N-Glycosylation of the I-like Domain of β1 Integrin Is Essential for β1 Integrin Expression and Biological Function

Abstract

N-Glycosylation of integrin ␣5␤1 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 (␣5S3-5) on the ␤-propeller of the ␣5 subunit are essential to the functional expression of the subunit. In particular, site 5 (␣5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin ␤1 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 ␤1 subunit (i.e. the ⌬4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the ␤1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either ␣5S3-5 or ␣5S5 mutant of the ␣5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the ␤1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the ␣5S3-5/ ␤1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the ␤1 subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an ␣ and a ␤ subunit (1). The interaction between integrin and the extracellular matrix is essential to both physiologic and pathologic events, such as cell migration, development, cell viability, immune homeostasis, and tumorigenesis (2, 3). Among the integrin superfamily, ␤1 integrin can combine with 12 distinct ␣ subunits (␣1-11, ␣v) to form heterodimers, thereby acquiring a wide variety of ligand specificity (1, 4). Integrins are thought to be regulated by inside-out signaling mechanisms that provoke conformational changes, which modulate the affinity of integrin for the ligand (5). However, an increasing body of evidence suggests that cell-surface carbohydrates mediate a variety of interactions between integrin and its extracellular environment, thereby affecting integrin activity and possibly tumor metastasis as well (6 -8).Guo et al. (9) reported that an increase in ␤1-6-GlcNAc sugar chains on the integrin ␤1 subunit stimulated cell migration. In addition, elevated sialylation of the ␤1 subunit, because of Ras-induced STGal-I transferase activity, also induced cell migration (10, 11). Conversely, cell migration and spreading were reduced by the addition of a bisecting GlcNAc, which is a product of N-acetylglucosaminyltransferase III (GnT-III), 2 to the ␣5␤1 and ␣3␤1 integrins (12, 13). Alterations of N-glycans on integrins might also regulate their cis interactions with membrane-associated proteins, including the epidermal ...

Potential roles of N-glycosylation in cell adhesion

“…We also found three N-glycosylation sites on I-like domain of integrin β1 subunit, which are important for α5 and β1 dimer formation and its expression on the cell surface [38]. Although the involvement of N-glycan in the αβ interaction remains unclear, it could be explained that an unknown lectin domain may exist on the each subunit, since the lectin domain of αMβ2 integrin is associated with GlcNAc on the non-reducing terminal of sugar chains on chilled platelets for its phagocytosis [39,40].…”

An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation

“…Immunoprecipitation and Western Blot-Immunoprecipitation was performed as described previously with minor modifications (5,33,35). Briefly, cells were rinsed twice with ice-cold PBS.…”

“…Virus Infections-Viral infection was performed as described previously (33,34). In brief, the lentivirus vectors (CSIV-TRERfA-CMV-KT or CS-RfA-ETBsd) were transfected into 293T cells with packaging plasmids by calcium phosphate.…”

“…Flow Cytometric Analysis-Flow cytometric analysis was performed as described previously with minor modifications (5,33,35). Briefly, semi-confluent cells were detached from the culture dishes using trypsin containing 1 mM EDTA and were subsequently stained with or without the primary mouse anti-␤1, followed by incubation with Alexa Fluor 647 goat anti-mouse IgG (Invitrogen).…”

N-Linked Glycosylation Is Essential for the Stability but Not the Signaling Function of the Interleukin-6 Signal Transducer Glycoprotein 130

“…Some proteins need N-linked glycans as chaperone-like structures during protein synthesis to ensure correct folding by increasing their solubility and masking hydrophobic patches, but the N-glycans can then be dispensable for protein function (1). In contrast, N-linked glycosylation is essential for ligand binding and stability of diverse growth factor, cytokine, peptide, and pattern recognition receptors as well as adhesion molecules (2)(3)(4)(5)(6)(7)(8).…”