Structural Characterization of a Novel Neuropeptide from the Central Nervous System of the Leech Erpobdella octoculata
THE LEECH OSMOREGULATOR FACTOR (*)
- Michel Salzet(1)(§),
- Phillipe Bulet(2),
- Wolf-Michael Weber(3),
- Wolfgang Clauss(3),
- Martine Verger-Bocquet(1) and
- Jean Malecha(1)
- From the (1)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, the
- (2)Institut de Biologie Moléculaire et Cellulaire, UPR 9022 CNRS, 15 rue Descartes, F-67084 Strasbourg Cédex, France, and the
- (3)Institut für Tierphysiologie, Justus-Liebig-Universität, Giessen, D-35392 Giessen, Germany
- § To whom correspondence should be addressed. Tel.: 33-2043-4054; Fax: 33-2043-4054.
Abstract
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, 
34 kDa (homodimer of 17 kDa) for the central nervous systems extracts and 
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.
Footnotes
-
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- CNS
-
central nervous system
- HPLC
-
high pressure liquid chromatography
- DIA
-
dot immunobinding assay
- ELISA
-
enzyme-linked immunosorbent assay
- HPGPC
-
high performance gel permeation chromatography
- PLGa
-
Pro-Leu-glycinamide
- OT
-
oxytocin
- a-OT
-
anti-oxytocin
- LORF
-
leech osmoregulator factor
- VP
-
vasopressin
- PAGE
-
polyacrylamide gel electrophoresis
- SG
-
segmental ganglia.
-
- Received October 2, 1995.
- Revision received December 27, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
