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 octoculata was performed by reversed-phase high performance liquid chromatography combined with both enzyme-linked immunosorbent and dot immunobinding assays for oxytocin. The amino acid sequence of the purified peptide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was established by Edman degradation and confirmed by electrospray mass spectrometry measurement. When injected in leeches, purified or synthetic peptides exert an anti-diuretic effect, the most effective ranged between 10 pmol and 1 nmol. They provoked an uptake of water 1-2 h post-injection. Furthermore, electrophysiological experiments conducted in the leech Hirudo medicinalis revealed an inhibition of the potency of Na+ conductances of leech skin by this peptide. Immunocytochemical studies with an antiserum against synthetic oxytocin-like molecule provided the cytological basis for existence of a neuropeptide, since large amounts of immunoreactive neurons were detected in the central nervous systems of E. octoculata. The purified molecule is both different to peptides of the oxytocin/vasopressin family and is a novel neuropeptide in the animal kingdom. It was named the leech osmoregulator factor (LORF). An identification of the proteins immunoreactive to an antiserum against oxytocin performed at the level of both central nervous systems extracts and in vitro central nervous system-translated RNA products indicated that in the two cases, a single protein was detected. These proteins with a molecular masses of, respectively, approximately 34 kDa (homodimer of 17 kDa) for the central nervous systems extracts and approximately 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.
Salzet
M
M
Laboratoire de Phylogénie moléculaire des Annélides ER 87 CNRS, Groupe de Neuroendocrinologie des Hirudinées, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq Cédex, France.
Bulet
P
P
Weber
W M
WM
Clauss
W
W
Verger-Bocquet
M
M
Malecha
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002490:Central Nervous System / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D007150:Immunohistochemistry; D007865:Leeches / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry* / Q000302:isolation & purification; D009842:Oligopeptides / Q000737:chemistry* / Q000302:isolation & purification; D009995:Osmosis
C099554:LORF protein, Erpobdella octoculata; D009479:Neuropeptides; D009842:Oligopeptides