Molecular cloning and expression of chondroitin 4-sulfotransferase.
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 approximately 29% identity and approximately 48% 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.
Yamauchi
S
S
Department of Life Science, Aichi University of Education, Kariya, Aichi 448-8542, Japan.
Mita
S
S
Matsubara
T
T
Fukuta
M
M
Habuchi
H
H
Kimata
K
K
Habuchi
O
O
D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002807:Chondroitin / Q000378:metabolism*; D003001:Cloning, Molecular; D003871:Dermatan Sulfate / Q000378:metabolism; D015723:Gene Library; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015238:Sulfotransferases / Q000096:biosynthesis / Q000235:genetics*; D014018:Tissue Distribution
D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003871:Dermatan Sulfate; D010724:Phosphoadenosine Phosphosulfate; D002807:Chondroitin; D015238:Sulfotransferases; C019893:chondroitin 4-sulfotransferase; D000116:Acetylgalactosamine