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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.


Related in: MedlinePlus

Epitope unmasking by knocking down MDC1. (A) MDC1 binds to the tyrosine at the 142nd residue of phosphorylated H2AX. H2AX wild type (B) or knockout (C) MEFs, were transfected with the constructs indicated and microirradiated with 405 nm laser 24 h post transfection. Recruitment to damage sites was measured before and after damage. (D) Kinetics of recruitment of MDC1 in the presence and absence of H2AX is shown. (E) Rationale for epitope masking by MDC1. In (F) and (G) MDC1 siRNA (or control siRNA) plus the indicated constructs were transfected into HEK293 cells and cells were microirradiated as described in detail in the methods. The scale bar represents 5 μm. (H) Kinetics of recruitment of the γ-H2AX-3 chromobody in cells transfected with MDC1 siRNA and control siRNA. (I) The percentage of cells that showed γ-H2AX-3 chromobody recruitment with MDC1 siRNA and control siRNA is shown. (J) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (L) and (K) Recovery curves of MDC1-GFP expressed in HeLa cells at damaged and undamaged sites (± micro IR, respectively) and the corresponding t-half values. (M) and (K) Recovery curves of γ-H2AX-3 chromobody expressed in HEK293 cells upon MDC1 knockdown in damaged and undamaged sites (± micro IR, respectively) and corresponding t-half values. In all the recruitment and recovery kinetics curves, mean values were plotted and the error bars (shaded and lined) denote standard deviation.
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f0020: Epitope unmasking by knocking down MDC1. (A) MDC1 binds to the tyrosine at the 142nd residue of phosphorylated H2AX. H2AX wild type (B) or knockout (C) MEFs, were transfected with the constructs indicated and microirradiated with 405 nm laser 24 h post transfection. Recruitment to damage sites was measured before and after damage. (D) Kinetics of recruitment of MDC1 in the presence and absence of H2AX is shown. (E) Rationale for epitope masking by MDC1. In (F) and (G) MDC1 siRNA (or control siRNA) plus the indicated constructs were transfected into HEK293 cells and cells were microirradiated as described in detail in the methods. The scale bar represents 5 μm. (H) Kinetics of recruitment of the γ-H2AX-3 chromobody in cells transfected with MDC1 siRNA and control siRNA. (I) The percentage of cells that showed γ-H2AX-3 chromobody recruitment with MDC1 siRNA and control siRNA is shown. (J) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (L) and (K) Recovery curves of MDC1-GFP expressed in HeLa cells at damaged and undamaged sites (± micro IR, respectively) and the corresponding t-half values. (M) and (K) Recovery curves of γ-H2AX-3 chromobody expressed in HEK293 cells upon MDC1 knockdown in damaged and undamaged sites (± micro IR, respectively) and corresponding t-half values. In all the recruitment and recovery kinetics curves, mean values were plotted and the error bars (shaded and lined) denote standard deviation.

Mentions: As the immunoprecipitation experiments demonstrated that the γ-H2AX-3 chromobody recognized the γ-H2AX in cell lysates, the question was how this was apparently not the case in living cells. We hypothesized that cellular proteins may mask the epitope. The most likely candidate is MDC1 (mediator of DNA damage checkpoint protein 1). MDC1 recognizes and binds the tyrosine 142 residue of the phosphorylated histone H2AX (Fig. 4A–D) [23]. MDC1 being a ∼250 kDa large protein could, by binding to tyrosine 142, mask the serine at position 139. The binding of MDC1 is known to facilitate the loading of repair proteins and initiate the DNA repair process [24]. Hence MDC1 knock down experiments were performed to check if this allows for epitope recognition by the γ-H2AX-3 chromobody. HEK 293 cells were transfected with MDC1 siRNA and the knockdown of MDC1 was controlled by immunostaining the cells. At 72 h, the siRNA treated cells were microirradiated with a 405 nm laser as before. Mild recruitment of the γ-H2AX-3 chromobody could be observed at the irradiated sites in MDC1 siRNA treated cells but not in the control siRNA cells (Fig. 4E-I). Notably, this occurs in the absence of ectopically expressed XRCC1. This confirms that MDC1 binding in living cells competitively inhibits likely by steric hindrance the binding of the γ-H2AX specific nanobody. Additionally, MDC1 knock down lead also to a mild decrease in the γ-H2AX foci numbers per se, which indicates that MDC1 reinforces γ-H2AX formation [25], maybe by facilitating spreading of the H2AX phosphorylation along the chromatin surrounding the DSB [2]. The lower amount of γ-H2AX upon MDC1 knock down explains the modest maximum amount of γ-H2AX-3 chromobody accumulation by contributing to the reduction of its epitope.


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)

Epitope unmasking by knocking down MDC1. (A) MDC1 binds to the tyrosine at the 142nd residue of phosphorylated H2AX. H2AX wild type (B) or knockout (C) MEFs, were transfected with the constructs indicated and microirradiated with 405 nm laser 24 h post transfection. Recruitment to damage sites was measured before and after damage. (D) Kinetics of recruitment of MDC1 in the presence and absence of H2AX is shown. (E) Rationale for epitope masking by MDC1. In (F) and (G) MDC1 siRNA (or control siRNA) plus the indicated constructs were transfected into HEK293 cells and cells were microirradiated as described in detail in the methods. The scale bar represents 5 μm. (H) Kinetics of recruitment of the γ-H2AX-3 chromobody in cells transfected with MDC1 siRNA and control siRNA. (I) The percentage of cells that showed γ-H2AX-3 chromobody recruitment with MDC1 siRNA and control siRNA is shown. (J) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (L) and (K) Recovery curves of MDC1-GFP expressed in HeLa cells at damaged and undamaged sites (± micro IR, respectively) and the corresponding t-half values. (M) and (K) Recovery curves of γ-H2AX-3 chromobody expressed in HEK293 cells upon MDC1 knockdown in damaged and undamaged sites (± micro IR, respectively) and corresponding t-half values. In all the recruitment and recovery kinetics curves, mean values were plotted and the error bars (shaded and lined) denote standard deviation.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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f0020: Epitope unmasking by knocking down MDC1. (A) MDC1 binds to the tyrosine at the 142nd residue of phosphorylated H2AX. H2AX wild type (B) or knockout (C) MEFs, were transfected with the constructs indicated and microirradiated with 405 nm laser 24 h post transfection. Recruitment to damage sites was measured before and after damage. (D) Kinetics of recruitment of MDC1 in the presence and absence of H2AX is shown. (E) Rationale for epitope masking by MDC1. In (F) and (G) MDC1 siRNA (or control siRNA) plus the indicated constructs were transfected into HEK293 cells and cells were microirradiated as described in detail in the methods. The scale bar represents 5 μm. (H) Kinetics of recruitment of the γ-H2AX-3 chromobody in cells transfected with MDC1 siRNA and control siRNA. (I) The percentage of cells that showed γ-H2AX-3 chromobody recruitment with MDC1 siRNA and control siRNA is shown. (J) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (L) and (K) Recovery curves of MDC1-GFP expressed in HeLa cells at damaged and undamaged sites (± micro IR, respectively) and the corresponding t-half values. (M) and (K) Recovery curves of γ-H2AX-3 chromobody expressed in HEK293 cells upon MDC1 knockdown in damaged and undamaged sites (± micro IR, respectively) and corresponding t-half values. In all the recruitment and recovery kinetics curves, mean values were plotted and the error bars (shaded and lined) denote standard deviation.
Mentions: As the immunoprecipitation experiments demonstrated that the γ-H2AX-3 chromobody recognized the γ-H2AX in cell lysates, the question was how this was apparently not the case in living cells. We hypothesized that cellular proteins may mask the epitope. The most likely candidate is MDC1 (mediator of DNA damage checkpoint protein 1). MDC1 recognizes and binds the tyrosine 142 residue of the phosphorylated histone H2AX (Fig. 4A–D) [23]. MDC1 being a ∼250 kDa large protein could, by binding to tyrosine 142, mask the serine at position 139. The binding of MDC1 is known to facilitate the loading of repair proteins and initiate the DNA repair process [24]. Hence MDC1 knock down experiments were performed to check if this allows for epitope recognition by the γ-H2AX-3 chromobody. HEK 293 cells were transfected with MDC1 siRNA and the knockdown of MDC1 was controlled by immunostaining the cells. At 72 h, the siRNA treated cells were microirradiated with a 405 nm laser as before. Mild recruitment of the γ-H2AX-3 chromobody could be observed at the irradiated sites in MDC1 siRNA treated cells but not in the control siRNA cells (Fig. 4E-I). Notably, this occurs in the absence of ectopically expressed XRCC1. This confirms that MDC1 binding in living cells competitively inhibits likely by steric hindrance the binding of the γ-H2AX specific nanobody. Additionally, MDC1 knock down lead also to a mild decrease in the γ-H2AX foci numbers per se, which indicates that MDC1 reinforces γ-H2AX formation [25], maybe by facilitating spreading of the H2AX phosphorylation along the chromatin surrounding the DSB [2]. The lower amount of γ-H2AX upon MDC1 knock down explains the modest maximum amount of γ-H2AX-3 chromobody accumulation by contributing to the reduction of its epitope.

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.


Related in: MedlinePlus