HNK-1 sulfotransferase modulates α-dystroglycan glycosylation by 3-O-sulfation of glucuronic acid on matriglycan.

Mutations in multiple genes required for proper O-mannosylation of α-dystroglycan are causal for congenital/limb-girdle muscular dystrophies and abnormal brain development in mammals. Previously, we and others further elucidated the functional O-mannose glycan structure that is terminated by matriglycan, [(-GlcA-ß3-Xyl-α3-)n]. This repeating disaccharide serves as a receptor for proteins in the extracellular matrix. Here, we demonstrate in vitro that HNK-1 sulfotransferase (HNK-1ST/carbohydrate sulfotransferase) sulfates terminal glucuronyl residues of matriglycan at the 3-hydroxyl and prevents further matriglycan polymerization by the LARGE1 glycosyltransferase. While α-dystroglycan isolated from mouse heart and kidney is susceptible to exoglycosidase digestion of matriglycan, the functional, lower molecular weight α-dystroglycan detected in brain, where HNK-1ST expression is elevated, is resistant. Removal of the sulfate cap by a sulfatase facilitated dual-glycosidase digestion. Our data strongly support a tissue specific mechanism in which HNK-1ST regulates polymer length by competing with LARGE for the 3-position on the nonreducing GlcA of matriglycan.

A prototype of the mammalian sulfotransferase 1 (SULT1) family in Xenopus laevis: molecular and enzymatic properties of XlSULT1B.S.

The cytosolic sulfotransferase 1 (SULT1) proteins are a family of highly divergent proteins that show variable expansion in different species during vertebrate evolution. To clarify the evolutionary origin of the mammalian lineage of the SULT1 family, we compiled Xenopus laevis and X. tropicalis SULT1 (XSULT1) sequences from public databases. The XSULT1 family was found to comprise at least six subfamilies, which corresponded in part to five mammalian SULT1 subfamilies but only poorly to zebrafish SULT1 subfamilies. SULT1C was most highly expanded, and could be divided into at least five subfamilies. A cDNA for X. laevis SULT1B (XlSULT1B.S), a homolog of mammalian SULT1B1, was cloned and its recombinant protein was expressed in a bacterial system. XlSULT1B.S, unlike mammalian SULT1B1, was found to be a monomeric protein of ~34 kDa, and displayed sulfonating activity toward 2-naphthol and p-nitrophenol (pNP). However, we could not detect such sulfonating activity toward any endogenous compounds including thyroid hormones, steroid hormones and dopamine, despite the fact that X. laevis and Rana catesbeiana liver cytosols contained sulfonating activity toward most of these endogenous compounds. At optimum pH (6.4), the Michaelis-Menten constant (Km) for pNP was two orders of magnitude greater in XlSULT1B.S (1.04 mM) than in the cytosol preparations (8-15 µM). Our results indicate that Xenopus possesses a prototype of the mammalian SULT1 family, and that XlSULT1B.S showed overall similarities in primary sequence to, and significant differences in molecular and enzymatic properties from, mammalian SULT1B1.

Sterol Sulfates and Sulfotransferases in Marine Diatoms.

Sterol sulfates are widely occurring molecules in marine organisms. Their importance has been so far underestimated although many of these compounds are crucial mediators of physiological and ecological functions in other organisms. Biosynthesis of sterol sulfates is controlled by cytosolic sulfotransferases (SULTs), a varied family of enzymes that catalyze the transfer of a sulfo residue (-SO3H) from the universal donor 3'-phosphoadenosine-5'-phosphosulfate to the hydroxyl function at C-3 of the steroid skeleton. The absence of molecular tools has been the main impediment to the development of a biosynthetic study of this class of compounds in marine organisms. In fact, there is very limited information about these enzymes in marine environments. SULT activity has, however, been reported in several marine species, and, recently, the production of sterol sulfates has been linked to the control of growth in marine diatoms. In this chapter, we describe methods for the study of sterol sulfates in this lineage of marine microalgae. The main aim is to provide the tools useful to deal with the biosynthesis and regulation of these compounds and to circumvent the bottleneck of the lack of molecular information. The protocols have been designed for marine diatoms, but most of the procedures can be used for other marine organisms.

Biological functions of sulfoglycolipids and the EMARS method for identification of co-clustered molecules in the membrane microdomains.

Two major sulfoglycolipids, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol) exist in mammals. Sulfatide is abundant in the myelin sheath and seminolipid is unique to the spermatogenic cells. The carbohydrate moiety of sulfatide and seminolipid is identical and synthesized by common enzymes: ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). We have purified CST homogenously, cloned the CST gene and generated CST-knockout mice. CST-null mice completely lack sulfoglycolipids all over the body. Analysis of CST-null mice has revealed that sulfatide is an essential component for the axo-glial junction at the paranode region and regulates terminal differentiation of oligodendrocytes, and that seminolipid is responsible for the formation of a functional lactate transporter assembly to take up the critical energy source for spermatocytes. We have developed a new analytical method termed EMARS to identify co-clustered molecules in the membrane microdomains in order to elucidate the functional molecules that collaborate with sulfoglycolipids.

Expression, purification and characterization of human cytosolic sulfotransferase (SULT) 1C4.

Human cytosolic sulfotransferase 1C4 (hSULT1C4) is a dimeric Phase II drug-metabolizing enzyme primarily expressed in the developing fetus. SULTs facilitate the transfer of a hydrophilic sulfonate moiety from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto an acceptor substrate altering the substrate's biological activity and increasing the compound's water solubility. While several of the hSULTs' endogenous and xenobiotic substrates have been identified, the physiological function of hSULT1C4 remains unknown. The fetal expression of hSULT1C4 leads to the hypothesis that the function of this enzyme may be to regulate metabolic and hormonal signaling molecules, such as estrogenic compounds, that may be generated or consumed by the mother during fetal development. Human SULT1C4 has previously been shown to sulfonate estrogenic compounds, such as catechol estrogens; therefore, this study focused on the expression and purification of hSULT1C4 in order to further characterize this enzyme's sulfonation of estrogenic compounds. Molecular modeling of the enzyme's native properties helped to establish a novel purification protocol for hSULT1C4. The optimal activity assay conditions for hSULT1C4 were determined to be pH 7.4 at 37°C for up to 10 min. Kinetic analysis revealed the enzyme's reduced affinity for PAPS compared to PAP. Human SULT1C4 sulfonated all the estrogenic compounds tested, including dietary flavonoids and environmental estrogens; however, the enzyme has a higher affinity for sulfonation of flavonoids. These results suggest hSULT1C4 could be metabolizing and regulating hormone signaling pathways during human fetal development.

High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System.

Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein-protein interactions and enzymatic activity. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases.

An engineered heterodimeric model to investigate SULT1B1 dependence on intersubunit communication.

Cytosolic sulfotransferases (SULTs) biotransform small molecules to polar sulfate esters as a means to alter their activities within the body. Understanding the molecular mechanism by which the SULTs perform their function is important for optimizing future therapeutic applications. Recent evidence suggests each SULT isoform acts by a half-site reaction (HSR) mechanism, in which a single SULT dimer subunit is active at any given time. HSR requires communication through the highly conserved KxxxTVxxxE dimerization motif. In this investigation, we sought to test the intersubunit interactions of SULT1B1 as it relates to enzyme activity. We generated two populations of SULT1B1 isoforms that efficiently heterodimerize upon mixing by targeted point mutation of the KxxxTVxxxE motif to KxxxTVxxxK or ExxxTVxxxE. The heterodimer exhibited wildtype-like activity with regard to native size, thermal integrity, PAP affinity, and PAPS Km, therefore serving as a valid model for investigating SULT1B1 dimer subunit interactions. The approach granted control over each independent subunit, permitting mutation of the critical 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding residue Arg258 and/or the catalytic base His109 in a single subunit of the dimer. Substitution of the dysfunctional subunits for fully active subunits yielded dimeric SULT1B1 with 50% the activity of the fully competent dimer, suggesting SULT1B1 intersubunit communication does not significantly contribute to the isoform's activity. These results are a testament to the unique properties of individual SULT isoforms. The dimerization system described in this manuscript can be used to study subunit interactions in other SULT isoforms as well as proteins in other families.

Controlling Sulfuryl-Transfer Biology.

In humans, the cytosolic sulfotransferases (SULTs) catalyze regiospecific transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate to thousands of metabolites, including numerous signaling small molecules, and thus regulates their activities and half-lives. Imbalances in the in vivo set points of these reactions leads to disease. Here, with the goal of controlling sulfonation in vivo, molecular ligand-recognition principles in the SULT and nuclear receptor families are integrated in creating a strategy that can prevent sulfonation of a compound without significantly altering its receptor affinity, or inhibiting SULTS. The strategy is validated by using it to control the sulfonation and estrogen receptor (ER) activating activity of raloxifene (a US Food and Drug Administration-approved selective estrogen receptor modulator) and its derivatives. Preventing sulfonation is shown to enhance ER-activation efficacy 10(4)-fold in studies using Ishikawa cells. The strategy offers the opportunity to control sulfuryl transfer on a compound-by-compound basis, to enhance the efficacy of sulfonated drugs, and to explore the biology of sulfuryl transfer with unprecedented precision.

Tyrosine sulfation as a protein post-translational modification.

Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS) is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST) through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

Biosynthesis and biological function of sulfoglycolipids.

Sulfation confers negative charge on glycolipids and the attached sulfate group presents a part of determinants for the molecular interactions. Mammalian sulfoglycolipids are comprised of two major members, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol). Sulfatide is abundant in the myelin sheath and seminolipid is unique to the spermatogenic cells. The carbohydrate moiety of sulfatide and seminolipid is biosynthesized via sequential reactions catalyzed by common enzymes: ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). To elucidate the biological function of sulfoglycolipids, we have purified CST, cloned the CST gene, and generated CST-knockout mice. CST-null mice completely lack sulfoglycolipids all over the body. CST-null mice manifest some neurological disorders due to myelin dysfunction, an aberrant enhancement of oligodendrocyte terminal differentiation, and an arrest of spermatogenesis. CST-deficiency ameliorates L-selectin-dependent monocyte infiltration in the renal interstitial inflammation, indicating that sulfatide is an endogenous ligand of L-selectin. Studies on the molecular mechanisms underlying the biological events for which sulfoglycolipids are essential are ongoing

Identification and characterization of zebrafish SULT1 ST9, SULT3 ST4, and SULT3 ST5.

By searching the GenBank database, we identified sequences encoding three new zebrafish cytosolic sulfotransferases (SULTs). These three new zebrafish SULTs, designated SULT1 ST9, SULT3 ST4, and SULT3 ST5, were cloned, expressed, purified, and characterized. SULT1 ST9 appeared to be mostly involved in the metabolism and detoxification of xenobiotics such as ß-naphthol, ß-naphthylamine, caffeic acid and gallic acid. SULT3 ST4 showed strong activity toward endogenous compounds such as dehydroepiandrosterone (DHEA), pregnenolone, and 17ß-estradiol. SULT3 ST5 showed weaker, but significant, activities toward endogenous compounds such as DHEA and corticosterone, as well as xenobiotics including mestranol, ß-naphthylamine, ß-naphthol, and butylated hydroxyl anisole (BHA). pH-dependency and kinetic constants of these three enzymes were determined with DHEA, ß-naphthol, and 17ß-estradiol as substrates. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine the expression of these three new zebrafish SULTs at different developmental stages during embryogenesis, through larval development, and on to maturity.

Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3'-phosphoadenosine-5'-phosphosulfate.

Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.

Molecular cloning, expression, and functional analysis of a predicted sulfotransferase STF9 from Mycobacterium avium.

Sulfotransferases catalyze the transfer of sulfate group from para-nitrophenyl sulfate (pNPS) or 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto acceptor molecules in the biosynthesis of sulfate esters. Human pathogenic mycobacteria are known to produce numerous sulfated molecules on their cell surface which have been implicated as important mediators in host-pathogen interactions. The open reading frame stf9, a predicted homologue of sulfotransferase in the Mycobacterium avium genomic data, was cloned and over expressed in Escherichia coli. The recombinant STF9 conserved the characteristic PAPS binding motif of sulfotransferase and was purified as a 44 kDa soluble protein which exhibited transfer of sulfate group from pNPS (K (m) 1.34 mM, V (max) 7.56 nmol/min/mg) onto 3'-phosphoadenosine-5'-phosphate (K (m) 0.24 mM, V (max) 10.36 nmol/min/mg). The recombinant STF9 protein was also capable of transferring sulfate group from PAPS onto certain acceptor substrates in E. coli, and showed binding affinity to the PAP-agarose resin, supporting the sulfotransferase activity of the recombinant STF9 protein. This is the first report of molecular evidence for sulfotransferase activity of a protein from M. avium. Mutation of Arg96 to Ala and Glu170 to Ala abolishes sulfotransferase activity, indicating the importance of Arg96 and Glu170 in STF9 activity catalysis.

Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates.

Chondroitin sulfate (CS) containing GlcA-GalNAc(4,6-SO(4)) (E unit) and CS containing GlcA(2SO(4))-GalNAc(6SO(4)) (D unit) have been implicated in various physiological functions. However, it has been poorly understood how the structure and contents of disulfated disaccharide units in CS contribute to these functions. We prepared CS libraries containing E unit or D unit in various proportions by in vitro enzymatic reactions using recombinant GalNAc 4-sulfate 6-O-sulfotransferase and uronosyl 2-O-sulfotransferase, and examined their inhibitory activity toward thrombin. The in vitro sulfated CSs containing disulfated disaccharide units showed concentration-dependent direct inhibition of thrombin when the proportion of E unit or D unit in the CSs was above 15-17%. The CSs containing both E unit and D unit exhibited higher inhibitory activity toward thrombin than the CSs containing either E unit or D unit alone, if the proportion of the total disulfated disaccharide units of these CSs was comparable. The thrombin-catalyzed degradation of fibrinogen, a physiological substrate for thrombin, was also inhibited by the CS containing both E unit and D unit. These observations indicate that the enzymatically prepared CS libraries containing various amounts of disulfated disaccharide units appear to be useful for elucidating the physiological function of disulfated disaccharide units in CS.

Crystal structure of human senescence marker protein 30: insights linking structural, enzymatic, and physiological functions .

Human senescence marker protein 30 (SMP30), which functions enzymatically as a lactonase, hydrolyzes various carbohydrate lactones. The penultimate step in vitamin-C biosynthesis is catalyzed by this enzyme in nonprimate mammals. It has also been implicated as an organophosphate hydrolase, with the ability to hydrolyze diisopropyl phosphofluoridate and other nerve agents. SMP30 was originally identified as an aging marker protein, whose expression decreased androgen independently in aging cells. SMP30 is also referred to as regucalcin and has been suggested to have functions in calcium homeostasis. The crystal structure of the human enzyme has been solved from X-ray diffraction data collected to a resolution of 1.4 A. The protein has a 6-bladed beta-propeller fold, and it contains a single metal ion. Crystal structures have been solved with the metal site bound with either a Ca(2+) or a Zn(2+) atom. The catalytic role of the metal ion has been confirmed by mutagenesis of the metal coordinating residues. Kinetic studies using the substrate gluconolactone showed a k(cat) preference of divalent cations in the order Zn(2+) > Mn(2+) > Ca(2+) > Mg(2+). Notably, the Ca(2+) had a significantly higher value of K(d) compared to those of the other metal ions tested (566, 82, 7, and 0.6 mum for Ca(2+), Mg(2+), Zn(2+), and Mn(2+), respectively), suggesting that the Ca(2+)-bound form may be physiologically relevant for stressed cells with an elevated free calcium level.

Identification of tyrosylprotein sulfotransferase in Arabidopsis.

Tyrosine sulfation is a posttranslational modification common in peptides and proteins synthesized by the secretory pathway in most eukaryotes. In plants, this modification is critical for the biological activities of a subset of peptide hormones such as PSK and PSY1. In animals, tyrosine sulfation is catalyzed by Golgi-localized type II transmembrane proteins called tyrosylprotein sulfotransferases (TPSTs). However, no orthologs of animal TPST genes have been found in plants, suggesting that plants have evolved plant-specific TPSTs structurally distinct from their animal counterparts. To investigate the mechanisms of tyrosine sulfation in plants, we purified TPST activity from microsomal fractions of Arabidopsis MM2d cells, and identified a 62-kDa protein that specifically interacts with the sulfation motif of PSY1 precursor peptide. This protein is a 500-aa type I transmembrane protein that shows no sequence similarity to animal TPSTs. A recombinant version of this protein expressed in yeast catalyzed tyrosine sulfation of both PSY1 and PSK precursor polypeptide in vitro, indicating that the newly identified protein is indeed an Arabidopsis (At)TPST. AtTPST is expressed throughout the plant body, and the highest levels of expression are in the root apical meristem. A loss-of-function mutant of AtTPST displayed a marked dwarf phenotype accompanied by stunted roots, pale green leaves, reduction in higher order veins, early senescence, and a reduced number of flowers and siliques. Our results indicate that plants and animals independently acquired tyrosine sulfation enzymes through convergent evolution.

Characterization and ontogenic study of novel steroid-sulfating SULT3 sulfotransferases from zebrafish.

In vertebrates, sulfation as catalyzed by members of the cytosolic sulfotransferase (SULT) family has been suggested to be involved in the homeostasis of steroids. To establish the zebrafish as a model for investigating how sulfation functions to regulate steroid metabolism during the developmental process, we have embarked on the identification of steroid-sulfating SULTs in zebrafish. By searching the GenBank database, we identified two putative cytosolic SULT sequences from zebrafish, designated SULT3 ST1 and ST2. The recombinant proteins of these two zebrafish SULT3 STs were expressed in and purified from BL21 (DE3) cells transformed with the pGEX-2TK expression vector harboring SULT3 ST1 or ST2 cDNA. Upon enzymatic characterization, purified SULT3 ST1 displayed the strongest sulfating activity toward 17beta-estradiol among the endogenous substrates tested, while SULT3 ST2 exhibited substrate specificity toward hydroxysteroids, particularly dehydroepiandrosterone (DHEA). The pH-dependence and kinetic constants of these two enzymes with 17beta-estradiol and DHEA were determined. A developmental expression study revealed distinct patterns of the expression of SULT3 ST1 and ST2 during embryonic development and throughout the larval stage onto maturity. Collectively, these results imply that these two steroid-sulfating SULT3 STs may play differential roles in the metabolism and regulation of steroids during zebrafish development and in adulthood.

Identification and characterization of KpsS, a novel polysaccharide sulphotransferase in Mesorhizobium loti.

Plants enter into symbiotic relationships with bacteria that allow survival in nutrient-limiting environments. The bacterium Mesorhizobium loti enters into a symbiosis with the legume host, Lotus japonicus, which results in the formation of novel plant structures called root nodules. The bacteria colonize the nodules, and are internalized into the cytoplasm of the plant cells, where they reduce molecular dinitrogen for the plant. Symbiosis between M. loti and L. japonicus requires bacterial synthesis of secreted and cell-surface polysaccharides. We previously reported the identification of an unusual sulphate-modified form of capsular polysaccharide (KPS) in M. loti. To better understand the physiological function of sulphated KPS, we isolated the sulphotransferase responsible for KPS sulphation from M. loti extracts, determined its amino acid sequence and identified the corresponding M. loti open reading frame, mll7563 (which we have named kpsS). We demonstrated that partially purified KpsS functions as a fucosyl sulphotransferase in vitro. Furthermore, mutants deficient for this gene exhibit a lack of KPS sulphation and a decreased rate of nodule formation on L. japonicus. Interestingly, the kpsS gene product shares no significant amino acid similarity with previously identified sulphotransferases, but exhibited sequence identity to open reading frames of unknown function in diverse bacteria that interact with eukaryotes.

Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1).

Substrate inhibition is a characteristic feature of many cytosolic sulfotransferases. The differences between the complex structures of SULT2A1/DHEA and SULT2A1/PAP or SULT2A1/ADT (Protein Data Bank codes are 1J99, 1EFH, and 1OV4, respectively) have enabled us to elucidate the specific amino acids responsible for substrate inhibition. Based on the structural analyses, substitution of the smaller residue alanine for Tyr-238 (Y238A) significantly increases the K(i) value for dehydroepiandrosterone (DHEA) and totally eliminates substrate inhibition for androsterone (ADT). In addition, Met-137 was proposed to regulate the binding orientations of DHEA and ADT in SULT2A1. Complete elimination or regeneration of substrate inhibition for SULT2A1 with DHEA or ADT as substrate, respectively, was demonstrated with the mutations of Met-137 on Y238A mutant. Analysis of the Met-137 mutants and Met-137/Tyr-238 double mutants uncovered the relationship between substrate binding orientations and inhibition in SULT2A1. Our data indicate that, in the substrate inhibition mode, Tyr-238 regulates the release of bound substrate, and Met-137 controls substrate binding orientation of DHEA and ADT in SULT2A1. The proposed substrate inhibition mechanism is further confirmed by the crystal structures of SULT2A1 mutants at Met-137. We propose that both substrate binding orientations exhibited substrate inhibition. In addition, a corresponding residue in other cytosolic sulfotransferases was shown to have a function similar to that of Tyr-238 in SULT2A1.

Tyrosine sulfation of statherin.

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.