Cell
Volume 162, Issue 1, 2 July 2015, Pages 198-210
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Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation

https://doi.org/10.1016/j.cell.2015.05.046Get rights and content
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Highlights

  • Sequence-independent monoclonal antibodies against phosphohistidine were developed

  • pHis mAbs are isomer specific (1-pHis or 3-pHis) and do not bind pTyr

  • pHis mAbs can be used in immunoblotting, IF, and immunoaffinity purification

  • Immunofluorescence reveals roles for 1-pHis and 3-pHis in phagocytosis and mitosis

Summary

Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.

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