title: The A-domain of beta 2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF.
abstract: The A-domain is a approximately 200-amino acid peptide present within structurally diverse proadhesive proteins including seven integrins. A recombinant form of the A-domain of beta 2 integrins CR3 and LFA-1 has been recently shown to bind divalent cations and to contain binding sites for protein ligands that play essential roles in leukocyte trafficking to inflammatory sites, phagocytosis and target cell killing. In this report we demonstrate that the neutrophil adhesion inhibitor, NIF produced by the hookworm Ancyclostoma caninium is a selective CD11b A-domain binding protein. NIF bound directly, specifically and with high affinity (Kd of approximately 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 antibody. No binding was observed to rCD11aA. The NIF-r11bA interaction required divalent cations, and was absent when the mutant r11bA D140GS/AGA (that lacks divalent cation binding capacity) was used. The NIF binding 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 with similar affinity in resting and activated human neutrophils, suggesting 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 opsonized particles. NIF thus represents the first example of a disintegrin that targets the integrin A-domain, and is likely to be used by the hookworm to evade the host's inflammatory response. The unique structure of NIF, which lacks a disintegrin motif, emphasizes basic structural differences in antagonists targeting A+ and A- integrins, that should be valuable in drug design efforts aimed at generating novel therapeutics. Identification of the region in NIF mediating A-domain binding should also be useful in this regard, and may, as in the case of disintegrins, unravel a new structural motif with cellular counterparts mediating important physiologic functions.
authors.affiliation: Department of Medicine, Massachusetts General Hospital, Charlestown 02129.
authors.forename: P
authors.initials: P
authors.lastname: Rieu
authors.forename: T
authors.initials: T
authors.lastname: Ueda
authors.forename: I
authors.initials: I
authors.lastname: Haruta
authors.forename: C P
authors.initials: CP
authors.lastname: Sharma
authors.forename: M A
authors.initials: MA
authors.lastname: Arnaout
mesh_terms: D000595:Amino Acid Sequence; D000722:Ancylostoma / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D018821:CD18 Antigens; D002448:Cell Adhesion / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D008565:Membrane Proteins; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010587:Phagocytosis / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D018821:CD18 Antigens; D006023:Glycoproteins; D015801:Helminth Proteins; D016023:Integrins; D016177:Macrophage-1 Antigen; D008565:Membrane Proteins; D008670:Metals; D011994:Recombinant Proteins; C086581:NIF protein, Ancylostoma caninum