Sulfonation is the transfer of a sulfonate group (SO3-1) from the “universal” sulfonate donor molecule 3-phosphoadensine 5-phosphosulfate (PAPS) to appropriate acceptor molecules. These molecules include a remarkable array of compounds that range in MW from less than 1,000 to three orders of magnitude higher and that undergo striking changes in their physicochemical properties upon the addition of the highly charged sulfonate group. Sulfonation increases water solubility and can lead to conformational changes in both low and high molecular weight molecules; lipophilic molecules are converted to amphiboles, and, with pKas near 1.5, sulfonates remain fully ionized at any pH found in biological systems. Importantly, sulfonation cannot occur in the absence of PAPS, which establishes PAPS as a strategic biological molecule and makes its availability of vital importance. Our laboratory is currently focusing on two aspects of the sulfonation process: the identification, characterization, and regulation of specific sulfotransferases and the effectors of post-translational modification and the molecular mechanisms regulating the availability of the sulfonate donor molecule.

PAPS Synthase Characterization and Expression
Fuda, Shimizu, Lee, Strott
The production of PAPS from ATP and inorganic sulfate is regulated by the bifunctional enzyme, PAPS synthase. Two isozymes of human PAPS synthase are encoded by genes located on chromosomes 4 (PAPS synthase 1) and 10 (PAPS synthase 2) and are 76 percent identical. In addition, PAPS synthase 2 exists in two forms (2a and 2b). We have cloned and characterized both gene products and determined the location of the catalytic domains for these bifunctional proteins. While all three isoforms demonstrate Michaelis/Menten kinetics, they are functionally distinct, i.e., the PAPS synthase 2 subtypes have a greater catalytic efficiency and are 15 to 20 times more active than PAPS synthase 1. Multiplex PCR reveals that PAPS synthase1 is expressed in all human tissues and is the predominant isoform except in the liver, thus suggesting that the gene for PAPS synthase 1 is constitutively expressed. In contrast to PAPS synthase 1, the PAPS synthase 2 subtypes are differentially expressed, suggesting that the differential splicing creates tissue-specific subtypes. Human PAPS synthase 2 was discovered during a search for the genetic basis of a developmental abnormality that causes a form of spondyloepimetaphyseal dysplasia. This dwarfing disorder presents with a skeletal phenotype involving the spine and long bones and is caused by a nonsense mutation located in the ATP sulfurylase domain of PAPS synthase 2. Discovery of the genetic disorder notwithstanding, a conundrum remains. Since human cartilage coexpresses PAPS synthase 1 and, to a greater degree, PAPS synthase 2, it is puzzling how the cartilage-specific dwarfing disorder arises. We determined that, in the growth plate of developing bones in contrast to what is found in adult or mature cartilage, PAPS synthase 2 is clearly the predominantly expressed isoform, whereas PAPS synthase 1 is poorly expressed, if at all.

PAPS Synthase Transcriptional Regulation
Shimizu, Fuda, Lee, Strott
In our initial studies regarding the transcriptional regulation of the genes for the human PAPS synthase isozymes, we identified the sites for initiation of RNA synthesis and determined the proximal promoter regions in both genes. The 5'-flanking regions upstream of the capping sites contain neither a canonical TATAAA box nor a CCAAT motif. There are, however, multiple GC/GT boxes present in the proximal promoter regions, and we determined that both genes are indeed under the influence of the Sp1 family of transcription factors, particularly Sp1 and Sp2. In addition, the transcription factor AP2 (aandb) may be involved in the regulation of the gene encoding PAPS synthase 2. Our transcriptional studies are ongoing, and we are investigating the developmental expression of the human PAPS genes in mice and guinea pigs by using a quantitative RT-PCR approach.

Cholesterol Sulfotransferase Functional Characterization
Javitt, Fuda, Lee, Shimizu, Strott
A recently cloned gene for a new member of the subfamily of hydroxysteroid sulfotransferases (HST2) encodes a protein that is 76 percent identical to the originally cloned human hydroxysteroid sulfotransferase (HST1). As a result of either different start sites or alternative splicing, the two subtypes of HST2 differ only at their extreme amino-termini. We have characterized the HST2a/b subtypes and discovered that they preferentially sulfonate cholesterol and specific oxysterols. It is noteworthy that, until this time, a specific cholesterol sulfotransferase had not been identified and characterized. Interestingly, the HST2b isoform exhibits over 10 times higher catalytic activity than the HST2a isoform. However, the physiological significance of this finding is not appreciated. HST1, which is commonly referred to as dehydroepiandrosterone (DHEA) sulfotransferase because DHEA is the preferred substrate, has a broad substrate specificity and, in addition to DHEA, will sulfonate a wide variety of steroids and sterols involving hydroxyl groups at different carbon locations and with different spatial orientations. For instance, a 3a-hydroxyl group (androsterone and bile acids), a 3b-hydroxyl group (DHEA, pregnenolone and cholesterol), a 17b-hydroxyl group (testosterone and estradiol), and a phenolic hydroxyl group (estradiol and estrone) can be sulfonated by human HST1. On the other hand, although human HST2b sulfonates pregnenolone with relatively high efficiency, it poorly sulfonates DHEA and does not sulfonate androsterone, bile acids, testosterone, or estrogens. Furthermore, HST2b, in contrast to HST1, demonstrates specific structural requirements regarding spatial orientation of the 3-hydroxyl group and planarity of the perhydrocyclopentanophenanthrene ring structure. That is, a planar arrangement of the fused rings and a beta orientation of the 3-hydroxyl group are essential structural conditions. The 5a-reduced form of cholesterol (cholestanol), which is a planar molecule like cholesterol, is about 70 percent as effective a substrate as cholesterol, whereas catalytic efficiency falls to 20 to 25 percent with the 5b-reduced form of cholesterol (coprostanol), which is a nonplanar molecule containing a severe bend in the fused ring structure. Spatial orientation of the 3-hydroxyl group is also crucial, as shown by the fact that the 3a -hydroxyl stereoisomer of cholesterol (epicholesterol) is an extremely poor substrate.

Cholesterol Sulfotransferase Structural Characterization
Fuda, Lee, Javitt, Shimizu, Strott
Although human HST2a/b is considered to be a hydroxysteroid sulfotransferase, it is nevertheless structurally unique and distinct from human HST1 as well as from HSTs cloned from other species; the outstanding distinction is the extended amino- and carboxy-termini. Overall, HST1 and HST2a/b have about 37 percent sequence identity. However, if the unique amino- and carboxy-terminal ends of the latter isoforms are excluded, identities increase to about 48 percent. All previously cloned members of the cytosolic sulfotransferase superfamily, i.e., estrogen and phenol sulfotransferases as well as the hydroxysteroid sulfotransferases, range in size from 282 to 295 amino acids, whereas the HST2a and b isoforms consist of 350 and 365 amino acids, respectively. Interestingly, the sizes of the HST2 isoforms are more closely aligned with members of the larger Golgi membrane–associated class of sulfotransferases. While it is assumed and most likely that the HST2 isoforms are soluble and not membrane-associated, such characteristics have not been demonstrated conclusively. However, the fact that a GxxGxxK P-loop motif present near the carboxy-terminus of all cytosolic sulfotransferases (but not membrane-associated sulfotransferases) is also conserved in the HST2 isoforms supports the conclusion that the proteins are soluble enzymes. The unique extended amino- and carboxy-terminal ends of the HST2a/b isoforms notwithstanding, the HST1 and HST2a/b proteins exhibit significant structural similarity in their core regions. Most notably, a PSB loop (another type of P-loop motif found in phosphate binding sites of nucleotide binding proteins), 5'PB (5'-phosphate binding site), and 3'PB (3'-phosphate binding site) motifs, along with specific amino acid residues important in protein-cofactor interaction of cytosolic sulfotransferases, are highly conserved. The functional significance of the extended amino- and carboxy-terminal ends of the HST2a/b isoforms is not known. One speculation is that the proline-enriched carboxy-terminal region might play a role in protein-protein interactions. Interestingly, the relatively long carboxy-terminal extension can be removed from HST2b without producing a significant change in catalytic activity, whereas removal of the shorter amino-terminal extension results in an almost complete loss of enzymatic function. Furthermore, we have identified a four–amino-acid sequence near the amino-terminus that is required for full catalytic activity. The results suggest that the amino-terminus plays a major role in catalysis and may be in part responsible for the substrate selectivity of this unique hydroxysteroid sulfotransferase. In collaboration with the Laboratory of Reproductive and Developmental Toxicology, NIEHS, we are undertaking crystallization before x-ray diffraction to determine the three-dimensional structure of human HST2a/b.

Cholesterol Sulfotransferase Transcriptional Regulation
Lee, Shimizu, Fuda, Javitt, Strott
Sulfonation of cholesterol and hydroxylated cholesterol 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., it stimulates protein kinase C isoforms, especially h isoform. Epidermal cornification involves the cross-linking of precursor proteins, a process dependent on the activity of transglutaminase-1, which in turn depends on the accumulation of cholesterol sulfonate, an activator of the transglutaminase-1 gene. Regarding oxysterols, sulfonation of these compounds is involved in the regulation of an important class of orphan nuclear receptors. Studies on the expression of HST2a/b during differentiation of normal human keratinocytes and correlation with expression of the genes for transglutaminase-1 and involucrin are under way. In addition, investigations into the molecular mechanisms and factors regulating expression of HST2a/b are in progress. The transcription start site and proximal promoter region of the HST2a/b gene have been identified. Similar to the genes for PAPS synthase, the promoter for the HST2a/b gene is rich in GC content and contains neither a TATAAA nor a CAATT box. An ortholog of human cholesterol sulfotransferase has been cloned in mice and is currently under investigation with the aim of developing a gene knock-out model.