Structural and functional characterization of EMF10, a heterodimeric disintegrin from Eristocophis macmahoni venom that selectively inhibits α5β1 integrin

alpha 5 beta 1, a major fibronectin receptor, is a widely distributed integrin that is essential for cell growth and organ development. Here, we describe a novel heterodimeric disintegrin named EMF10, isolated from the Eristocophis macmahoni venom, that is an extremely potent and selective inhibitor of alpha 5 beta 1. EMF10 inhibited adhesion of cells expressing alpha 5 beta 1 to fibronectin (IC50 = 1-4 nM) and caused expression of a ligand-induced 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 higher than 500 nM. Guinea pig megakaryocytes expressing alpha 5 beta 1 adhered to immobilized EMF10 and showed extensive spreading and cytoskeletal mobilization. As determined by electrospray mass spectrometry, EMF10 is composed of two species with molecular masses of 14 575 and 14 949 Da, respectively. 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 showed heterogeneity and may be present as EMF10B1 (M-r 7032) and EMF10B2 (M-r, 7405). In putative hairpin loops, EMF10A and EMF10B contained CKKGRGDNLNDYC and CWPAMGDWNDDYC motifs, respectively. The reduced and alkylated subunit B of EMF10 inhibited adhesion of K562 cells to fibronectin in a dose-dependent, saturable manner with IC50 of 3 mu M. The synthetic, cyclic CKKGRGDNLNDYC and CWPAMGDWNDDYC peptides expressed their inhibitory activity in the same system with IC50 of 100 mu M. We propose that alpha 5 beta 1 recognition of EMF10 is associated with the MGDW motif located in a putative hairpin loop of the B subunit and that the expression of activity may also depend an the RGDN motif in the subunit A and on the C-termini of both subunits.