Molecular cloning and expression of a novel chondroitin 6-O-sulfotransferase.
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 Galbeta1-4GlcNAc 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.
Kitagawa
H
H
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
Fujita
M
M
Ito
N
N
Sugahara
K
K
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000201:enzymology; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005091:Exons; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014960:X Chromosome; D000097811:Carbohydrate Sulfotransferases
D009844:Oligosaccharides; D011994:Recombinant Proteins; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases