STRUCTURAL CHARACTERIZATION OF A NOVEL NEUROPEPTIDE FROM THE CENTRAL-NERVOUS-SYSTEM OF THE LEECH ERPOBDELLA-OCTOCULATA - THE LEECH OSMOREGULATOR FACTOR

Purification of a material immunoreactive to an antiserum against the C-terminal part of the oxytocin (Pro-Leu-Gly-amide) and present in the central nervous system of the Pharyngobdellid leech Erpobdella octocu lata was performed by reversed-phase high performance liquid chromatog raphy combined with both enzyme-linked immunosorbent and dot immunobin ding assays for oxytocin. The amino acid sequence of the purified pept ide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was established by Edman degrada tion and confirmed be electrospray mass spectrometry measurement. When injected in leeches, purified or synthetic peptides exert an antidiur etic effect, the most effective ranged between 10 pmol and 1 nmol. The y provoked an uptake of water 1-2 h post-injection. Furthermore, elect rophysiological experiments conducted in the leech Hirudo medicinalis revealed an inhibition of the potency of Na+ conductances of leech ski n by this peptide. Immunocytochemical studies with an antiserum agains t synthetic oxytocin-like molecule provided the cytological basis for existence of a neuropeptide, since large amounts of immunoreactive neu rons were detected ih the central nervous systems of E. octoculata. Th e purified molecule is both different to peptides of the oxytocin/vaso pressin family and is a novel neuropeptide in the animal kingdom, It w as named the leech osmoregulator factor (LORF). An identification of t he proteins immunoreactive to an antiserum against oxytocin performed at the level of both central nervous systems extracts and in vitro cen tral nervous system-translated RNA products indicated that in the two cases, a single protein was detected. These proteins with a molecular masses of, respectively, similar to 34 kDa (homodimer of 17 kDa) for t he central nervous systems extracts and similar to 19 kDa for in vitro central nervous system-translated RNA products were not recognized by the antiserum against MSEL- and VLDV-neurophysin (proteins associated to oxytocin and vasopressin), confirming that LORF did not belong to the oxytocin/vasopressin family.