The importance of N-glycosylation on β(3) integrin ligand binding and conformational regulation

N-glycosylations can regulate the adhesive function of integrins. Great variations in both the number and distribution of N-glycosylation sites are found in the 18 α and 8 β integrin subunits. Crystal structures of α(IIb)β(3) and α(V)β(3) have resolved the precise structural location of each N-glycan site, but the structural consequences of individual N-glycan site on integrin activation remain unclear. By site-directed mutagenesis and structure-guided analyses, we dissected the function of individual N-glycan sites in β(3) integrin activation. We found that the N-glycan site, β(3)-N320 at the headpiece and leg domain interface positively regulates α(IIb)β(3) but not α(V)β(3) activation. The β(3)-N559 N-glycan at the β(3)-I-EGF3 and α(IIb)-calf-1 domain interface, and the β(3)-N654 N-glycan at the β(3)-β-tail and α(IIb)-calf-2 domain interface positively regulate the activation of both α(IIb)β(3) and α(V)β(3) integrins. In contrast, removal of the β(3)-N371 N-glycan near the β(3) hybrid and I-EGF3 interface, or the β(3)-N452 N-glycan at the I-EGF1 domain rendered β(3) integrin more active than the wild type. We identified one unique N-glycan at the βI domain of β(1) subunit that negatively regulates α(5)β(1) activation. Our study suggests that the bulky N-glycans influence the large-scale conformational rearrangement by potentially stabilizing or destabilizing the domain interfaces of integrin.