currently trying to identify what NHEK cellular membranes are involved in the incorporation of cholesterol sulfate. Strott CA, Higashi Y. Cholesterol sulfate in human physiology: what's it all about? J Lipid Res 2003;44:1268-1278. Expression of cholesterol sulfotransferase (SULT2Bb) in human platelets Yanai, Javitt, Higashi, Fuda, Strott Among the many regulatory actions of cholesterol sulfate is its ability to influence blood clotting and brinolysis. In addition, cholesterol sulfate has been shown to support platelet aggregation. We have documented the presence of the enzyme (SULT2B1b) that sulfonates cholesterol in human platelets and examined the influence of plasma lipoproteins on the expression and activity of this enzyme. We detected SULT2B1b mRNA by RT-PCR and found it to be the only steroid/sterol sulfotransferase expressed in these discoid anucleate particles. Using real-time PCR for quantification, we found that, at 4°C, the level of SULT2B1b mRNA in platelets was maintained but markedly diminished over a period of four hours at 37°C. The loss of SULT2B1b mRNA, however, was significantly reduced in the presence of HDL but not LDL. The stabilizing influence of HDL was attributable specifically to its apolipoprotein A-I (apoA-I) component, whereas apolipoprotein A-II (apoA-II) and apolipoprotein E (apoE) were without effect. Importantly, there was a direct correlation between platelet SULT2B1b mRNA and protein levels in the presence or absence of lipoprotein that was reflected in enzymatic activity and cholesterol sulfate production. An exciting recent finding is that platelet membranes contain a specific and saturable cholesterol sulfate-binding activity. We are currently embarking on the purification, characterization, and cloning of this binding factor. Mouse ortholog of the human SULT2B1 gene Fuda, Yanei, Strott; in collaboration with Shimizu We have cloned a novel mouse hydroxysteroid sulfotransferase cDNA and determined the organization of its gene structure. The new mouse sulfotransferase (SULT2B1a) and its closely related isoform (SULT2B1b) are derived from a single gene as a result of an alternative exon 1 and differential splicing. Thus, the only structural distinction between the SULT2B1 isoforms lies in their amino-terminal ends. The mouse Sult2B1 gene structure is identical to that of the human SULT2B1 gene, indicating that the gene and its products are highly conserved. Furthermore, the mouse SULT2B1 isoforms, similar to human SULT2B1, have a predilection for cholesterol and pregnenolone over DHEA, which is ordinarily sulfonated by the SULT2A1 subfamily. Differential expression of mouse SULT2B1a, SULT2B1b, and SULT2A1, along with their distinct substrate preferences, has physiologic relevance. For instance, the exclusive expression of SULT2B1a in the central nervous system and its preference for pregnenolone as a substrate would be in keeping with the importance of pregnenolone sulfate as a neurosteroid involved in learning and memory processes. The sole expression of SULT2B1b in skin and the fact that it clearly has a predilection for cholesterol as a substrate are in keeping with the importance of cholesterol sulfate as a regulatory molecule in keratinocyte differentiation and development of the epidermal barrier. On the other hand, the overwhelming expression of SULT2A1 in the liver would be in keeping with its role in general metabolism involving both xenobiotics and endobiotics. Interestingly, the mouse Sult2B1 and Sult2A1 genes are differentially expressed during embryonic development, with the former expressed at all stages from E8.5-E19 and the latter not expressed until E19. The results suggest that expression of the mouse Sult2B1 gene is more significant during early development than the Sult2A1 gene, especially the expression of SULT2B1a during the early stages of brain development and the expression of SULT2B1b during growth of epithelia. Shimizu C, Fuda H, Yanai H, Strott CA. Conservation of the hydroxysteroid sulfotransferase SULT2B1 gene structure in the mouse: pre- and postnatal expression, kinetic analysis of isoforms, and comparison with prototypical SULT2A1. Endocrinology 2003;144:1186-1193. COLLABORATORS For further information, contact chastro@box-c.nih.gov |
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