Functional Regulation of Integrin Affinity by Divalent Cations, Glycosylation and Simvastatin

Integrins are heterodimeric metalloproteins cell surface receptors that are expressed on every nu- cleated cell in the body. Integrin dysfunctions are implicated in many diseases—including bleeding disorders, inflammation, and cancer. Thus integrins are critical and proven drug targets. Integrins are unique in their bidirectional signaling and the transmission of the signal between the α and β subunits. β2 integrins are composed of a variable α (αL, αM, αX, αD) and a constant β2 subunit and are exclusively expressed on leukocytes. The α subunit contains an inserted I domain with a metal- dependent adhesion site (MIDAS) that acts as a ligand-binding site thereby defining the functional property of the integrin. Divalent cations are indispensable for integrin functions, stabilizing the integrin structure and regulate integrin-ligand interaction. In this study, the effect of divalent cations on integrin function was investigated for both αXβ2 and αDβ2 integrins. Locking the β2 I-domain into the binding-incompetent state using either inhibitory monoclonal mAb, TS1/18, or small-molecule inhibitor XVA143 enabled us to assess the effect of the divalent cations on the αI- domain. The tested divalent cations including Mg2+, Co2+, Cd2+, and Ni2+ induced an increase in the αI-domain affinity towards physiological ligands iC3b and fibrinogen. In this study, the effect of individual N-glycans on αXβ2 expression, conformation and affinity was investigated. This study suggests that individual N-glycan sites exhibit different roles on the affinity of the αXβ2, emphasizing the importance of studying the functional role of individual N-glycans in integrin-ligand affinity and conformation. Better understanding integrin conformation and ligand affinity could lead to improved ther- apeutics for the treatment of inflammatory diseases. In this study, we identify simvastatin as a competitive antagonist of αXβ2. Optimizing full length integrin purification is critical to investi- gate the structural and functional regulation of integrins in the presence of small molecules such as simvastatin. The cloning and purification strategies developed can be applied for that purpose.