Vernier zone residue of 4 mouse subgroup II kappa light chains is a critical determinant for antigen recognition

H. de Haard, B. Kazemier, A. van der Bent, P. Oudshoorn, P. Boender, J.W. Arends, B. van Gemen

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    Background: During the conversion of murine monoclonal antibodies directed against the human chorionic gonadotropin (hCG) into bacterially expressed single chain fragments (scFv), we found a major reduction of binding activity upon introduction of a primer encoded mutation. Objectives: In this study we tried to determine which mutation was responsible and on what manner this mutation affected antigen binding (structural effect versus direct involvement of the residue in binding). Results: No binding could be detected, when the wild type residue methionine at position 4 within the Framework region 1 of the Vκ light chain was substituted by serine in two antibodies with a subgroup II kappa light chain. However, a similar replacement within an anti-hCG antibody with a subgroup IV kappa light chain and thereby having leucine as wild type residue, did not affect the binding characteristics. The mutant scFv's derived from both AB-s sensitive for substitution by serine never reacted with antigen in ELISA. Analysis with surface plasmon resonance revealed a residual binding only on a sensorchip with a high density coating of antigen; however, an increased dissociation, relative to that of the wild type scFv and the absence of reactivity in ELISA suggest a drastically altered affinity. Conclusion: A structural explanation for the changed binding characteristics can be the influence of the position 4 residue, as being a constituent of the Vernier zone, on the position of the CDR1 loop of Vκ, which might harbour residues that directly bind to antigen, or indirectly positions other variable loops of the binding pocket. An increased sensitivity for trypsin digestion supported the hypothesis of a local conformational change in the serine mutant of the subgroup II kappa containing antibody.
    Original languageUndefined/Unknown
    Pages (from-to)203-215
    Issue number3-4
    Publication statusPublished - 1999

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