Role of Site-Specific Glycosylation in the I-Like Domain of Integrin β1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation

Abstract

Integrin β1 plays an essential role in the crosstalk between tumor cells and their microenvironment. Aberrant N-glycosylation of integrin β1 was documented to alter integrin β1 expression, dimerization, and biological function. However, the biological function of site-specific N-glycosylation of integrin β1 on extracellular vesicles is not fully understood. In this study, we mutated putative N-glycosylation sites in different domains of integrin β1. Removal of the N-glycosylation sites on the I-like domain of integrin β1 (termed the Δ4–6 β1 mutant) suppressed focal adhesion kinase (FAK) signaling, cell migration, and adhesion compared with other β1 mutants. Cell adhesion, migration, and activation of FAK were suppressed in recipient MCF7 cells co-cultured with Δ4–6 mutant cells and treated with small extracellular vesicles (sEVs) from Δ4–6 mutant cells. Notably, the wild-type and β1 mutant were both present in sEVs, and could be transferred to recipient cells via sEVs, resulting in changes of cell behavior. Our findings demonstrate the important roles of N-glycosylation of the I-like domain of integrin β1. Moreover, the vesicular Δ4–6 β1 mutant can regulate integrin-mediated functions in recipient cells via sEVs.

Sialylation on vesicular integrin β1 determined endocytic entry of small extracellular vesicles into recipient cells

“…Integrin β1 is a typical glycoprotein characterized by its distinct structural domains, including a plexin-semaphorin-integrin (PSI) domain, an integrin-epidermal growth factor (I-EGF) domain, an I-like domain, both upstream and downstream of the hybrid domain, and a β-tail domain [ 29 ]. We further explored which domain of integrin β1 mainly bearing sialic acids using our previous established breast cancer cells expressing integrin β1 with mutant N -glycosylation sites in distinct domains (Additional file 1 : Figure S14) [ 22 ]. Lectin blotting analysis unveiled a markedly lower α2,6-linked sialic acid level in hybrid domain mutant (termed Δ7–8 mutant) compared to which in the other three mutants (Additional file 1 : Figure S15).…”

“…Human full-length and site-directed mutant integrin β1 were constructed in our laboratory [ 22 ]. Full-length and mutant integrin β1 with flag tag were cloned into the pLVX-AcGFP-N1 plasmid (Takara; Shiga, Japan), and transfected into YTS-1 cells as described previously [ 18 ].…”

Site-specific N-glycosylation of integrin α2 mediates collagen-dependent cell survival

“…Introducing an N -glycosylation sequence (N303-I304-T305) located at the hybrid-I-like domain on β1 or β3 integrin stabilized the open conformation of integrins and activated the metal ion-dependent binding site, thereby increasing their affinity for ligands ( Luo et al., 2003 ). More recently, removal of N -glycosylation sites on the I-like domain of integrin β1 (N212, N269, and N363) suppressed focal adhesion kinase signaling and cell migration in both donor and recipient cells through extracellular vesicles ( Cao et al, 2021 ). As a result, we were interested to know whether modulation of N -glycosylation sites on α2 subunit may affect ligand binding affinity, heterodimeric formation of α2β1 integrin, and downstream signaling.…”

Extracellular vesicles in the breast cancer brain metastasis: physiological functions and clinical applications

“…Additionally, there are examples where cancer cells modify EV surface glycosylation to impart various properties. Cao et al (2021) demonstrated that N-glycosylation of integrin β1 on EVs derived from highly metastatic triple-negative breast cancer cells (MDA-MB-231) enhances the migratory ability of recipient cells that take up EVs through FAK signaling, promoting metastasis through glycosylated proteins on EVs. Moreover, breast cancer cell-derived MVs carrying highly glycosylated extracellular MMP inducer (EMMPRIN) were found to stimulate cancer cell invasion through the activation of the p38/MAPK signaling pathway in recipient cancer cells ( Menck et al, 2015 ).…”