| Summary: | Tachykinins are signalling peptides released by the processing of preprotachykinin precursors, which are subject to post-translational modifications including amidation. Tachykinins act on the three tachykinin receptors NKl, NK2 and NK3. Differential processing of the preprotachykinin precursors can lead to the formation of a diverse range of tachykinins (including extended forms). Our previous findings have shown increased proteolytic cleavage of the human TAC3 and TAC4 preprotachykinin precursors by the placenta in pathological conditions such as pre-eclampsia comparable to that found in the brain. This study has developed an experimental strategy for the capture and detection of tachykinins combining peptide extraction, enrichment by immunoaffinity purification, RP-HPLC separation and MALDI-TOF. The combined application of these methods in rat brain identified mHK-l as an amidated decapeptide and also captured and detected SP. The detection of a modified form of mHK-l in the brain matching the mass of an additional acetyl group (+42 a.m.u.) indicates the existence of a neuropeptide-specifie post-translational modification. The biological role of acetylation is to provide greater stability for the peptide and affinity of binding for its receptor. The methodology described in this research could be applied for the capture of tachykinins expressed in normal, pre-eclamptic and IUGR placentae, in order to investigate the changes that occur in precursor processing during disease states such as pre-eclampsia and to identify post-translational modifications. A parallel in silico analysis of the publicly accessible NCBI and Ensembl databases was conducted to identify tachykinin precursors. Multiple sequence alignment of retrieved preprotachykinin sequences was conducted and the phylogenetic relationship between the identified species investigated. Collectively, the results expand the number of known or predicted tachykinins and tachykinin gene-related peptides. Moreover, they separate the preprotachykinin precursors into three distinct groups. The analysis also sheds lights on the evolution of the tachykinin precursor cleavage sites (e.g. the N-terminal monobasic cleavage site of human EKA/B). Overall, this study has developed technologies for identifying tachykinin precursor post-translation modifications that may serve as a tool for determining different peptide physiologies between neuronal and peripheral tissues and different disease states.
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