Abstract
Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.
Highlights
To generate methylsite-specific antibodies, rabbits were immunized with a MAP3K2 peptide sequence surrounding trimethylated Lys260 (Fig. 1, A–B), N-terminally conjugated to keyhole limpet hemocyanin (KLH)
We created antibodies that recognize the peptide antigen in a methylation-dependent manner by using rabbit immunization followed by antibody library construction and phage display-based selection
Traditional rabbit monoclonal antibody (mAb) platforms rely on hybridoma technology or B cell cloning to generate lead candidates and these systems suffer from limited library sizes (104–106, compared with 1010–1011 in phage display)
Summary
To generate methylsite-specific antibodies, rabbits were immunized with a MAP3K2 peptide sequence surrounding trimethylated Lys260 (Fig. 1, A–B), N-terminally conjugated to keyhole limpet hemocyanin (KLH). The F9 structure showed that the methylated lysine was completely thrown out from the cage at several time points during the simulations, and the antibody retained the peptide only through interaction with the C terminus (Fig. S8) This exclusion of the methylated lysine residue from the aromatic cage was confirmed by the calculation of the solvent accessible surface area of the methylated lysine residue (Fig. S9). This result appears to be concordant with the SPR results, which showed that C9, E6, and D6 have higher preference toward methylated peptide compared with F9, indicating that our simulations appropriately recaptures antigen recognition observed in the in vitro experiments. The results showing large fluctuation of RMSD values in the apo structures indicate that the relative position of aromatic residues to each other continuously changed; i.e., the cage collapsed in the absence of peptide antigen, suggesting that the aromatic cages would stably form only in the presence of the antigen even in the C9 and D6 antibodies
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
