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 membrane proteins, CD38 and bone marrow stromal cell antigen 1 (BST-1)/CD157. 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. Previously, a soluble form of BST-1/CD157 (sBST-1), which lacked the glycosylphosphatidylinositol-anchored portion, was expressed 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-1 contains four putative N-glycosylation sites. Tunicamycin treatment reduced sBST-1 expression in the culture medium, indicating that N-glycosylation is essential for secretion. Site-directed mutagenesis was performed to generate sBST-1 mutants (N1-N4), each preserving a single N-glycosylation site. N1, N3 and N4 were well secreted into 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 facilitates 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 effective method to generate homogeneous material for structural studies.
Yamamoto-Katayama
S
S
Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita-City, Osaka 565-0874, Japan.
Sato
A
A
Ariyoshi
M
M
Suyama
M
M
Ishihara
K
K
Hirano
T
T
Nakamura
H
H
Morikawa
K
K
Jingami
H
H
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000378:metabolism; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016367:Baculoviridae; D002460:Cell Line; D018360:Crystallography, X-Ray; D058851:GPI-Linked Proteins; D005786:Gene Expression Regulation / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D008322:Mammals; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009244:NAD+ Nucleosidase / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D015703:Antigens, CD; D000943:Antigens, Differentiation; D000954:Antigens, Surface; D058851:GPI-Linked Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014415:Tunicamycin; D036541:ADP-ribosyl Cyclase; C087521:ADP-ribosyl cyclase 2; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1