N-glycosylation of the β-propeller domain of the integrin α5 subunit is essential for α5β1 heterodimerization, expression on the cell surface, and its biological function

The N-glycosylation of integrin alpha 5 beta 1 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-glycosylationsitesonthe alpha 5 integrin. Removal of the putative N-glycosylation sites on the beta-propeller, Thigh, Calf-1, or Calf-2 domains of the alpha 5 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 (sites1-5) 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 alpha 5 subunit carrying only three potential N-glycosylationsites(3-5sites) on the beta-propellerwerecomparablewiththose 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 Delta 3-5 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 alpha 5 subunit is essential for heterodimerization and biological functions of alpha 5 beta 1 integrin and might also be useful for studies of the molecular structure.