The importance of N-glycosylation on β3 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.