Role of the Trp-disulfide Triads in the UV Light Induced Degradation of a Monoclonal Antibody scFv
Eszter Illyés
Laboratory for Chiroptical Structure Analysis, Institute of Chemistry, Eötvös Loránd University, Budapest, H-1518, Budapest 112, P.O. Box 32, Hungary
Stephanie Staelens
Laboratory for Thrombosis Research, Interdisciplinary Research Center, KU Leuven Campus Kortrijk, B-5300 Kortrijk, E. Sabbelaan 53, Belgium
Vanhooren .
Laboratory for Protein Biophysics, Interdisciplinary Research Center, KU Leuven Campus Kortrijk, B-5300 Kortrijk, E. Sabbelaan 53, Belgium
Hans Deckmyn
Laboratory for Thrombosis Research, Interdisciplinary Research Center, KU Leuven Campus Kortrijk, B-5300 Kortrijk, E. Sabbelaan 53, Belgium
Ignace Hanssens
Laboratory for Protein Biophysics, Interdisciplinary Research Center, KU Leuven Campus Kortrijk, B-5300 Kortrijk, E. Sabbelaan 53, Belgium
Zsuzsa Majer *
Laboratory for Chiroptical Structure Analysis, Institute of Chemistry, Eötvös Loránd University, Budapest, H-1518, Budapest 112, P.O. Box 32, Hungary
*Author to whom correspondence should be addressed.
Abstract
Aims: Proteins are targets for photodegradation due to absorption of incident light by endogenous chromophores, e.g aromatic side chains. In this work we study the role of Trp-disulfide triads in the light induced loss of immunoglobulin activity.
Study Design: We investigated a single chain variable fragment (scFv) of the Trp-disulfide triad containing monoclonal antibody 82D6A3. The scFv binds to von Willebrand factor (VWF) and upon illumination with near UV-B-light the scFv partially loses its binding capacity to VWF. In order to relate this observed degeneration to the specific Trp-disulfide triads, we mutated W35(VL) and W36(VH) which are in direct contact with the disulfide bridge of the VL and VH domain respectively, to Phe and compared the effects upon illumination of these mutants and the wild type scFv.
Methodology: We constructed and expressed three mutants, then tested the binding affinity of wild type and mutants to VWF. To study whether illumination caused protein fragmentation (rapture of disulfide bridge, structural changes, number of evolved thiols) we performed fluorescence spectroscopy, western blot and SDS-PAGE.
Results: Upon illumination with near UV-B-light the scFv partially lost its capacity to bind to VWF, indicating that the structure, orientation or the accessibility of the paratope is changed; while disulfide bonds were broken in the wild type and the monosubstituted mutants and dimers and multimers/higher aggregates were formed.
Conclusion: The results indicate that light induced excitation of W35(VL) and W36(VH) mediates photolysis of the vicinal disulfide bonds. And although the more distant W47(VH), W50(VH) and W108(VH) do not contribute to photolysis of the disulfide bonds, the simultaneous substitution of W35(VL) and of W36(VH) nevertheless did not protect the scFv’s affinity for VWF against illumination.
Keywords: Immunoglobulin, Trp-Phe mutants, tryptophan fluorescence, disulfide bonds, photolysis, aggregation, scFv