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.