Preparation and high-performance liquid chromatography of 3′-phosphoadenosine-5′-phospho[35S]sulfate with a predetermined specific activity

We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO 4 )-GlcA(2SO 4 )-GalNAc(6SO 4 ), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO 4 )-GlcA(2SO 4 )-GalNAc(6SO 4 ), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3-phosphoadenosine 5-phosphosulfate (PAPS) to position 2 of the GlcA residue of CS, is expected to be involved in synthesis of these structures; however, the specificity of 2OST concerning recognition of the sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of the sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C, and desulfated dermatan sulfate. When

show abstract

“…[ 35 S]PAPS was prepared as described (24). Chondroitin (squid skin) was prepared as described (25).…”

Section: Methodsmentioning

confidence: 99%

Recognition of Sulfation Pattern of Chondroitin Sulfate by Uronosyl 2-O-Sulfotransferase

Ohtake

Kimata

Habuchi

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…[ 35 S]PAPS was prepared as described (25). Chondroitin (squid skin) was prepared previously described (26).…”

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of Chondroitin 4-Sulfotransferase from the Culture Medium of a Rat Chondrosarcoma Cell Line

Yamauchi

Hirahara

Usui

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

Chondroitin 4-sulfotransferase, which transfers sulfate from 3-phosphoadenosine 5-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3,5-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.Chondroitin sulfate proteoglycan is found in various tissues and thought to play important roles in various cellular interactions involving cell adhesion (1, 2), regulation of neurite outgrowth (3-6), migration of neural crest cells (7), binding of phospholipase A 2 (8), and an adherence receptor for Plasmodium falciparum-infected erythrocytes (9). In most of mammalian and avian chondroitin sulfate proteoglycans, the glycosaminoglycan chains bear sulfate at position 4 or 6 of N-acetylgalactosamine residues. The ratio of chondroitin 4-sulfate/chondroitin 6-sulfate has been reported to vary with the development of animals (10 -12), malignant change (13), and leukocyte differentiation (14). Sulfation of positions 4 and 6 of GalNAc residue was shown to be catalyzed by different sulfotransferases (15); chondroitin 4-sulfotransferase (C4ST) 1 and chondroitin 6-sulfotransferase (C6ST). Characterization of the purified sulfotransferases and molecular cloning of these cDNAs are basically important to reveal the functional roles of these chondroitin sulfate isomers. We have purified C6ST from the culture medium of chick chondrocytes (16) and cloned the cDNA (17). Unexpectedly, the purified C6ST was found to catalyze not only chondroitin but also keratan sulfate (18) and sialyl N-acetyllactosamine oligosaccharides (19). We previously observed that C4ST was also secreted to the culture medium of chick chondrocytes (20), but the purification of C4ST from the culture medium of chick chondrocytes has been hampered by the presence of an excess amount of C6ST. Unlike chick chondrocytes, rat chondrosa...

show abstract

“…We also describe the expression patterns in various tissues of the three isoforms. (66). Chondroitin (squid skin) was prepared as described previously (67).…”

mentioning

confidence: 99%

The Occurrence of Three Isoforms of Heparan Sulfate 6-O-Sulfotransferase Having Different Specificities for Hexuronic Acid Adjacent to the TargetedN-Sulfoglucosamine

Habuchi¹,

Tanaka²,

Habuchi³

et al. 2000

Journal of Biological Chemistry

209

188

View full text Add to dashboard Cite

We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208 -9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.Heparan sulfate proteoglycans (HSPGs) 1 are ubiquitously present on cell surface and in extracellular matrix including basement membrane and have divergent structures and functions (1-3). The heparan sulfate (HS) chains in HSPGs are known to interact with a variety of proteins such as heparinbinding growth factors, extracellular matrix components, protease inhibitors, protease, and lipoprotein lipase (4 -8). These interactions are implicated not only in various dynamic cellular behaviors including cell proliferation, differentiation, adhesion, migration, and morphology during development (9 -16), but also in various physiological phenomena such as inflammation (17), blood coagulation (18 -20), and tumor cell invasion and malignancy (21-23). Moreover, the pathogens such as bacteria, parasites, and viruses are known to infect host cells through the interactions between the cell surface HS on host cell and the coat proteins or cell surface proteins of pathogens (24,25).Recently, genetic screens and analyses are suggesting not only in Drosophila but also in mammals that these interactions also play pivotal roles in embryonic development. For example, the sugarless mutant (10 -12), which is deficient in UDP-glucose dehydrogenas...

show abstract

Preparation and high-performance liquid chromatography of 3′-phosphoadenosine-5′-phospho[35S]sulfate with a predetermined specific activity

Cited by 37 publications

References 6 publications

Recognition of Sulfation Pattern of Chondroitin Sulfate by Uronosyl 2-O-Sulfotransferase

Recognition of Sulfation Pattern of Chondroitin Sulfate by Uronosyl 2-O-Sulfotransferase

Purification and Characterization of Chondroitin 4-Sulfotransferase from the Culture Medium of a Rat Chondrosarcoma Cell Line

The Occurrence of Three Isoforms of Heparan Sulfate 6-O-Sulfotransferase Having Different Specificities for Hexuronic Acid Adjacent to the TargetedN-Sulfoglucosamine

Contact Info

Product

Resources

About