Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins

Abstract

The crustacean stomatogastric ganglion (STG) is modulated by numerous neuropeptides that are released locally in the neuropil or that reach the STG as neurohormones. Using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides followed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem mass spectrometric (MS/MS) analysis, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), pQIRYHQCYFNPISCF-COOH, present in the pericardial organs of the crab, Cancer borealis. Another C-type allatostatin (CbAST-C2), SYWKQCAFNAVSCFamide, was discovered using the expressed sequence tag (EST) database search strategy in both C. borealis and the lobster, Homarus americanus, and further confirmed with de novo sequencing using ESI-Q-TOF tandem MS. Electrophysiological experiments demonstrated that both CbAST-C1 and CbAST-C2 inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10 −6 M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8 Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies < 0.7Hz. Surprisingly, these state-dependent actions are similar to those of the structurally unrelated allatostatin A and allatostatin B families of peptides.

Inter-animal variability in the effects of C-type allatostatin on the cardiac neuromuscular system in the lobster Homarus americanus

“…Although it was initially thought to exist solely among holometabolous insects, several recent studies have predicted and/or identified the presence of related peptides from the C-AST family in other insects and crustaceans, including decapod crustaceans Gard et al, 2009;Ma et al, 2009;Stemmler et al, 2010;Veenstra, 2009;Weaver and Audsley, 2009). Here, we found that the native H. americanus C-AST (pQIRYHQCYFNPISCF), like the C-AST-like peptide (SYWKQCAFNAVSCFamide) that was examined previously , modulates rhythmic pattern generation in the H. americanus cardiac neuromuscular system.…”

“…The modulatory effects of C-AST in the lobster cardiac system clearly differ in detail from these effects, as they involve changes in the cycle frequency and duration of action potential bursts in pattern-generating neurons of the lobster CG, but not changes at the muscle or neuromuscular junction. Inhibitory effects of all three AST types (A-, B-and C-types) have been documented in the pyloric pattern generator of the stomatogastric system, but details of the mechanisms used to cause the observed decreases in cycle frequency have not yet been examined Dircksen et al, 1999;Fu et al, 2007;Ma et al, 2009;Szabo et al, 2011). Additionally, inhibitory effects of A-type allatostatins have been recorded in the crab CG (Cruz-Bermudez and Marder, 2007).…”

Variability, compensation, and modulation in neurons and circuits

“…Responses of individual neurons, synapses, and circuits to neuromodulation are often quite variable (50,51). In some instances, it is clear that the effect of the modulator depends on some obvious feature of the state of the preparation (52)(53)(54)(55)(56). In some instances, this variability arises from unknown sources and is likely a consequence of differing underlying cell or circuit parameters.…”

Crustacean neuropeptides

“…More recently, in silico database searches for putative peptide precursors or molecular cloning approaches, coupled with predictions of peptide processing, have led to the MS confirmation of the structures of novel neuropeptides, including members of the C-type allatostatin (AST) [59][60][61], orcokinin [62], pigment dispersing hormone (PDH) [63], and SIFamide [64] families (see ''Crustacean neuropeptide families'' for descriptions of these peptide groups). As mentioned above, bioinformaticsaided MS techniques are also playing an important role in large-scale neuropeptidome studies [23,25].…”