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 alpha and 8 beta integrin subunits. Crystal structures of alpha(IIb)beta(3) and alpha(V)beta(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 beta(3) integrin activation. We found that the N-glycan site, beta(3)-N320 at the headpiece and leg domain interface positively regulates alpha(IIb)beta(3) but not alpha(V)beta(3) activation. The beta(3)-N559 N-glycan at the beta(3)-I-EGF3 and alpha(IIb)-calf-1 domain interface, and the beta(3)-N654 N-glycan at the beta(3)-beta-tail and alpha(IIb)-calf-2 domain interface positively regulate the activation of both alpha(IIb)beta(3) and alpha(V)beta(3) integrins. In contrast, removal of the beta(3)-N371 N-glycan near the beta(3) hybrid and I-EGF3 interface, or the beta(3)-N452 N-glycan at the I-EGF1 domain rendered beta(3) integrin more active than the wild type. We identified one unique N-glycan at the beta I domain of beta(1) subunit that negatively regulates alpha(5)beta(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.