Molecular Cloning and Expression of Chondroitin 4-Sulfotransferase

Abstract

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3-phosphoadenosine 5-phosphosulfate to position 4 of N-acetylgalactosamine residue of chondroitin. The enzyme has been previously purified to apparent homogeneity from the serum-free culture medium of rat chondrosarcoma cells (Yamauchi, A., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J. H., and Habuchi, O. (1999) J. Biol. Chem. 274, 2456 -2463). The purified enzyme also catalyzed the sulfation of partially desulfated dermatan sulfate. We have now cloned the cDNA of the mouse C4ST on the basis of the amino acid sequences of peptides obtained from the purified enzyme by protease digestion. This cDNA contains a single open reading frame that predicts a protein composed of 352 amino acid residues. The protein predicts a Type II transmembrane topology. The predicted sequence of the protein contains all of the known amino acid sequence and four potential sites for N-glycosylation, which corresponds to the observation that the purified C4ST is an N-linked glycoprotein. The amino acid sequence of mouse C4ST showed significant sequence homology to HNK-1 sulfotransferase. Comparison of the sequence of mouse C4ST with human HNK-1 sulfotransferase revealed ϳ29% identity and ϳ48% similarity at the amino acid level. When the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that catalyzes the transfer of sulfate to position 4 of GalNAc residue of both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis showed that, among various mouse adult tissues, 5.7-kilobase message of C4ST was mainly expressed in the brain and kidney.Chondroitin sulfate proteoglycans are found in various tissues as molecules having divergent molecular architecture (1, 2). Chondroitin sulfate chains attached to chondroitin sulfate proteoglycans appear to play important roles in the formation and maintenance of cartilage tissue, because undersulfation of chondroitin sulfate resulted from the defective synthesis of PAPS 1 (3, 4) or defective sulfate transport (5) was found to cause underdevelopment of skeleton. Various chondroitin sulfate proteoglycans have been reported to be present in the brain (6, 7) and to function in the regulation of neurite outgrowth and neural cell adhesion (8 -12), neuronal migration (13), and the survival of neurons (14). Chondroitin sulfate chains are also shown to be involved in the interaction with CD44 (15, 16), phospholipase A 2 (17), Plasmodium falciparuminfected erythrocytes (18), and L-selectin (19). Chondroitin sulfates have sulfate group at various positions of the component sugars; position 6 and/or 4 of GalNAc residues and position 2 or 3 of GlcA residues. The pattern of sulfation of chondroitin sulfate chains varies with the source of the proteoglycans, development of animal (20 -22), and malignant change (23), suggesting that sulfate moieties attached to the specific position of the component sugars may be related to ...

Determination of the Glycosaminoglycan-Protein Linkage Region Oligosaccharide Structures of Proteoglycans from Drosophila melanogaster and Caenorhabditis elegans

“…No sulfation of the Gal residues in the linkage region of C. elegans chondroitin may be related to the finding that chondroitin is not sulfated in this organism. No homologs of human CS/DS 4-O-sulfotransferases (63,64) are found in the C. elegans genome. In Drosophila, for the synthesis of chondroitin 4-sulfate, the 4-O-sulfation of the Gal residue in the linkage region may not be required as a prerequisite.…”

Molecular Cloning and Characterization of Chondroitin-4-O-sulfotransferase-3

“…dermatan) or to terminal GalNAc in the sequence GalNAc␤1,4GlcNAc␤1,2Man found at the terminus of certain N-linked oligosaccharides. Based on the levels of mRNA expression, C4ST-1 is widely expressed in tissues (8,9), whereas C4ST-3 has a restricted pattern of expression. Since C4ST-3 and C4ST-1 have similar, if not identical, specificities and are both expressed in liver, kidney, and lymph nodes, the biological role played by C4ST-3 is not clear.…”

“…GalNAc-4-ST1 and GalNAc-4-ST2 both transfer sulfate to the C-4 hydroxyl of terminal ␤1,4-linked GalNAc on N-linked oligosaccharides such as those found on the glycoprotein hormones lutropin and thyrotropin (10,11). Whereas C4ST-1, C4ST-2, and D4ST-1 are also GalNAc-4-O-sulfotransferases, they only transfer sulfate to the C-4 hydroxyl of internal ␤1,4-linked GalNAc moieties within the repeating disaccharide sequences found in chondroitin and dermatan (7)(8)(9). We now report the cloning of another member of this family of sulfotransferases.…”

“…The putative open reading frame (ORF) encodes a protein that shows homology to the luminal domain of C4ST-1, a member of the HNK-1 family of sulfotransferases (8,9). Subsequent BLASTN queries of the dbEST data set using this region of homology identified two matching EST sequences (GenBank TM accession numbers BF448098 and AI074149, respectively).…”

“…These include human HNK-1 sulfotransferase (HNK-1 ST) 1 itself (1, 2), GalNAc-4-ST1 (3-5), GalNAc-4-ST2 (5, 6), dermatan-4-sulfotransferase-1 (D4ST-1) (7), chondroitin-4-sulfotransferase-1 (C4ST-1) (8,9), and chondroitin-4-sulfotransferase-2 (C4ST-2) (8). With the exception of HNK-1 ST itself, which transfers sulfate to the C-3 hydroxyl of terminal glucuronic acid in the sequence GlcUA␤1,3Gal␤1,4GlcNAc-R to produce the HNK-1 epitope SO 4 -3-GlcUA␤1,3Gal␤1,4GlcNAc-R, the members of this family of sulfotransferases are all GalNAc-4-sulfotransferases.…”