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First published online March 31, 2009
Journal of Experimental Biology 212, 1140-1152 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.028621
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Identification of SYWKQCAFNAVSCFamide: a broadly conserved crustacean C-type allatostatin-like peptide with both neuromodulatory and cardioactive properties

Patsy S. Dickinson1,*, Teerawat Wiwatpanit1, Emily R. Gabranski1, Rachel J. Ackerman2, Jake S. Stevens1, Christopher R. Cashman1,2, Elizabeth A. Stemmler2 and Andrew E. Christie3

1 Department of Biology, Bowdoin College, Brunswick, ME 04011, USA
2 Department of Chemistry, Bowdoin College, Brunswick, ME 04011, USA
3 Center for Marine Functional Genomics, Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA


Figure 1
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  		Fig. 1. Deduced amino acid sequence of a Homarus americanus C-AST-like peptide-containing prepro-hormone. Accession numbers of the ESTs from which the prepro-hormone was predicted are shown on the left with the deduced amino acid sequences of the precursor protein shown on the right. Signal peptides (when present) are shown in grey, with prohormone convertase cleavage loci shown in black. The C-AST-like peptide isoform is shown in red, with an additional precursor-related peptide shown in blue. Asterisks indicate the position of the stop codon.

 

Figure 2
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  		Fig. 2. (A) Direct MALDI-FTMS spectrum of a small piece of freshly dissected Homarus americanus supraoesophageal ganglion (brain). This spectrum was measured using DHB as the matrix, with the conditions optimized for accumulation of m/z 2500. Calibration was done using the known peptide peaks, APSGFLGMRamide (CabTRP I) and VYRKPPFNGSIFamide (Val1-SIF) at m/z 934.4927 and m/z 1423.7845, respectively. As can be seen from this spectrum, a peak corresponding to that of the [M+H]+ ion for SYWKQCAFNAVSCFamide (a disulfide bridge present between the cysteine residues) was present at m/z 1650.7158 (–2.0 p.p.m. error from the theoretical m/z of 1650.7192). (B) Predicted mass and isotopic distribution for the [M+H]+ ion for SYWKQCAFNAVSCFamide (a disulfide bridge present between the cysteine residues). (C) An expansion of the measured isotopic distribution for putative SYWKQCAFNAVSCFamide, showing that the measured mass and isotopic distribution strongly support the existence of this peptide in the lobster brain.

 

Figure 3
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  		Fig. 3. Direct tissue MALDI-FTMS spectra of freshly dissected commissural ganglia from (A) Petrolisthes cinctipes (infraorder Anomura), (B) Cancer magister (infraorder Brachyura), (C) Panulirus versicolor (infraorder Achelata) and (D) Callianassa californiensis (infraorder Thalassinidea). All spectra were measured using DHB as the matrix, with conditions optimized for m/z 1500. The inverted triangle shows the location of the m/z of the [M+H]+ ion for SYWKQCAFNAVSCFamide (disulfide bridge present between the cysteine residues; calculated m/z=1650.7192). The inserts show an expansion of the [M+H]+ peak region to show the measured mass and the isotopic distributions. Spectra were calibrated using known peptide peaks, including APSGFLGMRamide (CabTRP I) and GYRKPPFNGSIFamide (Gly1-SIF) at m/z 934.4927 and m/z 1381.7375, respectively.

 

Figure 4
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  		Fig. 4. SYWKQCAFNAVSCFamide decreased the frequency of the pyloric motor pattern in preparations in which all modulatory input to the stomatogastric ganglion (STG) was eliminated by blocking conduction in the single input nerve to the STG, the stomatogastric nerve (stn). (A) In control saline, the pattern consisted of bursts in the pyloric dilator (PD) neurons (seen here on the pyloric dilator nerve (pdn) and the ventral lateral ventricular nerve (vlvn). The lateral posterior gastric (LPG) neurons [recorded on the dorsal lateral ventricular nerve (dlvn)] fired several spikes with each PD neuron burst. (B) During bath application of 10–6 mol l–1 SYWKQCAFNAVSCFamide, cycle frequency decreased, and firing in the LPG neurons became tonic rather than linked to the PD neuron bursts.

 

Figure 5
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  		Fig. 5. Bath application of 10–6 mol l–1 SYWKQCAFNAVSCFamide significantly decreased pyloric cycle frequency when conduction in the single input nerve to the stomatogastric ganglion, the stomatogastric nerve (stn), was blocked and cycle frequency was relatively low (average 0.34 Hz) (A), but had no effect on pyloric cycle frequency (B) or gastric cycle frequency (C) when the stn was intact and both patterns were spontaneously active.

 

Figure 6
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  		Fig. 6. Bath application of 10–6 mol l–1 SYWKQCAFNAVSCFamide had no apparent effects on the activity of either the pyloric or gastric motor patterns when modulatory inputs from the anterior ganglia [commissural ganglia (CoGs) and oesophageal ganglion (OG)] were intact and the patterns were both active. (A) Control; (B) in the presence of SYWKQCAFNAVSCFamide. Pyloric pattern seen in the firing of the pyloric dilator (PD), pyloric (PY) and ventricular dilator (VD) neurons; gastric activity is recorded in the firing of the lateral gastric (LG), lateral posterior gastric (LPG) and gastric mill (GM) neurons. Activity was recorded on the pyloric dilator (pdn), ventral lateral ventricular (vlvn), medial ventricular (mvn) and dorsal lateral gastric (dlvn) nerves.

 

Figure 7
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  		Fig. 7. SYWKQCAFNAVSCFamide significantly increase the amplitude, but not the frequency, of spontaneous heartbeat in Homarus americanus. (A) Pooled data from 16 preparations show that SYWKQCAFNAVSCFamide significantly increased the amplitude of heart contractions (t-test, P=0.015). However, it had no consistent effect on heartbeat frequency. (B) The extent of increase was larger in preparations with initially low amplitudes, suggesting that the effect of the peptide is to some extent state dependent. (C) It is probable that the lack of significance for frequency change is due, at least in part, to a state-dependent effect of the peptide, where the rate of heartbeat was increased when the baseline frequency was low, but decreased when baseline rate was high.

 

Figure 8
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  		Fig. 8. SYWKQCAFNAVSCFamide increased the amplitude and altered the frequency of heartbeat in Homarus americanus. Heartbeat was recorded with a force transducer in control saline and during perfusion with 10–6 mol l–1 SYWKQCAFNAVSCFamide. (A) In some preparations, the frequency of heartbeat decreased, whereas the amplitude of contractions increased in the presence of SYWKQCAFNAVSCFamide. (B) In other preparations, both the frequency and the amplitude of the heartbeat increased in the presence of the peptide.

 

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© The Company of Biologists Ltd 2009