PMID:15628971
| Citation |
Yusa, A, Kitajima, K and Habuchi, O (2005) N-linked oligosaccharides are required to produce and stabilize the active form of chondroitin 4-sulphotransferase-1. Biochem. J. 388:115-21 |
|---|---|
| Abstract |
C4ST-1 (chondroitin 4-sulphotransferase-1) transfers sulphate to position 4 of N-acetylgalactosamine in chondroitin. We showed previously that purified C4ST-1 from the culture medium of rat chondrosarcoma cells was a glycoprotein containing approx. 35% N-linked oligosaccharides. In the present paper, we investigated the functional role of the N-linked oligosaccharides attached to C4ST-1. We found that (i) treatment of recombinant C4ST-1 with peptide N-glycosidase F caused a marked decrease in activity, (ii) production of the active form of C4ST-1 by COS-7 cells transfected with cDNA of C4ST-1 was inhibited by tunicamycin, (iii) deletion of the N-glycosylation site located at the C-terminal region of C4ST-1 abolished activity, (iv) attachment of a single N-glycan at the C-terminal region supported production of the active form of C4ST-1, but the resulting recombinant enzyme was much more unstable at 37 degrees C than the control recombinant protein, and (v) truncation of C-terminal region up to the N-glycosylation site at the C-terminal region resulted in total loss of activity. These observations strongly suggest that N-linked oligosaccharides attached to C4ST-1 contribute to the production and stability of the active form of C4ST-1. In addition, the N-linked oligosaccharide at the C-terminal region appears to affect the glycosylation pattern of recombinant C4ST; a broad protein band of the wildtype protein resulting from microheterogeneity of N-linked oligosaccharides disappeared and four discrete protein bands with different numbers of N-linked oligosaccharides appeared when the N-linked oligosaccharide at the C-terminal region was deleted. |
| Links |
PubMed PMC1186699 Online version:10.1042/BJ20041573 |
| Keywords |
Animals; CHO Cells; COS Cells; Cercopithecus aethiops; Cricetinae; Cricetulus; Enzyme Activation; Mutagenesis, Site-Directed; Mutation; Oligosaccharides/chemistry; Oligosaccharides/metabolism; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism; Recombinant Proteins; Sulfotransferases/chemistry; Sulfotransferases/genetics; Sulfotransferases/metabolism; Tunicamycin |
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