Protein phosphorylation is central to many cellular processes.1
Consequently, tools that permit phospho-protein levels to be monitored as a function of cell type or state have proven invaluable in biomedical research.2,8
While strategies are now well established for cataloguing pSer, pThr and pTyr levels in native proteomes, little progress has been made in the analogous study of protein histidine phosphorylation. The latter represents a very serious analytical blind-spot, given that pHis plays a critical role in signaling cascades in bacteria and lower eukaryotes and occurs commonly in metabolic enzymes in all cell types.3,7
At the heart of this problem lies the chemical nature of the pHis modification. The intrinsic instability of the phosphoramidate linkage within pHis has frustrated the development of even the most basic biochemical tools, in particular a modification specific antibody. Thus, our ability to study the extent and dynamics of the pHis modification in native proteomes has lagged far behind that of other phospho-protein modifications.3
In this study, we employed a pHis mimetic approach to develop the first pan-specific antibody against the modification. This research tool was used to directly detect pHis-containing proteins through ELISA, dot blot, Western blot and immunoprecipitation experiments. Our pan antibody, utilized in conjunction with mass spectrometry, also enabled the detection and identification of endogenously phosphorylated pHis proteins from cell lysates.
The potential utility of anti-pHis antibodies has long been appreciated and efforts to develop such reagents date back over ten years.4
The short half-life of native phosphohistidine in serum makes it of little value as a hapten for raising modification-specific antibodies.4,7
Faced with this situation, alternative strategies based on the use of more stable pHis analogs or mimetics have been considered.3
These include the use of the more stable thiophosphorylated analog of pHis, which can be generated chemically or enzymatically, the latter using ATP-γ-S.10,24
While pHis-specific antibodies have yet to be reported using this approach, the unique nucleophilicity of the thiophosphoryl group can be used to install a secondary immunogenic site via attachment of a suitable hapten.10
Replacement of the labile phosphoramidate linkage with a more inert chemical bond has also been explored. Stable phosphonate linkages can be incorporated into various five-membered heterocyclic frameworks, leading to a range of presumptive pHis mimetics.11,12,13,25
Previously, we described one such mimetic based on a triazole framework, phosphoryltriazolylalanine.11
Antibodies raised against this unnatural residue in the context of a specific peptide sequence, cross-react with native pHis in the same sequence context. This study is important since it establishes that pHis mimetics can be used in antibody development. Still, it remained unclear whether sequence-independent pHis antibodies, which are far more valuable for proteomics studies, could be generated using this mimetic approach. Indeed, it has been questioned whether phosphoryltriazole-based molecules can be used to generate sequence-independent pHis antibodies since such antibodies should rely solely on the recognition of the pHis sidechain, which modeling indicates differs, albeit subtly, in its electrostatic surface potentials from the phosphoryltriazole.11,12
In this report, we showed that it is indeed possible to develop pan pHis antibodies using phosphoryltriazole as the hapten. We speculate that electrostatic differences in the imidazole and triazole ring systems in pHis and the mimetic, respectively, are outweighed, at least in terms of antibody recognition, by the anionic phosphoryl group common to both.
One limitation of the current pan-pHis antibody is its mild cross-reactivity with pTyr residues. Fortunately, pHis and pTyr can be biochemically distinguished from each other based on the stability towards acid or hydroxylamine (Supplementary Figs. 4 and 19
). In addition, we observed that commercial pan pTyr antibodies show little affinity towards pHis proteins. Taking advantage of the affinity differences, we were able to selectively immunodeplete pTyr-containing proteins in the presence of pHis proteins. Parenthetically, this result argues against the idea of using a pTyr antibody as a surrogate for the pHis antibody.26,27
A practical drawback of our antibody is its finite supply, being a polyclonal antibody. Generation of monoclonal antibodies, potentially carried out with next-generation pHis analogs to minimize pTyr cross-reactivity, will address those issues in the future.
The availability of pan-pHis antibodies opens the way to a number of investigations both biochemical and cell-based. Monitoring protein histidine phosphorylation in a test tube currently involves the use of radiolabeling, with for example 32
P-ATP, which has its attendant safety and cost (i.e. disposal) issues.28
As demonstrated herein, the availability of a robust immuno-detection platform overcomes many of these problems, potentially allowing higher throughput studies of biochemical systems where pHis is involved, for example two-component signaling.29
Complementing the in vitro
applications, we imagine that pan-pHis antibodies will also find broad use in cell-based studies by allowing global pHis levels to be monitored in different contexts, for instance various cell types or as a function of some cellular perturbation. As an illustration of the latter, we were able to probe global protein histidine phosphorylation in E. coli
under different metabolic states. This led to the discovery that nitrogen availability and carbon source dramatically affects the pHis levels in a number of proteins. Among those proteins, PEP synthase, PpsA, was found to undergo rapid dephosphorylation in vivo
upon the relief of nitrogen limitation. Subsequent in vitro
PpsA phosphorylation and dephosphorylation assays showed that the dephosphorylation to form PEP is inhibited by α-KG, a key metabolite tightly regulated by the nitrogen availability. These observations clarified a long-standing question about the mechanism of PpsA inhibition by α-KG. PpsA is a key enzyme in E. coli
metabolic engineering efforts geared towards channeling carbon flux for production of shikimic acid and downstream aromatic metabolites.30
Thus, we believe our findings on the regulation of PEP synthase activity will provide further insights on the bioengineering of the bacterial central metabolic pathway.
In conclusion, we have successfully developed the first pan-pHis antibody and used this reagent in variety of assays that commonly employ modification-specific antibodies. These applications of our pan-pHis antibody help address the long-standing dearth of adequate tools in histidine phosphorylation research, which has been the major roadblock to progress in the field. Further studies, including the investigation of histidine phosphorylation in mammalian systems and antibody development for the other pHis isomer, are currently underway.