title: Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation.
abstract: Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation.
authors.affiliation: Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Chinestra
authors.affiliation: Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France; Université Toulouse III-Paul Sabatier, Faculté des Sciences Pharmaceutiques, Toulouse, France.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Olichon
authors.affiliation: Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France; Université Toulouse III-Paul Sabatier, Faculté des Sciences Pharmaceutiques, Toulouse, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Medale-Giamarchi
authors.affiliation: Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France; Université Toulouse III-Paul Sabatier, Faculté des Sciences Pharmaceutiques, Toulouse, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Lajoie-Mazenc
authors.affiliation: Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France; Université Toulouse III-Paul Sabatier, Faculté des Sciences Pharmaceutiques, Toulouse, France.
authors.forename: Rémi
authors.initials: R
authors.lastname: Gence
authors.affiliation: Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Inard
authors.affiliation: CRCT, plateau de protéomique, Toulouse, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Ligat
authors.affiliation: Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Faye
authors.affiliation: Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France; Université Toulouse III-Paul Sabatier, Faculté des Sciences Pharmaceutiques, Toulouse, France; Institut Claudius Regaud, Toulouse, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Favre
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D061505:Cell Surface Display Techniques; D004789:Enzyme Activation; D015723:Gene Library; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020743:rhoB GTP-Binding Protein / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
chemical_list: D057127:Single-Chain Antibodies; D020743:rhoB GTP-Binding Protein