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Generation of an alpaca-derived nanobody recognizing γ-H2AX.

Rajan M, Mortusewicz O, Rothbauer U, Hastert FD, Schmidthals K, Rapp A, Leonhardt H, Cardoso MC - FEBS Open Bio (2015)

Bottom Line: In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody.We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function.These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technische Universitaet Darmstadt, Germany.

ABSTRACT
Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

No MeSH data available.


Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the γ-H2AX-3 chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.
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f0015: Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the γ-H2AX-3 chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.

Mentions: To determine why the chromobody is recruited to sites of DNA damage in the presence of the overexpressed XRCC1, mutants of XRCC1 were developed (Fig. 3). XRCC1 has been found to have multiple phosphorylation sites containing serine residues (Fig. 3A), which are modified by casein kinase 2, a highly conserved protein serine/threonine kinase. Hence, we used multi-phospho site point mutants as well as a deletion mutant containing only the N-terminal domain excluding most of the phosphorylation sites. These mutants were compared with the full length XRCC1 (Fig. 3) to test for alternative phospho epitope recognition. The XRCC1 CKM mutant retaining some of the phosphorylation sites showed only a mild difference in the recruitment kinetics of the γ-H2AX-3 chromobody, when compared to XRCC1 full length. The XRCC1-NTD mutant, on the other hand, could no longer support γ-H2AX-3 chromobody recruitment. This was further substantiated by comparing the maximal accumulation of both proteins (Fig. 3F). Hence, it could be concluded that the chromobody’s recruitment is proportional to the XRCC1 accumulation and XRCC1 recruitment is slightly affected by the phosphosite mutation and completely affected by the deletion mutation. This indicates that either alternative XRCC1 phospho-epitope recognition allows the chromobody to accumulate at damage sites or a phospho-protein whose accumulation is dependent on XRCC1.


Generation of an alpaca-derived nanobody recognizing γ-H2AX.

Rajan M, Mortusewicz O, Rothbauer U, Hastert FD, Schmidthals K, Rapp A, Leonhardt H, Cardoso MC - FEBS Open Bio (2015)

Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the γ-H2AX-3 chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588710&req=5

f0015: Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the γ-H2AX-3 chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.
Mentions: To determine why the chromobody is recruited to sites of DNA damage in the presence of the overexpressed XRCC1, mutants of XRCC1 were developed (Fig. 3). XRCC1 has been found to have multiple phosphorylation sites containing serine residues (Fig. 3A), which are modified by casein kinase 2, a highly conserved protein serine/threonine kinase. Hence, we used multi-phospho site point mutants as well as a deletion mutant containing only the N-terminal domain excluding most of the phosphorylation sites. These mutants were compared with the full length XRCC1 (Fig. 3) to test for alternative phospho epitope recognition. The XRCC1 CKM mutant retaining some of the phosphorylation sites showed only a mild difference in the recruitment kinetics of the γ-H2AX-3 chromobody, when compared to XRCC1 full length. The XRCC1-NTD mutant, on the other hand, could no longer support γ-H2AX-3 chromobody recruitment. This was further substantiated by comparing the maximal accumulation of both proteins (Fig. 3F). Hence, it could be concluded that the chromobody’s recruitment is proportional to the XRCC1 accumulation and XRCC1 recruitment is slightly affected by the phosphosite mutation and completely affected by the deletion mutation. This indicates that either alternative XRCC1 phospho-epitope recognition allows the chromobody to accumulate at damage sites or a phospho-protein whose accumulation is dependent on XRCC1.

Bottom Line: In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody.We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function.These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technische Universitaet Darmstadt, Germany.

ABSTRACT
Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

No MeSH data available.