Molecular cloning and expression of chick chondrocyte chondroitin 6-sulfotransferase.
Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. The enzyme has been purified previously to apparent homogeneity from the serum-free culture medium of chick chondrocytes. The purified enzyme also catalyzed the sulfation of keratan sulfate. We have now cloned the cDNA of the enzyme. This cDNA contains a single open reading frame that predicts a protein composed of 458 amino acid residues. The protein predicts a Type II transmembrane topology similar to other glycosyltransferases and heparin/heparan sulfate N-sulfotransferase/N-deacetylases. Evidence that the predicted protein corresponds to the previously purified C6ST was the following: (a) the predicted sequence of the protein contains all of the known amino acid sequence, (b) when the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, both the C6ST activity and the keratan sulfate sulfotransferase activity were overexpressed, (c) a polyclonal antibody raised against a fusion peptide, which was expressed from a cDNA containing the sequence coding for 150 amino acid residues of the predicted protein, cross-reacted to the purified C6ST, and (d) the predicted protein contained six potential sites for N-glycosylation, which corresponds to the observation that the purified C6ST is an N-linked glycoprotein. The amino-terminal amino acid sequence of the purified protein was found in the transmembrane domain, suggesting that the purified protein might be released from the chondrocytes after proteolytic cleavage in the transmembrane domain.
Fukuta
M
M
Department of Life Science, Aichi University of Education, Kariya, Japan.
Uchimura
K
K
Nakashima
K
K
Kato
M
M
Kimata
K
K
Shinomura
T
T
Habuchi
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002356:Cartilage / Q000166:cytology / Q000201:enzymology*; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D015151:Immunoblotting; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D015238:Sulfotransferases / Q000096:biosynthesis / Q000235:genetics*; D000097811:Carbohydrate Sulfotransferases
D015342:DNA Probes; D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases