THE A-DOMAIN OF BETA-2 INTEGRIN CR3 (CD11B CD18) IS A RECEPTOR FOR THE HOOKWORM-DERIVED NEUTROPHIL ADHESION INHIBITOR NIF/

The A-domain is a similar to 200-amino acid peptide present within str ucturally diverse proadhesive proteins including seven integrins. A re combinant form of the A-domain of beta 2 integrins CR3 and LFA-1 has b een recently shown to bind divalent cations and to contain binding sit es for protein ligands that play essential roles in leukocyte traffick ing to inflammatory sites, phagocytosis and target cell killing. In th is report we demonstrate that the neutrophil adhesion inhibitor, NIF p roduced by the hookworm Ancylostoma caninum is a selective CD11b A-dom ain binding protein. NIF bound directly, specifically and with high af finity (K-d of similar to 1 nM) to recombinant CD11b A-domain (r11bA). The binding reaction was characterized by rapid association and very slow dissociation, and was blocked by an anti-r11bA monoclonal antibod y. No binding was observed to rCD11aA. The NIF-r11bA interaction requi red divalent cations, and was absent when the mutant r11bA D140GS/AGA (that lacks divalent cation binding capacity) was used. The NIF bindin g site in r11bA was mapped to four short peptides, one of which being an iC3b binding site. The interaction of NIF with CR3 in intact cells followed similar binding kinetics to those with r11bA, and occurred wi th similar affinity in resting and activated human neutrophils, sugges ting that the NIF epitope is activation independent. Binding of NIF to CR3 blocked its ability to bind to its ligands iC3b, fibrinogen, and CD54, and inhibited the ability of human neutrophils to ingest serum o psonized particles. NIF thus represents the first example of a disinte grin that targets the integrin A-domain, and is likely to be used by t he hookworm to evade the hosts inflammatory response. The unique struc ture of NIF, which lacks a disintegrin motif, emphasizes basic structu ral differences in antagonists targeting A(+) and A(-) integrins, that should be valuable in drug design efforts aimed at generating novel t herapeutics. Identification of the region in NIF mediating A-domain bi nding should also be useful in this regard, and may, as in the case of disintegrins, unravel a new structural motif with cellular counterpar ts mediating important physiologic functions.