PMID: 11439087

 

    Legend: Sugar

Title : Site-directed removal of N-glycosylation sites in BST-1 / CD157 : effects on molecular and functional heterogeneity

Abstract :

1. Cyclic ADP ribose (cADPR) is a novel second messenger that releases calcium from intracellular calcium stores, but works independently of inositol 1,4,5-trisphosphate
2. In mammals ADP-ribosyl cyclase function is found in two membrane proteins , CD38 and bone marrow stromal cell antigen 1 ( BST-1 )/ CD157
3. These enzymes are exposed extracellularly and also possess cADPR hydrolase activity, but an intracellular soluble ADP-ribosyl cyclase has been reported in human T-cells
4. Previously, a soluble form of BST-1 / CD157 ( sBST-1 ), which lacked the glycosylphosphatidylinositol-anchored portion, was expressed by a baculovirus-insect-cell system
5. In this study, we have purified the sBST-1 , and it migrated as two major bands by SDS /PAGE, suggesting that it is post-translationally modified
6. BST-1 contains four putative N-glycosylation sites
7. Tunicamycin treatment reduced sBST-1 expression in the culture medium, indicating that N-glycosylation is essential for secretion
8. Site-directed mutagenesis was performed to generate sBST-1 mutants ( N1-N4 ), each preserving a single N-glycosylation site
9. N1, N3 and N4 were well secreted into the medium, and were each detected as a single band
10. Although N3 and N4 retained the ADP-ribosyl cyclase activity, the cADPR-hydrolase activity was retained only in N4
11. We conclude that N-glycosylation of sBST-1 facilitates the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and enzymic activity
12. Furthermore a crystal has been obtained using the N4 mutant, but not the wild-type sBST-1
13. Thus the artificial engineering of N-glycosylation sites could be an effective method to generate homogeneous material for structural studies