Sulfation of Quercitrin, Epicatechin and Rutin by Human Cytosolic Sulfotransferases (SULTs): Differential Effects of SULT Genetic Polymorphisms.

Radix Bupleuri is one of the most widely used herbal medicines in China for the treatment of fever, pain, and/or chronic inflammation. Quercitrin, epicatechin, and rutin, the flavonoids present in Radix Bupleuri, have been reported to display anti-inflammatory, antitumor, and antioxidant biological activities among others. Sulfation has been reported to play an important role in the metabolism of flavonoids. In this study, we aimed to systematically identify the human cytosolic sulfotransferase enzymes that are capable of catalyzing the sulfation of quercitrin, epicatechin, and rutin. Of the thirteen known human cytosolic sulfotransferases, three (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1C2, and cytosolic sulfotransferase 1C4) displayed sulfating activity toward quercitrin, three (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1A3, and cytosolic sulfotransferase 1C4) displayed sulfating activity toward epicatechin, and six (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1A2, cytosolic sulfotransferase 1A3, cytosolic sulfotransferase 1B1, cytosolic sulfotransferase 1C4, and cytosolic sulfotransferase 1E1) displayed sulfating activity toward rutin. The kinetic parameters of the cytosolic sulfotransferases that showed the strongest sulfating activities were determined. To investigate the effects of genetic polymorphisms on the sulfation of quercitrin, epicatechin, and rutin, individual panels of cytosolic sulfotransferase allozymes previously prepared were analyzed and shown to display differential sulfating activities toward each of the three flavonoids. Taken together, these results provided a biochemical basis underlying the metabolism of quercitrin, epicatechin, and rutin through sulfation in humans.

In Silico Prediction of Human Sulfotransferase 1E1 Activity Guided by Pharmacophores from Molecular Dynamics Simulations.

Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model, and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28% of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.

Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.

Post-translational protein modification by tyrosine sulfation has an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalysed by the Golgi enzyme called the tyrosylprotein sulfotransferase. To date, no crystal structure is available for tyrosylprotein sulfotransferase. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human tyrosylprotein sulfotransferase isoform 2 complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3'-phosphoadenosine-5'-phosphate at 1.9 Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. Tyrosylprotein sulfotransferase isoform 2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel ß-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the tyrosylprotein sulfotransferase isoform 2 structure resembles that observed for the receptor type tyrosine kinases.

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.

Evaluation of virtual screening as a tool for chemical genetic applications.

A collection of over 50,000 functionally annotated drugs, clinical candidates, and endogenous ligands was docked in silico against nine binding sites from seven protein targets, representing diverse function and structure, namely the sulfotransferases SULT1E1 and SULT1A3, the histone methyltransferase EHMT1, the histone acetyltransferase MYST3, and the nuclear hormone receptors ERalpha, PPARgamma, and TRbeta. For 5 of the 9 virtual screens, compounds that docked best to the receptors clearly recapitulated known biological functions of the genes or identified novel biology subsequently validated in a separate experimental study. In two cases, the hit list indicated some relevant but isolated biological functions which would probably have been ignored a priori, and selected compounds were completely unrelated to gene function for the last two virtual screens. This study demonstrates that virtual screening of pharmacologically annotated compound libraries can be used to derive target biology.

In-vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases.

OBJECTIVES: The aim of this study was to investigate the concentration-dependent sulfation of piceatannol, a dietary polyphenol present in grapes and wine and known for its promising anticancer and anti-inflammatory activity. METHODS: Sulfation of piceatannol was investigated in human liver cytosol as well as using a panel of recombinant sulfotransferase isoforms. Furthermore, the chemical structures of novel sulfates were identified by liquid chromatography/mass spectrometry (LC/MS). KEY FINDINGS: In the presence of 3'-phosphoadenosine-5'-phosphosulfate, three metabolites could be detected whose structures were identified by LC/MS/MS as piceatannol disulfate (M1) and two monosulfates (M2, M3). The kinetics of M1 formation exhibited a pattern of substrate inhibition with a Ki of 21.8 +/- 11.3 microm and a Vmax/Km of 7.63 +/- 1.80 microl/mg protein per min. Formation of M2 and M3 showed sigmoidal kinetics with apparent Km and Vmax values of 27.1 +/- 2.90 microm and 118.4 +/- 4.38 pmol/mg protein per min, respectively, for M2; and 35.7 +/- 2.70 microm and 81.8 +/- 2.77 pmol/mg protein per min, respectively, for M3. Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 was formed equally by SULT1A1*1 and SULT1B1 and to a lesser extent by SULT1A1*2. M2 was preferentially catalysed by SULT1A1*2, 1A3 and 1E1. The formation of M3, however, was mainly catalysed by SULT1A2*1 and SULT1A3. CONCLUSIONS: Our results elucidate the importance of piceatannol sulfation in human liver, which must be taken into account in humans after dietary intake of piceatannol.

Human chorionic gonadotropin-dependent up-regulation of genes responsible for estrogen sulfoconjugation and export in granulosa cells of luteinizing preovulatory follicles.

Estrogen sulfotransferase (EST) is responsible for the sulfoconjugation of estrogens, thereby changing their physical properties and preventing their action via the estrogen receptors. These sulfoconjugated steroids no longer diffuse freely across the lipid bilayer; instead, they are exported by members of the ATP-binding cassette family, such as ABCC1. The objective of this study was to investigate the regulation of EST and ABCC1 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The transcripts for EST and ABCC1 were cloned by RT-PCR, and the regulation of their mRNAs was studied in preovulatory follicles obtained during estrus at 0, 12, 24, 30, 33, 36, and 39 h after hCG. Results obtained from RT-PCR/Southern blot analyses showed significant changes in steady-state levels of both EST and ABCC1 mRNA after hCG treatment (P < 0.05). In granulosa cells, a significant increase in EST transcript was observed 30-39 h after hCG. Similarly, ABCC1 transcript levels were induced in granulosa cells 12-39 h after hCG. In contrast, no significant changes in either EST or ABCC1 were detected in theca interna samples after hCG. The increase in EST and ABCC1 transcripts observed in granulosa cells was reflected in preparations of intact follicle walls, suggesting that the granulosa cell layer contributes the majority of EST and ABCC1 expression in preovulatory follicles. The present study demonstrates that follicular luteinization is accompanied not only by a decrease in 17 beta-estradiol biosynthesis but also by an increase in expression of genes responsible for estrogen inactivation and elimination from granulosa cells, such as EST and ABCC1, respectively.

Regulation of the chondroitin/dermatan fine structure by transforming growth factor-beta1 through effects on polymer-modifying enzymes.

The chondroitin/dermatan sulfate proteoglycans (CS/DSPGs), biglycan, decorin, and versican play several important roles in extracellular matrix influencing matrix organization, cell proliferation, and recruitment. Moreover, they bind and regulate growth factors in the extracellular matrix. We have previously shown that cultured human lung fibroblasts treated with transforming growth factor-beta (TGF-beta) alone or in combination with epidermal growth factor and platelet-derived growth factor, increase the production of these PGs. In this report, we describe that the structure of their galactosaminoglycan side chains is altered, albeit there is no alteration of polysaccharide length. The findings showed that iduronic acid content is reduced by 50% in decorin and biglycan, whereas 4-O-sulfation is increased 2-fold in versican. To unravel the mechanism behind these changes, the activities of chondroitin C-5 epimerase and of O-sulfotransferases in cellular fractions prepared from fibroblasts were quantitated, and transcript levels of the relevant sulfotransferases were measured by real time polymerase chain reaction (RT-PCR). The C-5 epimerase activity was reduced by 25% in TGF-beta1 treated cells and 50% in fibroblasts treated with the growth factor combination. No change in activity in dermatan 4-O sulfotransferase was observed, and only a minor decrease in dermatan 4-O-sulfotransferase-1 (D4ST-1) mRNA was observed. On the other hand, chondroitin 4-O sulfotransferase activity increased 2-fold upon TGF-beta1 treatment and 3-fold upon treatment with the growth factor combination. This is in agreement with a 2-fold up-regulation of chondroitin-4-O-sulfotransferase 1 (C4ST-1) mRNA, and no changes in chondroitin-4-O-sulfotransferase 2 (C4ST-2) mRNA. Thus, cellular activity and transcript level correlated well with the changes in the structure of the dermatan/chondroitin sulfate chains.

Inhibitory effects of various beverages on ritodrine sulfation by recombinant human sulfotransferase isoforms SULT1A1 and SULT1A3.

PURPOSE: Ritodrine is known to undergo extensive presystemic sulfation in the intestinal mucosa, and its bioavailability is as low as 30%. Accordingly, inhibition of intestinal sulfation may lead to an increase in the bioavailability of ritodrine. In this study, we aimed to investigate the activities of ritodrine sulfation by SULT1A1, which is expressed predominantly in the liver, and SULT1A3, which is expressed predominantly in the intestine, as well as the inhibitory effects of beverages on their activities. METHODS: We investigated ritodrine sulfation by using recombinant human sulfotransferase (SULT) 1A1 and SULT1A3 in an in vitro study. Next, we investigated the inhibitory effects of grapefruit juice, orange juice, green tea, and black tea on ritodrine sulfation. RESULTS: Sulfation of ritodrine by SULT1A3 was much higher than that by SULT1A1, suggesting that the bioavailability of ritodrine may be limited by intestinal SULT1A3. The ritodrine sulfation activities of SULT1A1 and SULT1A3 were significantly inhibited by all beverages examined at a concentration of 10%. Green tea and black tea exhibited potent inhibition; even at a concentration of 5%, they both inhibited SULT1A1 by 100% and SULT1A3 by >or=95%. CONCLUSION: Our results suggest that concomitant ingestion of beverages such as green tea and black tea may increase the bioavailability of orally administered ritodrine, and perhaps other beta2-agonists, and lead to an increase in the clinical effects or adverse reactions.

Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure.

The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.

Zebrafish tyrosylprotein sulfotransferase: molecular cloning, expression, and functional characterization.

By employing the reverse transcriptase-polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.

Phase I and phase II metabolic activities are retained in liver slices from mouse, rat, dog, monkey and human after cryopreservation.

Precision-cut liver slices are described as a valuable tool for in vitro metabolism studies of potential drug candidates. Recently, some papers reported successful cryopreservation conditions for liver slices, facilitating a broader and more efficient use of the tissue (particularly of human origin). The aim of this study is to evaluate the effect of cryopreservation on both phase I and phase II metabolism in liver slices prepared from mouse, rat, dog, monkey and human, using rapid freezing in the presence of 18% DMSO. Glucuronidation and sulfation activities (phase II) in both freshly prepared and cryopreserved liver slices were determined by rapid LC-MS/MS analyses using 7-hydroxycoumarin as a marker substrate. Testosterone was used as a marker substrate for cytochrome P450 mediated drug metabolism (phase I). Although the metabolic patterns and rates varied among the different species, the phase I and phase II metabolic capacities of the liver slices were well maintained after cryopreservation. Despite the good biotransformation capacity of cryopreserved slices a decrease in viability, expressed as ATP content and LDH leakage, was observed. MTT reduction was well maintained after cryopreservation. The possibility to cryopreserve liver slices will allow a more efficient utilisation of tissue, in particular from human, but also from dog and monkey. Finally, cryopreserved liver slices from mouse, rat, dog, monkey and human with good phase I and II metabolism activities are a useful in vitro tool to compare metabolite profiles of new chemical entities between species.

Sulfonation of environmental estrogens by zebrafish cytosolic sulfotransferases.

Environmental estrogen-like chemicals are increasingly recognized as a potential hazardous factor for wildlife as well as humans. We have recently embarked on developing a zebrafish model for investigating the role of sulfonation in the metabolism and adverse functioning of environmental estrogens. Here, we report on a systematic investigation of the sulfonation of representative environmental estrogens (bisphenol A, 4-n-octylphenol, 4-n-nolylphenol, diethylstilbestrol, and 17 alpha-ethynylestradiol) by zebrafish cytosolic sulfotransferases (STs). Of the seven enzymes tested, four zebrafish STs (designated ZF ST #2, ZF ST #3, ZF ST #4, and ZF DHEA ST) exhibited differential sulfonating activities toward the five environmental estrogens tested, with ZF ST #3 being more highly active than the other three. It was further demonstrated that bisphenol A, 4-n-octylphenol, and 4-n-nonylphenol exerted concentration-dependent inhibition of the sulfonation of 17 beta-estradiol, implying a potential role of these environmental estrogens in interfering with the sulfonation, and possibly homeostasis, of endogenous estrogens. Kinetic studies revealed that the mechanism underlying the inhibition by bisphenol A or 4-n-nonylphenol to be of the competitive type.

Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermatan sulfate biosynthesis.

4-O-Sulfation of GalNAc is a high frequency modification of chondroitin sulfate and dermatan sulfate (DS), and three major GalNAc 4-O-sulfotransferases including dermatan 4-O-sulfotransferase-1 (D4ST-1) and chondroitin 4-O-sulfotransferases-1 and -2 (C4ST-1 and -2) have been identified. 4-O-Sulfation of GalNAc during DS biosynthesis had long been postulated to be a prerequisite for iduronic acid (IdoUA) formation by C5-epimerization of GlcUA. This hypothesis has recently been argued based on enzymological studies using microsomes that C5-epimerization precedes 4-O-sulfation, which was further supported by the specificity of the cloned D4ST-1 with predominant preference for IdoUA-GalNAc flanked by GlcUA-GalNAc over IdoUA-GalNAc flanked by IdoUA-GalNAc in exhaustively desulfated dermatan. Whereas the counterproposal explains the initial reactions, apparently it cannot rationalize the synthetic mechanism of IdoUA-GalNAc(4-O-sulfate)-rich clusters typical of mature DS chains. In this study, we examined detailed specificities of the three recombinant human 4-O-sulfotransferases using partially desulfated DS as an acceptor. Enzymatic analysis of the transferase reaction products showed that D4ST-1 far more efficiently transferred sulfate to GalNAc residues in -IdoUA-Gal-NAc-IdoUA-than in -GlcUA-GalNAc-GlcUA-sequences. In contrast, C4ST-1 showed the opposite preference, and C4ST-2 used GalNAc residues in both sequences to comparable degrees, being consistent with its phylogenetic relations to D4ST-1 and C4ST-1. Structural analysis of the oligosaccharides, which were isolated after chondroitinase AC-I digestion of the 35S-labeled transferase reaction products, revealed for the first time that D4ST-1, as compared with C4ST-1 and C4ST-2, most efficiently utilized GalNAc residues located not only in the sequence -IdoUA-GalNAc-IdoUA- but also in -GlcUA-Gal-NAc-IdoUA- and -IdoUA-GalNAc-GlcUA-. The isolated oligosaccharide structures also suggest that 4-O-sulfation promotes subsequent 4-O-sulfation of GalNAc in the neighboring disaccharide unit.

Arginine residues in the active site of human phenol sulfotransferase (SULT1A1).

Cytosolic sulfotransferases (STs) catalyze the sulfation of hydroxyl containing compounds. Human phenol sulfotransferase (SULT1A1) is the major human ST that catalyzes the sulfation of simple phenols. Because of its broad substrate specificity and lack of endogenous substrates, the biological function of SULT1A1 is believed to be an important detoxification enzyme. In this report, amino acid modification, computer structure modeling, and site-directed mutagenesis were used for studies of Arg residues in the active site of SULT1A1. The Arg-specific modification reagent, 2,3-butanedione, inactivated SULT1A1 in an efficient, time- and concentration-dependent manner, suggesting Arg residues play an important role in the catalytic activity of SULT1A1. According to the computer model, Arg78, Arg130, and Arg257 may be important for SULT1A1 catalytic activity. Site-directed mutagenesis results demonstrated that the positive charge on Arg78 is not critical for SULT1A1 because R78A is still active. In contrast, a negative charge at this position, R78E, completely inactivated SULT1A1. Arg78 is in close proximity to the site of sulfuryl group transfer. Arg257 is located very close to the 3'-phosphate in adenosine 3'-phosphate 5'-phosphosulfate (PAPS). Site-directed mutagenesis demonstrated that Arg257 is critical for SULT1A1: both R257A and R257E are inactive. Although Arg130 is also located very close to the 3'-phosphate of PAPS, R130A and R130E are still active, suggesting that Arg130 is not a critical residue for the catalytic activity of SULT1A1. Computer modeling suggests that the ionic interaction between the positive charge on Arg257, and the negative charge on 3'-phosphate is the primary force stabilizing the specific binding of PAPS.

A unique nonreducing terminal modification of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase.

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO4)). We previously identified human GalNAc4S-6ST cDNA and showed that the recombinant GalNAc4S-6ST could transfer sulfate efficiently to the nonreducing terminal GalNAc(4SO4) residues. We here present evidence that GalNAc4S-6ST should be involved in a unique nonreducing terminal modification of chondroitin sulfate A (CSA). From the nonreducing terminal of CS-A, a GlcA-containing oligosaccharide (Oligo I) that could serve as an acceptor for GalNAc4S-6ST was obtained after chondroitinase ACII digestion. Oligo I was found to be GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) because GalNAc(4SO4) and deltaHexA(2SO4)-GalNAc(6SO4) were formed after chondroitinase ABC digestion. When Oligo I was used as the acceptor for GalNAc4S-6ST, sulfate was transferred to position 6 of GalNAc(4SO4) located at the nonreducing end of Oligo I. Oligo I was much better acceptor for GalNAc4S-6ST than GalNAc(4SO4)-GlcAGalNAc(6SO4). An oligosaccharide (Oligo II) whose structure is identical to that of the sulfated Oligo I was obtained from CS-A after chondroitinase ACII digestion, indicating that the terminal modification occurs under the physiological conditions. When CS-A was incubated with [35S]PAPS and GalNAc4S-6ST and the 35S-labeled product was digested with chondroitinase ACII, a 35S-labeled trisaccharide (Oligo III) containing [35S]GalNAc(4,6-SO4) residue at the nonreducing end was obtained. Oligo III behaved identically with the sulfated Oligos I and II. These results suggest that GalNAc4S-6ST may be involved in the terminal modification of CS-A, through which a highly sulfated nonreducing terminal sequence is generated.

Molecular cloning and characterization of chondroitin-4-O-sulfotransferase-3. A novel member of the HNK-1 family of sulfotransferases.

We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated chondroitin-4-sulfotransferase-3 (C4ST-3) (GenBank accession number AY120869) based on its homology to HNK-1 sulfotransferase (HNK-1 ST). The cDNA predicts an open reading frame encoding a type II membrane protein of 341 amino acids with a 12-amino acid cytoplasmic domain and a 311-amino acid luminal domain containing a single potential N-linked glycosylation site. C4ST-3 has the greatest amino acid sequence identity when aligned with chondroitin-4-O-sulfotransferase 1 (C4ST-1) (45%) but also shows significant amino acid identity with chondroitin-4-O-sulfotransferase 2 (C4ST-2) (27%), dermatan-4-O-sulfotransferase 1 (29%), HNK-1 ST (26%), N-acetylgalactosamine-4-O-sulfotransferase 1 (26%), and N-acetylgalactosamine-4-O-sulfotransferase 2 (23%). C4ST-3 transfers sulfate to the C-4 hydroxyl of beta1,4-linked GalNAc that is substituted with a beta-linked glucuronic acid at the C-3 hydroxyl. The open reading frame of C4ST-3 is encoded by three exons located on human chromosome 3q21.3. Northern blot analysis reveals a single 2.1-kilobase transcript. C4ST-3 message is expressed in adult liver and at lower levels in adult kidney, lymph nodes, and fetal liver. Although C4ST-3 and C4ST-1 have similar specificities, the highly restricted pattern of expression seen for C4ST-3 suggests that it has a different role than C4ST-1.

Molecular cloning and expression of two distinct human chondroitin 4-O-sulfotransferases that belong to the HNK-1 sulfotransferase gene family.

Using an expression cloning strategy, the cDNA encoding the human HNK-1 sulfotransferase (HNK-1ST) has been cloned. During this cloning we found that HNK-1ST and other Golgi-associated sulfotransferases cloned before share homologous sequences including the RDP motif (Ong, E., Yeh, J.-C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) J. Biol. Chem. 223, 5190-5195). Using this conserved sequence in HNK-1ST as a probe, we identified two expressed sequence tags in EST data base which have 31.6 and 30.7% identity with HNK-1ST at the amino acid levels. Expression of these two full-length cDNAs failed to form HNK-1 glycan nor to add sulfate to CD34 or NCAM. Surprisingly, proteins expressed by these cDNAs transferred sulfate to the C-4 position of N-acetylgalactosamine in chondroitin and desulfated dermatan sulfate, thus we named these two enzymes, chondroitin 4-O-sulfotransferase 1 and -2 (C4ST-1 and C4ST-2). Both C4ST-1 and C4ST-2, however, did not form 4, 6-di-O-sulfated N-acetylgalactosamine when chondroitin sulfate C was used as an acceptor. Moreover, analysis of (35)S-labeled dermatan sulfate formed by C4ST-1 indicate that sulfation preferentially took place in GlcA-->GalNAc unit than in IdoA-->GalNAc unit, suggesting that 4-O-sulfation at N-acetylgalactosamine may precede epimerization of glucuronic acid to iduronic acid during dermatan sulfate biosynthesis. Northern analysis demonstrated that the transcript for C4ST-1 is predominantly expressed in peripheral leukocytes and hematopoietic tissues while the C4ST-2 transcript is more widely expressed in various tissues. These results indicate C4ST-1 and C4ST-2 play complementary roles in chondroitin and dermatan sulfate synthesis in different tissues.

Expression of heparan sulfate D-glucosaminyl 3-O-sulfotransferase isoforms reveals novel substrate specificities.

The 3-O-sulfation of glucosamine residues is an important modification during the biosynthesis of heparan sulfate (HS). Our previous studies have led us to purify and molecularly clone the heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1), which is the key enzyme converting nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact). In this study, we expressed and characterized the full-length cDNAs of 3-OST-1 homologous genes, designated as 3-OST-2, 3-OST-3A, and 3-OST-3B as described in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5170-5184). All these cDNAs were successfully expressed in COS-7 cells, and heparan sulfate sulfotransferase activities were found in the cell extracts. We demonstrated that 3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate D-glucosaminyl 3-O-sulfotransferases because the enzymes transfer sulfate from adenosine 3'-phosphophate 5'-phospho-[35S]sulfate ([35S]PAPS) to the 3-OH position of glucosamine. 3-OST-3A and 3-OST-3B sulfate an identical disaccharide. HSact conversion activity in the cell extract transfected by 3-OST-1 was shown to be 300-fold greater than that in the cell extracts transfected by 3-OST-2 and 3-OST-3A, suggesting that 3-OST-2 and 3-OST-3A do not make HSact. The results of the disaccharide analysis of the nitrous acid-degraded [35S]HS suggested that 3-OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3A transfers sulfate to IdoA2S-GlcNS. Our results demonstrate that the 3-O-sulfation of glucosamine is generated by different isoforms depending on the saccharide structures around the modified glucosamine residue. This discovery has provided evidence for a new cellular mechanism for generating a defined saccharide sequence in structurally complex HS polysaccharide.

Multiple isoforms of heparan sulfate D-glucosaminyl 3-O-sulfotransferase. Isolation, characterization, and expression of human cdnas and identification of distinct genomic loci.

3-O-Sulfated glucosaminyl residues are rare constituents of heparan sulfate and are essential for the activity of anticoagulant heparan sulfate. Cellular production of the critical active structure is controlled by the rate-limiting enzyme, heparan sulfate D-glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) (EC 2.8.2.23). We have probed the expressed sequence tag data base with the carboxyl-terminal sulfotransferase domain of 3-OST-1 to reveal three novel, incomplete human cDNAs. These were utilized in library screens to isolate full-length cDNAs. Clones corresponding to predominant transcripts were obtained for the 367-, 406-, and 390-amino acid enzymes 3-OST-2, 3-OST-3A, and 3-OST-3B, respectively. These type II integral membrane proteins are comprised of a divergent amino-terminal region and a very homologous carboxyl-terminal sulfotransferase domain of approximately 260 residues. Also recovered were partial length clones for 3-OST-4. Expression of the full-length enzymes confirms the 3-O-sulfation of specific glucosaminyl residues within heparan sulfate (Liu, J., Shworak, N. W., Sinaÿ, P., Schwartz, J. J. Zhang, L., Fritze, L. M. S., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5185-5192). Southern analyses suggest the human 3OST1, 3OST2, and 3OST4 genes, and the corresponding mouse isologs, are single copy. However, 3OST3A and 3OST3B genes are each duplicated in humans and show at least one copy each in mice. Intriguingly, the entire sulfotransferase domain sequence of the 3-OST-3B cDNA (774 base pairs) was 99.2% identical to the same region of 3-OST-3A. Together, these data argue that the structure of this functionally important region is actively maintained by gene conversion between 3OST3A and 3OST3B loci. Interspecific mouse back-cross analysis identified the loci for mouse 3Ost genes and syntenic assignments of corresponding human isologs were confirmed by the identification of mapped sequence-tagged site markers. Northern blot analyses indicate brain exclusive and brain predominant expression of 3-OST-4 and 3-OST-2 transcripts, respectively; whereas, 3-OST-3A and 3-OST-3B isoforms show widespread expression of multiple transcripts. The reiteration and conservation of the 3-OST sulfotransferase domain suggest that this structure is a self-contained functional unit. Moreover, the extensive number of 3OST genes with diverse expression patterns of multiple transcripts suggests that the novel 3-OST enzymes, like 3-OST-1, regulate important biologic properties of heparan sulfate proteoglycans.