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Research Article Open access CC BY 3.0

Role of the Trp-disulfide Triads in the UV Light Induced Degradation of a Monoclonal Antibody scFv

Eszter Illyés, Stephanie Staelens, Vanhooren ., Hans Deckmyn, Ignace Hanssens, Zsuzsa Majer

International Journal of Biochemistry Research & Review · pp. 367–385 · Published 16 Apr 2014

10.9734/IJBCRR/2014/8453

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.

Immunoglobulin Trp-Phe mutants tryptophan fluorescence disulfide bonds photolysis aggregation scFv

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