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[摘要]：The alpha 2 beta 1 antagonist rhodocetin from Calloselasma rhodostoma is a heterotetrameric CLRP (C-type lectin-related protein) 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 mAbs (monoclonal antibodies) 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, the third group recognized the gamma delta subunit only when separated from the alpha beta subunit, whereas a fourth group interacted with the gamma delta subunit both in the heterotetrameric molecule and complexed with the integrin alpha 2 A-domain. Using the specific mAbs, we have shown that the rhodocetin heterotetramer dissociates into the alpha beta and gamma delta subunit upon binding to the integrin alpha 2 A-domain at both the molecular and cellular levels. After dissociation, the gamma delta subunit firmly interacts with the alpha 2 beta 1 integrin. thereby blocking it, whereas the rhodocetin alpha beta subunit is released from the complex. The small molecular interface between the alpha beta and gamma delta subunits within rhodocetin is mostly mediated by charged residues, which causes the two dissociated subunits to have hydrophilic surfaces. |
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