PMID: 15628971

 

    Legend: Sugar

Title : N-linked oligosaccharides are required to produce and stabilize the active form of chondroitin 4-sulphotransferase-1

Abstract :

1. C4ST-1 ( chondroitin 4-sulphotransferase-1 ) transfers sulphate to position 4 of N-acetylgalactosamine in chondroitin
2. We showed previously that purified C4ST-1 from the culture medium of rat chondrosarcoma cells was a glycoprotein containing approx
3. 35% N-linked oligosaccharides
4. In the present paper, we investigated the functional role of the N-linked oligosaccharides attached to C4ST-1
5. 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, (i v ) 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
6. 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
7. 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