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Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation
Dorothea Höpfner1,2, Joel Fauser1,2, Marietta S Kaspers1
1Department of Biochemistry and Signaltransduction, University Medical Center Hamburg-Eppendorf (UKE), Martinistr. 52, 20246 Hamburg, Germany.
Abstract:
AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recently, a role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis has emerged, driving the demand for universal tools to study this modification. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse that are capable of protein backbone-independent recognition of AMPylation, in denatured (western blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, these anti-AMP mABs will further advance the current understanding of AMPylation and the spectrum of modified targets.
Insights
Researchers developed novel monoclonal anti-adenosine monophosphate (AMP) antibodies. These tools detect AMPylation, a key post-translational modification, aiding in understanding cellular processes and identifying new cancer targets.
Area of Science:
- Biochemistry and Molecular Biology
- Immunology
- Cancer Research
Background:
- AMPylation is a post-translational modification involving adenosine monophosphate (AMP) attachment.
- AMPylation plays a crucial role in regulating infection and maintaining cellular homeostasis.
- There is a growing need for universal tools to study AMPylation due to its emerging significance.
Purpose of the Study:
- To develop and characterize novel monoclonal antibodies (mAbs) for detecting AMPylation.
- To assess the performance of these antibodies in various biochemical applications.
- To explore the AMPylation patterns in cancer cell lines using the developed mAbs.
Main Methods:
- Generation of three mouse monoclonal anti-AMP antibodies (mAbs).
- Validation of mAbs using Western blot, ELISA, and Immunoprecipitation (IP).
- Application of mAbs to analyze AMPylation in diverse cancer cell lines.
Main Results:
- Developed highly sensitive and specific anti-AMP mAbs capable of protein backbone-independent AMPylation recognition.
- Demonstrated antibody efficacy in both denatured (Western blot) and native (ELISA, IP) conditions.
- Revealed a broad and previously undescribed AMPylation pattern across various cancer cell lines.
Conclusions:
- The novel anti-AMP mAbs are superior tools for studying AMPylation compared to existing methods.
- These antibodies facilitate new target identification and validation in AMPylation research.
- The findings advance the understanding of AMPylation's role and its diverse targets, particularly in cancer.
