Hyposialylation of Integrins Stimulates the Activity of Myeloid Fibronectin Receptors

Semel, Alexis C.; Seales, Eric C.; Singhal, Anuj; Eklund, Elizabeth A.; Colley, Karen J.; Bellis, Susan L.

doi:10.1074/jbc.m202493200

Cited by 101 publications

(95 citation statements)

References 41 publications

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“…However, this is not always the case. The expression of hyposialylated integrin ␣5␤1 was induced by phorbol esterstimulated differentiation in myeloid cells in which the expression of the ST6GalI was down-regulated by the treatment, increasing FN binding (19). A similar phenomenon was also observed in hematopoietic or other epithelial cells.…”

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confidence: 48%

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

Sato

Isaji

Tajiri

et al. 2009

Journal of Biological Chemistry

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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...

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confidence: 48%

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

Sato

Isaji

Tajiri

et al. 2009

Journal of Biological Chemistry

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“…3 These observations suggest that site-specific modulation of N-glycans on integrin affects its biological functions. Therefore, a detailed mutagenesis study of N-glycans on integrins is indispensable for the elucidation of the complicated mechanisms that are involved in its functional regulation by glycosyltransferases, such as GnT-III, ␣2,6-sialyltransferase, and GnT-V (9,12,13,41).…”

Section: Discussionmentioning

confidence: 99%

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

Isaji

Sato

Fukuda

et al. 2009

Journal of Biological Chemistry

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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 ...

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“…However, some studies have reported conflicting results. For example, terminal sialylation reduces attachment to ECM glycoproteins in melanoma [7] and in myeloid cells [8] , but enhances adhesion in erythroleukemia K562 cells [9] and colon epithelial SW948 cells [10] . These observations support the possibility that integrins are not the only relevant targets of ST6Gal-I-mediated sialylation in cancer cell adhesion and suggest that other surface glycoproteins may also be involved.…”

Section: Discussionmentioning

confidence: 99%

“…c-Met is hyposialylated in ST6Gal-I-KD HCT116 cells It has been postulated that changes in sialic acid structure on membrane glycoconjugates can modulate the adhesion of cancer cells to ECM components in an integrin-dependent manner [7][8][9][10] . Because we did not find any change in ST6Gal-I-KD HCT116 cell adhesion, we suggest that integrins alone cannot fully explain this observation.…”

Section: Knockdown Of St6gal-i Reduced Endogenous St6gal-i Expressionmentioning

confidence: 99%

α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells

et al. 2009

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Aim: We aimed to investigate the potential modification of previously unrecognized surface glycoprotein(s) by α2,6-sialylation other than by integrins. Methods: The expression of β-galactoside α2,6-sialyltransferase (ST6Gal-I) in the colon cancer cell line HCT116 was reduced by siRNA. The adhesion and Boyden chamber assay were used to detect the variation in cell motility. α2,6-Sialylation proteins were detected with lectin affinity assay. The mRNA expression, protein expression and downstream signaling modulation with siRNA were detected using reverse transcription-polymerase chain reaction, flow cytometry analysis, and Western blot. Results: In HCT116 cells, the knockdown of ST6Gal-I inhibited cell motility, but did not affect cell adhesion. This selectively altered cell migration was caused by the loss of α2,6-sialic acid structures on c-Met. Moreover, STAT3 was dephosphorylated at tyrosine 705 in ST6Gal-I-knockdown (ST6Gal-I-KD) HCT116 cells. Conclusion: c-Met is the substrate of ST6Gal-I. The hyposialylation of c-Met can abolish cell motility in ST6Gal-I-KD HCT116 cells.

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Hyposialylation of Integrins Stimulates the Activity of Myeloid Fibronectin Receptors

Cited by 101 publications

References 41 publications

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

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

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

α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells

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