Processing of the tachykinin precursor TAC3 in the human placenta and characterisation of the peptide product, neurokinin B (NKB)

• Main Author: Brayley, Kerensa
• Published: University of Reading 2005
• Subjects: 618
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485703

Excessive secretion of the peptide, neurokinin B (NKB) into the circulation during the third trimester of pregnancy has been reported in women with the gestational disease, pre-eclampsia (PE). Therefore, the objective of this work was to develop a clinic friendly two-site enzyme-linked immunosorbent assay (ELISA) to detect levels of NKB directly from plasma samples of pregnant women and to determine the mechanism's of precursor processing. !t was hoped to confIrm the link between elevated levels of this peptide and PE. Initial assays of pregnancy plasmas using the two-site NKB ELISA were inconclusive in determining a link, as were those conducted using an ELISA developed to the adjacent C-terminal portion of the precursor peptide. Consequently, extraction and purification ofNKB from the human placenta was undertaken, with separation on the basis of size and hydrophobicity. A commercial one-site competitive NKB radioimmunoassay was used at each stage to identify immunoreactive fractions and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) ,was performed on the purified samples. These experiments indicated that NKB extracted from the placenta had a higher molecular weight than NKB reported from the brain. The mass difference indicated one of two potential posttranslational modifications in the placenta of either a di-methylation or phosphocholine (PC) modification. These hypotheses were investigated through the development of specific two-site ELISAs. Putative processing mechanlsms were also explored through synthetic peptide digests with the enzyme carboxypeptidase B. Analysis of the digests via thin layer chromatography and MS proved that the dimethylation hampered exopeptidase processing. Quantitative PCR for the closely-related membrane-bound enzyme carboxypeptidase M, showed highest expression in the placenta. Synthetic NKB peptides carrying a dirnethylation however, had no biological activity either in in vitro ligand binding studies at the NK3 recepto~ or in in vivo models involving both rats and mice. Larger scale placental purification disproved the dimethylation theory and provided evidence for the PC modification thus far unprecedented in mammals, where pilot assays detected signals in both placental extracts and plasma samples. Collectively, these results provide evidence for the existence ofa unique placental NKB peptide, the structure of which has yet to be fully elucidated, as does the exact nature of the involvement ofthis peptide in PE.