Peptides 34 (2012) 82–87 Contents lists available at SciVerse ScienceDirect Peptides j our na l ho me p age : www.elsevier.com/locate/peptides A pharmacological study of NLP-12 neuropeptide signaling in free-living and parasitic nematodes Lise Peeters a,1 , Tom Janssen a,∗,1 , Wouter De Haes a , Isabel Beets a , Ellen Meelkop a , Warwick Grant b , Liliane Schoofs a a Functional Genomics and Proteomics Lab, K.U.Leuven, Naamsestraat 59, B-3000 Leuven, Belgium b Department of Genetics, School of Molecular Sciences, La Trobe University, Victoria 3086, Australia a r t i c l e if o Article history: Received 2 September 2011 Received in revised form 6 October 2011 Accepted 6 October 2011 Available online 14 October 2011 Keywords: Neuropeptide, C. elegans, Structure–activity relationship, Strongyloides ratti a b s t r a c t NLP-12a and b have been identified as cholecystokinin/sulfakinin-like neuropeptides in the free-living nematode Caenorhabditis elegans. They are suggested to play an important role in the regulation of diges- tive enzyme secretion and fat storage. This study reports on the identification and characterization of an NLP-12-like peptide precursor gene in the rat parasitic nematode Strongyloides ratti. The S. ratti NLP- 12 peptides are able to activate both C. elegans CKR-2 receptor isoforms in a dose-dependent way with affinities in the same nanomolar range as the native C. elegans NLP-12 peptides. The C-terminal RPLQ- Famide sequence motif of the NLP-12 peptides is perfectly conserved between free-living and parasitic nematodes. Based on systemic amino acid replacements the Arg-, Leu- and Phe- residues appear to be critical for high-affinity receptor binding. Finally, a SAR analysis revealed the essential pharmacophore in C. elegans NLP-12b to be the pentapeptide RPLQFamide. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Neuropeptides represent a vast and chemically diverse family of signaling molecules, which play an important role in neu- romodulation and neurotransmission. They nearly always act via G protein-coupled receptors and are said to represent the most abundant signaling molecules in the Caenorhabditis elegans nervous system [15,23]. In 2008 Janssen et al. identified two CCK receptor-like neuropeptides, CKI (DYRPLQFamide) and CKII (DGYRPLQFamide) (NLP-12a and NLP-12b, respectively) as the endogenous ligands of two CCK receptor-like receptor isoforms (CKR-2a/2b) found in C. elegans [12,13]. This neuropeptide sig- naling system shares a high degree of sequence similarity with its vertebrate (CCK) and arthropod (sulfakinin) counterparts and also displays similar biological activities with respect to digestive enzyme secretion and fat storage. NLP-12 peptides are expressed in a single tail neuron, identified as the interneuron DVA, which is known to integrate mechanosensory information. Recently, ∗ Corresponding author at: Functional Genomics and Proteomics Lab, Zoological Institute K.U.Leuven, Naamsestraat 59, B-3000 Leuven, Belgium. Tel.: +32 16 32 39 00; fax: +32 16 32 39 02. E-mail addresses: Lise.Peeters@bio.kuleuven.be (L. Peeters), Tom.Janssen@bio.kuleuven.be (T. Janssen), Wouter.DeHaes@bio.kuleuven.be (W. De Haes), Isabel.Beets@bio.kuleuven.be (I. Beets), Ellen.Meelkop@bio.kuleuven.be (E. Meelkop), W.Grant@latrobe.edu.au (W. Grant), Schoofs@bio.kuleuven.be (L. Schoofs). 1 Equally contributing authors. NLP-12 signaling was shown to be involved in the mediation of a mechanosensory feedback loop that couples muscle con- traction to changes in presynaptic release, thereby providing a mechanism for proprioceptive control of locomotion [10]. This emphasizes the pleitropic nature of its biological functions in C. elegans. NLP-12 peptides are widely conserved within nematodes and almost identical in at least 11 species, belonging to three dif- ferent clades, of which eight are parasitic [11,13]. We have now identified CK-like peptides in one additional nematode species; Strongyloides ratti, a gastro-intestinal parasitic nematode of rats. S. ratti serves as a model organism for two important parasites of humans, S. stercoralis and S. fuelleborni, which cause strongy- loidiasis in an estimated 30–200 million people worldwide [2]. Other species of the Strongyloides genus are also common para- sites of wild and domestic animals [26,27]. They have a complex life cycle with both parasitic and one facultative free-living gen- eration [25]. Eggs pass out of the host via the feces and develop into iL3 (infective larval stage 3) or free-living adult males and females [26]. Because iL3 (infective larval stage 3) and dauer lar- vae of C. elegans are both arrested, long lived, resistant stages, they are thought to be analogous stages. They both resume devel- opment under appropriate conditions: low population densities and abundant food for C. elegans, and entering a host for S. ratti [4,8,19]. In this study we discuss the similarities between the NLP- 12 peptides of C. elegans and other nematodes. Because NLP-12 peptides are common to other nematode pest species, these pep- tides could be of great importance in broad spectrum anthelmintics [18]. 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.10.014