title: N-glycosylation of the beta-propeller domain of the integrin alpha5 subunit is essential for alpha5beta1 heterodimerization, expression on the cell surface, and its biological function.
abstract: The N-glycosylation of integrin alpha5beta1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the alpha5 integrin. Removal of the putative N-glycosylation sites on the beta-propeller, Thigh, Calf-1, or Calf-2 domains of the alpha5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1-5) on the beta-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the alpha5 subunit carrying only three potential N-glycosylation sites (3-5 sites) on the beta-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Delta3-5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the beta-propeller domain of the alpha5 subunit is essential for heterodimerization and biological functions of alpha5beta1 integrin and might also be useful for studies of the molecular structure.
authors.affiliation: Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Isaji
authors.forename: Yuya
authors.initials: Y
authors.lastname: Sato
authors.forename: Yanyang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Eiji
authors.initials: E
authors.lastname: Miyoshi
authors.forename: Yoshinao
authors.initials: Y
authors.lastname: Wada
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
authors.forename: Jianguo
authors.initials: J
authors.lastname: Gu
mesh_terms: D000818:Animals; D016466:CHO Cells; D037281:Calnexin / Q000378:metabolism; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism*; D002465:Cell Movement; D006224:Cricetinae; D019281:Dimerization; D005353:Fibronectins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D039482:Integrin alpha5 / Q000235:genetics / Q000378:metabolism*; D019012:Integrin beta1 / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism
chemical_list: D005353:Fibronectins; D039482:Integrin alpha5; D019012:Integrin beta1; D021122:Protein Subunits; D037281:Calnexin