Monoclonal antibodies reveal the alteration of the rhodocetin structure upon α2β1 integrin binding

Abstract

The α2β1 antagonist rhodocetin from Calloselasma rhodostoma is a heterotetrameric CLRP (C-type lectin-related protein) consisting of four distinct chains, α, β, γ and δ. Via their characteristic domain-swapping loops, the individual chains form two subunits, αβ and γ δ. To distinguish the four chains which share similar molecular masses and high sequence homologies, we generated 11 mAbs (monoclonal antibodies) with different epitope specificities. Four groups of distinct mAbs were generated: the first targeted the rhodocetin βchain, the second group bound to the αβ subunit mostly in a conformation-dependent manner, the third group recognized the γ δ subunit only when separated from the αβ subunit, whereas a fourth group interacted with the γ δ subunit both in the heterotetrameric molecule and complexed with the integrin α2 A-domain. Using the specific mAbs, we have shown that the rhodocetin heterotetramer dissociates into the αβ and γ δ subunit upon binding to the integrin α2 A-domain at both the molecular and cellular levels. After dissociation, the γ δ subunit firmly interacts with the α2β1 integrin, thereby blocking it, whereas the rhodocetin αβ subunit is released from the complex. The small molecular interface between the αβ and γ δ subunits within rhodocetin is mostly mediated by charged residues, which causes the two dissociated subunits to have hydrophilic surfaces. © The Authors Journal compilation © 2011 Biochemical Society.