Brain Research 862 (2000) 162–170 www.elsevier.com / locate / bres Research report HPLC and electrospray ionization mass spectrometry as tools for the identification of APGWamide-related peptides in gastropod and bivalve mollusks: comparative activities on Mytilus muscles * ¨ ´ Joel Henry , Celine Zatylny, Pascal Favrel ´ Laboratoire de Biologie et Biotechnologies Marines, Universite de Caen, 14032 Caen Cedex, France Accepted 1 February 2000 Abstract The APGWamide-related neuropeptides, predicted by the cDNA of the APGWamide precursor of Mytilus edulis, have been sought by means of HPLC and electrospray mass ionization. The three predicted peptides KPGWamide, RPGWamide and TPGWamide were detected in the three main muscles and surprisingly an ion at m / z 429 corresponding to the gastropod peptide APGWamide was also demonstrated. Similar investigations performed in Lymnaea stagnalis central nervous system (CNS) revealed the occurrence of mussel APGWamide-related peptides (APGWamide-RPs) demonstrating for the first time the presence and the expression of the two precursors in both gastropod and bivalve mollusks. The absence of homologous domain in the Mytilus precursor [P. Favrel, M. Mathieu, Molecular cloning of a cDNA encoding the precursor of Ala-Pro-Gly-Trp-amide related neuropeptides from the bivalve Mytilus edulis. Neurosci. ´ Lett. 1996;205:210–214] and the Lymnaea precursor [A.B. Smit, C.R. Jimenez, R.W. Dirks, R.P. Croll,W.P.M. Geraerts, Characterization of cDNA clone encoding multiple copies of the neuropeptide APGWamide in the molluscs Lymnaea stagnalis. J. Neurosci. 1992;12:1709–1715] eliminates the hypothesis of an alternative splicing of a single gene and suggests the likelihood of two genes probably resulting from duplication of an ancestral gene before the divergence between gastropods and bivalves. The similar potency observed on contraction assay and the differential distribution of the various peptides suggest that they may exert distinct activities on multiple targets.  2000 Elsevier Science B.V. All rights reserved. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Peptides: biosynthesis, metabolism, and biochemical characterization Keywords: APGWamide; Neuropeptides; ESI–MS; HPLC; Lymnaea stagnalis; Mytilus edulis 1. Introduction byssus retractor muscle (ABRM) and the pedal retractor muscle [9]. On the other hand, in the pond snail Lymnaea An APGWamide-related neuropeptide precursor has stagnalis, the peptide APGWamide processed from the been recently cloned from Mytilus edulis predicting the unique precursor characterized harbors ten copies of the three new tetrapeptides: KPGWamide, RPGWamide and tetrapeptide [10]. The mature peptide was shown to be TPGWamide [5]. The full processing of the precursor leads involved in the control of male mating behavior, it indeed to the synthesis of five RPGWamide, one KPGWamide and relaxes serotonin-induced contractions of the penis retrac- one TPGWamide. The biological activity of these neuro- tor muscle [7]. APGWamide is co-expressed with Lymnaea peptides has not yet been investigated in the Mussel, even neuropeptide Y (LNPY), pedal peptide and conopressin in though the gastropod neuropeptide APGWamide as well as neurons located in the anterior lobe of the right cerebral multiple truncated and elongated forms were shown to ganglion which projects into the penial nerve [3]. This induce contraction potentiating modulations of the anterior peptide cocktail controls the different muscles involved in the ejaculation of semen during copulation [12]. Beside this peripheral effect, APGWamide was also shown to *Corresponding author. Tel.: 133-2-3156-5596; fax: 133-2-3156- serve as central neurotransmitter, where it affects both 5596. E-mail address: henry@ibba.unicaen.fr (J. Henry) reproductive and non-reproductive controlling neurons [2]. 0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993(00)02108-9