Effects Of N-glycosylation Sites Mutations On The Biological Function Of Integrin β1 In Breast Cancer Cells

Integrin,one typical transmembrane receptor,mediates cytoskeleton remodeling and intracellular signal transduction by binding with extracellular matrix components.There are18 α and 8 β subunits consist of integrins.Integrin β1,one of the β subfamily,is able to bind with each of 12 α subunits to form different types of heterodimers.Integrin β1 plays an essential role in the tumor microenvironment.Integrin β1 is modified with N-glycosylation,and aberrant N-glycosylation on integrin β1 was documented to alter its expression,dimerization and biological function.Small extracellular vesicles are wrapped in a lipid bilayer and carry various biological molecules,including proteins,nucleic acids and lipids,which can mediate the communication of substances and signals between cells.The previous study in our group showed that migration ability of recipient cells was suppressed by vesicular integrin β1bearing high levels of bisecting Glc NAc structures.However,the effect of N-glycosylation in different domain of integrin β1 on biological function of small extracellular vesicles is not elucidated.According to the characteristics of different domains of integrin β1,in this study,we mutated putative N-glycosylation sites in different domain of integrin β1,and transfected MDA-MB-231 cells with lentivirus to obtain WT-β1 cell,Δ1-3,Δ4-6,Δ7-8,and Δ9-12 mutants.Removal of the N-glycosylation sites on the I-like domain of integrin β1(termed as Δ4-6mutant)reduced focal adhesion kinase(FAK)signaling,cell migration,and adhesion compared to other β1 mutants.Compared with the untreated group,the conditioned medium(CM)of WT-β1 cells or Δ1-3,Δ7-8,Δ9-12 mutants,significantly enhanced the adhesion and the activation of FAK of breast cancer cells MCF7.The results of the CM treated group of Δ4-6 mutant were similar to the untreated group,and the adhesion of MCF7 cells and the activation of FAK were not significantly altered.Compared with the untreated group,after co-cultured with the transfectant cells,the adhesion and migration ability of MCF7 cells were significantly enhanced by WT-β1 cells or Δ1-3,Δ7-8,Δ9-12 mutant,while after co-cultured with Δ4-6mutant,adhesion and migration ability of MCF7 cells did not show significant change.Similarly,compared with small extracellular vesicles derived from WT-β1 cells,small extracellular vesicles derived from Δ4-6 mutant cells significantly reduced the migration ability of recipient cells.Futhermore,the mutation of the N-glycosylation site of integrin β1does not affect the release of small extracellular vesicles,nor its entry into recipient cells.In conclusion,this study proves that at 4-6 N-glycosylation sites on I-like domain of integrinβ1 can significantly affect the function of the protein,and determines the adhesion and migration of receptor cells affected by small extracellular vesicles mediated by integrin β1.