Resurrecting an antibody against an alternative conformation of cytochrome c: the recombinant mAb 1D3

Cytochrome c can undergo reversible conformational changes under biologically-relevant conditions including oxidative post-translational modifications, interactions with phospholipids and electric fields. Revealing the existence and relevance of these alternative cytochrome c proteoforms at the cell and tissue level is challenging and has benefitted from immunochemical approaches. Indeed, a monoclonal antibody that recognizes a major alternative conformation of cytochrome c, 1D3, was previously developed and characterized. The alternative conformation involves the destabilization of the Ω-loops encompassing residues 70 to 85 and 40 to 57 and the displacement of the Met80 heme ligand. However, the 1D3 producing hybridoma was lost and several attempts to reproduce it failed. Thus, with the remaining amounts of mAb 1D3 we aimed to “resurrect” 1D3 by the generation of a recombinant version of it. We employed the Valens algorithm for proteomic sequencing of 1D3. Briefly, the antibody was digested with multiple proteases and analyzed via LC-MS/MS using multiple fragmentation modes. By combining de novo peptide sequencing and homology mapping, we were able to obtain full-length sequences for the heavy and light chains of 1D3. Gene fragments representing the predicted heavy and light chain variable domains were synthesized using gBLOCK technology (IDT Inc., http://www.idtdna.com/). These were cloned into the heavy and light chain frameworks, respectively, using standard molecular biology techniques. Expression of light and heavy chains of 1D3 and its molecular assembly in HEK293 and CHO cells resulted in a functional antibody as tested by competitive ELISA with, for example, nitrotyrosine-containing cytochrome c species (NO274Tyr-Cyt c), known to adopt an alternative conformation. The recombinant 1D3 has the same specificity and affinity as the “original” 1D3 and is being successfully applied to various cellular models; enhanced levels of the 1D3-detectable alternative conformation of cytochrome c and their nuclear translocation are observed under conditions of altered cell homeostasis.