title: 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 γδ. 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 β chain, the second group bound to the αβ subunit mostly in a conformation-dependent manner, the third group recognized the γδ subunit only when separated from the αβ subunit, whereas a fourth group interacted with the γδ 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 γδ subunit upon binding to the integrin α2 A-domain at both the molecular and cellular levels. After dissociation, the γδ 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 γδ subunits within rhodocetin is mostly mediated by charged residues, which causes the two dissociated subunits to have hydrophilic surfaces.
authors.affiliation: Excellence Cluster Cardio-Pulmonary System, Center for Molecular Medicine, Vascular Matrix Biology, Frankfurt University Hospital, Frankfurt am Main 60590, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Bracht
authors.forename: Flávia
authors.initials: F
authors.lastname: Figueiredo de Rezende
authors.forename: Jörg
authors.initials: J
authors.lastname: Stetefeld
authors.forename: Lydia M
authors.initials: LM
authors.lastname: Sorokin
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Eble
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D003435:Crotalid Venoms / Q000737:chemistry* / Q000276:immunology*; D000939:Epitopes; D057927:Hydrophobic and Hydrophilic Interactions; D038982:Integrin alpha2beta1 / Q000276:immunology* / Q000378:metabolism*; D037181:Lectins, C-Type / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D000939:Epitopes; D038982:Integrin alpha2beta1; D037181:Lectins, C-Type; D021122:Protein Subunits; C119730:rhodocetin