An N-Glycosylation Site on theβ-Propeller Domain of the Integrin α5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification byβ1,4-N-Acetylglucosaminyltransferase III

Abstract

Recently we reported that N-glycans on the ␤-propeller domain of the integrin ␣5 subunit (S-3,4,5) are essential for ␣5␤1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type ␣5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the ␣5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type ␣5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the ␣5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin ␣5␤1 is well known as a fibronectin (FN) 3 receptor. The interaction between integrin ␣5 and FN is essential for cell migration, cell survival, and development (5-8). In addition, integrins are N-glycan carrier proteins. For example, ␣5␤1 integrin contains 14 and 12 putative N-glycosylation sites on the ␣5 and ␤1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin ␣5␤1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature ␣5␤1 integrin appeared on the cell surface, an...

Potential roles of N-glycosylation in cell adhesion

“…Since the α5 subunit has a 47 % homology to αV, Mould, et al speculate that the structural environment of the αβ interfaces could be affected by the presence of Nglycans by a homology modeling structure of α5β1 [34]. To further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the α5 subunit in detail, and found that site-4 was a key site that could be specifically modified by GnT-III [19]. Furthermore, the deletion of site-4 abolished the suppression of cell spread induced by GnT-III in the transfectant.…”

“…Similarly, characterization of carbohydrate moieties of integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that β1-6 GlcNAc branched structures were highly expressed in metastatic cells compared with nonmetastatic cells [11]. In contrast to GnT-V, the overexpression of GnT-III resulted in an inhibition of α5β1 integrin-mediated cell spreading and migration, and the phosphorylation of the focal adhesion kinase [19,20]. The affinity of the binding of integrin α5β1 to FN was significantly reduced as a result of the introduction of a bisecting GlcNAc to the α5 subunit.…”

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

“…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.…”

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).…”