Molecular Cloning and Expression of a Novel Chondroitin 6-O-Sulfotransferase

• Published in: The Journal of biological chemistry 2000-07, Vol.275 (28), p.21075-21080
• Main Authors: Kitagawa, Hiroshi, Fujita, Masaki, Ito, Nobue, Sugahara, Kazuyuki
• Format: Article
• Language: English
• Subjects: Adult; Amino Acid Sequence; Animals; Base Sequence; Carbohydrate Sequence; Carbohydrate Sulfotransferases; Cell Membrane - enzymology; Chromosome Mapping; Cloning, Molecular; COS Cells; Exons; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Humans; Molecular Sequence Data; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Open Reading Frames; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Homology, Amino Acid; Spleen - enzymology; Substrate Specificity; Sulfotransferases - chemistry; Sulfotransferases - genetics; Sulfotransferases - metabolism; Transcription, Genetic; Transfection; X Chromosome
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M002101200

A novel human chondroitin 6-O-sulfotransferase, designated C6ST-2, was identified by BLAST analysis of expressed sequence tag using the sequence of a previously described human chondroitin 6-O-sulfotransferase (C6ST-1) as a probe. The new cDNA sequence revealed an open reading frame coding for a protein of 486 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 24% identity to the human C6ST-1, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase with marked specificity for polymer chondroitin. In contrast, keratan sulfate and oligosaccharides containing the Galβ1–4GlcNAc sequence, which are good acceptor substrates for the C6ST-1, hardly served as acceptors. The identification of the reaction product indicated that the enzyme is a novel chondroitin 6-O-sulfotransferase (C6ST-2) that mainly transfers sulfate to N-acetylgalactosamine. The coding region of C6ST-2 was contained in a single exon and localized to chromosome Xp11. Northern blot analysis of human brain poly(A)+ RNA revealed a single transcript of 2.4 kilobase pairs. Reverse transcription-polymerase chain reaction analysis showed that C6ST-2 is developmentally regulated in various tissues with expression persisting through adulthood in the spleen. Thus, we demonstrated the redundancy in chondroitin 6-O-sulfotransferases capable of forming chondroitin 6-sulfate, which is important for understanding the mechanisms leading to specific changes in the sulfation profile of chondroitin sulfate chains in various tissues during development and malignant transformation.