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

Abstract

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...

Functions of Chondroitin Sulfate/Dermatan Sulfate Chains in Brain Development

“…Sulfotransferase activity toward CS/DS was assayed by a method described previously (44). Briefly, the standard reaction mixture (60 l) contained 10 l of the resuspended beads, 50 mM imidazole-HCl, pH 6.8, 2 mM dithiothreitol, 10 M [ 35 S]PAPS (1 or 3 ϫ 10 5 dpm), and CS-B from porcine skin, which had been pretreated with nitrous acid as the acceptor polysaccharide (2 g as GlcUA).…”

Molecular Cloning and Characterization of GalNAc 4-Sulfotransferase Expressed in Human Pituitary Gland

“…Assay of C4ST Activity-C4ST activity was assayed by the method described previously (20). The standard reaction mixture contained 50 mM imidazole-HCl, pH 6.8, 0.0025% protamine chloride, 2 mM dithiothreitol, 25 nmol (as glucuronic acid) chondroitin, 50 pmol of [ , and enzyme in a final volume of 50 l. For determining the activity toward desulfated dermatan sulfate, chon-droitin was replaced with 25 nmol (as galactosamine) of desulfated dermatan sulfate and the amount of protamine chloride was increased to 0.02%.…”

“…1A). C4ST also contains four potential glycosylation sites (21), and the contents of N-linked oligosaccharide of the purified C4ST was estimated as 35% as judged from the decrease in molecular size after digestion with N-glycosidase F (20). If the content of N-linked oligosaccharides of the GalNAc4ST is nearly equal to that of C4ST, molecular size of the glycosylated form of the recombinant GalNAc4ST could be estimated as about 64 kDa.…”

“…Various sulfotransferases involved in the sulfation of glycosaminoglycans (14) and oligosaccharides (15)(16)(17) have been cloned. We have purified and cloned chondroitin-6-sulfotransferase (C6ST) 1 (18,19) and chondroitin-4-sulfotransferase (C4ST) (20,21), which are involved in the sulfation of position 6 and position 4, respectively, of GalNAc residues of chondroitin. C6ST also transfers sulfate to position 6 of Gal residue of keratan sulfate and sialyl N-acetyllactosamine oligosaccharides (22,23).…”

CHST11 (carbohydrate (chondroitin 4) sulfotransferase 11)

“…However, CHST11 was initially identified as a protein secreted from chondrocytes and chondrosarcoma cells (Habuchi et al, 1991;Yamauchi et al, 1999).…”