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

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. In mammals ADP-ribosyl cyclase function is found in two m embrane proteins, CD38 and bone marrow stromal cell antigen 1 (BST-1)/CD157 . These enzymes are exposed extracellularly and also possess cADPR hydrolas e activity, but an intracellular soluble ADP-ribosyl cyclase has been repor ted in human T-cells. Previously, a soluble form of BST-1/CD157 (sBST-1), w hich lacked the glycosylphosphatidylinositol-anchored portion, was expresse d by a baculovirus-insect-cell system. 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. BST-I contains four putative N-glycosylat ion sites. Tunicamycin treatment reduced sBST-1 expression in the culture m edium, indicating that N-glycosylation is essential for secretion. Site-dir ected mutagenesis was performed to generate sBST-1 mutants (N1-N4), each pr eserving a single N-glycosylation site. Nl, N3 and N4 were well secreted in to the medium, and were each detected as a single band. Although N3 and N4 retained the ADP-ribosyl cyclase activity, the cADPR-hydrolase activity was retained only in N4. We conclude that N-glycosylation of sBST-1 facilitate s the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and enzymic activity. Furthermore a crystal has been obtained using the N4 mutant, but not the wild-type sBST-1 . Thus the artificial engineering of N-glycosylation sites could be an effe ctive method to generate homogeneous material for structural studies.