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

Abstract

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 _IIbβ ₃ and α _Vβ 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 _IIbβ ₃ but not α _Vβ 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 _IIbβ ₃ and α _Vβ 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 ₅β 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.