Monoclonal antibodies reveal the alteration of the rhodocetin structure upon alpha2beta1 integrin binding
Résumé
The alpha2beta1 integrin antagonist rhodocetin from Calloselasma rhodostoma is a heterotetrameric C-type lectin-related protein (CLRP) consisting of four distinct chains, alpha, beta, gamma, and delta. Via their characteristic domain-swapping loops, the individual chains form two subunits, alpha beta and gamma delta. To distinguish the four chains which share similar molecular masses and high sequence homologies, we generated 11 monoclonal antibodies (mAbs) with different epitope specificities. Four groups of distinct mAbs were generated: the first targeted the rhodocetin beta chain, the second group bound to the alpha beta subunit mostly in a conformation-dependent manner, and the third group recognized the gamma delta subunit only when separated from the alpha beta subunit, while a fourth group interacted with the gamma delta subunit both in the heterotetrameric molecule and complexed with the integrin alpha2 A-domain. Using the specific mAbs, we showed that the rhodocetin heterotetramer dissociates into the alpha beta and gamma delta subunit upon binding to the integrin alpha2 A-domain at both molecular and cellular levels. After dissociation, the gamma delta subunit firmly interacts with the alpha2beta1 integrin thereby blocking it, while the rhodocetin alpha beta is released from the complex. The small molecular interface between the alpha beta and gamma delta subunit within rhodocetin is mostly mediated by charged residues, which renders the two dissociated subunits with hydrophilic surfaces.
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