Structural and functional characterization of EMF10, a heterodimeric disintegrin from Eristocophis macmahoni venom that selectively inhibits alpha 5 beta 1 integrin

alpha 5 beta 1, a major fibronectin receptor, is a widely distributed integ rin that is essential for cell growth and organ development. Here, we descr ibe a novel heterodimeric disintegrin named EMF10, isolated from the Eristo cophis macmahoni venom, that is an extremely potent and selective inhibitor of alpha 5 beta 1. EMF10 inhibited adhesion of cells expressing alpha 5 be ta 1 to fibronectin (IC50 = 1-4 nM) and caused expression of a ligand-induc ed binding site (LIBS) on the beta 1 subunit of alpha 5 beta 1 integrin. It partially inhibited adhesion of cells expressing alpha II beta 3, alpha v beta 3, and alpha 4 beta 1 to appropriate Ligands only at concentration hig her than 500 nM. Guinea pig megakaryocytes expressing alpha 5 beta 1 adhere d to immobilized EMF10 and showed extensive spreading and cytoskeletal mobi lization. As determined by electrospray mass spectrometry, EMF10 is compose d of two species with molecular masses of 14 575 and 14 949 Da, respectivel y. EMF10 is a heterodimer containing two subunits: EMF10A (M-r 7544 Da) and EMF10B (M-r 7405 and 7032 Da) linked covalently by S-S bonds. Subunit B sh owed heterogeneity and may be present as EMF10B1 (M-r 7032) and EMF10B2 (M- r, 7405). In putative hairpin loops, EMF10A and EMF10B contained CKKGRGDNLN DYC and CWPAMGDWNDDYC motifs, respectively. The reduced and alkylated subun it B of EMF10 inhibited adhesion of K562 cells to fibronectin in a dose-dep endent, saturable manner with IC50 of 3 mu M. The synthetic, cyclic CKKGRGD NLNDYC and CWPAMGDWNDDYC peptides expressed their inhibitory activity in th e same system with IC50 of 100 mu M. We propose that alpha 5 beta 1 recogni tion of EMF10 is associated with the MGDW motif located in a putative hairp in loop of the B subunit and that the expression of activity may also depen d an the RGDN motif in the subunit A and on the C-termini of both subunits.