title: N-glycosylation of the I-like domain of beta1 integrin is essential for beta1 integrin expression and biological function: identification of the minimal N-glycosylation requirement for alpha5beta1.
abstract: N-Glycosylation of integrin alpha5beta1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (alpha5S3-5) on the beta-propeller of the alpha5 subunit are essential to the functional expression of the subunit. In particular, site 5 (alpha5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin beta1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the beta1 subunit (i.e. the Delta4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the beta1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either alpha5S3-5 or alpha5S5 mutant of the alpha5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the beta1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the alpha5S3-5/beta1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the beta1 subunit, it might also be useful for the study of molecular structures.
authors.affiliation: Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, 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: Tomohiko
authors.initials: T
authors.lastname: Fukuda
authors.forename: Jianguo
authors.initials: J
authors.lastname: Gu
mesh_terms: D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D039081:Integrin alpha5beta1 / Q000235:genetics / Q000378:metabolism*; D019012:Integrin beta1 / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D046188:Protein Modification, Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000502:physiology
chemical_list: D039081:Integrin alpha5beta1; D019012:Integrin beta1