Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms
Graphical abstract
Introduction
Tachykinins are a highly conserved group of peptides found in both invertebrate and vertebrate animals. These peptides function as neurotransmitters and neuromodulators of both the central and peripheral nervous systems [55]. The mammalian tachykinins neurokinin A, neurokinin B and Substance P are sensory neuropeptides with roles in both nociception and inflammation. Tachykinins exhibit both afferent and efferent functions and participate in the regulation of several physiological processes including peripheral sensory mechanisms such as nociception and inflammation as well as autonomic functions such as smooth muscle contractility in the vascular, gastrointestinal and genitourinary systems [34], [44]. In addition, tachykinins are involved in central nervous system pathways mediating pain, anxiety, motor coordination and cognition [34].
The actions of tachykinin peptides are mediated by one or more tachykinin receptors. Three subtypes of vertebrate tachykinin receptors, known as neurokinin receptor 1 (NK1R), neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R), as well as numerous subtypes of invertebrate tachykinin receptors, have been described to date [37], [55]. Neurokinin receptors have been shown to act via Gq/11 coupling proteins increasing inositol phosphate 3 and diaceylglycerol (DAG) levels within cells bound by an agonist [41]. To date, a number of characteristic tachykinin amino acid motifs have been found to be crucial to the structure–activity relationships of tachykinins and tachykinin-like peptides. Vertebrate tachykinins are characterized by a FXGLM-amide motif while invertebrate tachykinins are characterized by a C-terminal FXGXR-amide motif (Table 1).
Octopuses live in habitats ranging from pelagic to benthic zones of all of the world's oceans ranging from Arctic to Antarctic, with some species specialists to certain habitats [53]. Octopuses secrete a variety of bioactive molecules from their posterior venom glands in order to feed on both vertebrate and invertebrate prey [15], [24], [26]. Upon envenomation rapid immobilization due to hypotensive effects as well as complete, irreversible, flaccid paralysis is observed in crustaceans [21], [40]. The consequent evolutionary selection pressure has resulted in a wide diversity of bioactive substances present in octopus and other coleoid venoms including small molecules such as acetylcholine, histamine, octopamine, tserotonin (aka: enteramine), taurine, tetrodotoxin and tyramine [7], [8], [10], [11], [12], [13], [14], [16], [17], [20], [29], [47] and proteins [1], [3], [18], [19], [22], [23], [25], [33], [36], [38], [45], [48], [51], [52]. Included in this tremendous molecular biodiversity are the tachykinin peptides Oct-TK-I and Oct-TK-II from Octopus vulgaris [32], Oct-TK-III from Octopus kaurna [19], and eledoisin from Eledone cirrhosa [1], [23]. These tachykinin forms are interesting in that even though they are from an invertebrate venom, the C-terminal amide motif is that of the vertebrate form (Table 2).
As octopuses prey upon a wide diversity of vertebrate and invertebrate species, one might predict their tachykinin type toxins to target the vertebrate as well as the invertebrate receptors but this prediction has never been experimentally tested. Structurally, only vertebrate-type tachykinins containing a C-terminal FXGLM-amide moiety have thus far been identified in octopus venom. Consistent with this observation, the tachykinins Oct-TK-I and Oct-TK-II from octopus venom have been shown to be vertebrate active [32]. However a comparison of the relative effects upon vertebrate and vertebrate tissues has not been undertaken. Further, a novel tachykinin we previously sequenced [19], which differs significantly from Oct-TK-I and Oct-TK-II in nature and distribution of charged residues, has not been functionally characterized. Therefore, the aim of this study was to compare the differential effects of octopus venom tachykinin peptides upon invertebrate and vertebrate tissue preparations.
Section snippets
Peptide synthesis and purification
In order to explore potential neofunctionalization derivations, we constructed Oct-TK-I (KPPSSSEFIGLM), Oct-TK-II (KPPSSSEFVGLM) and Oct-TK-III (DPPSDDEFVSLM) peptides in both amide and non-amide forms. Protected Fmoc-amino acid derivatives were purchased from Auspep (Melbourne, Australia). The following side chain protected amino acids were used: His(Trt), Hyp(tBu), Tyr(tBu), Lys(Boc), Trp(Boc), Arg(Pbf), Asn(Trt), Asp(OtBu), Glu(OtBu), Gln(Trt), Ser(tBu), Thr(tBu), and Tyr(tBu). All other
Results
In the vertebrate-isolated ileum tissue assay, all three amide-peptides showed classic tachykinin responses: after addition to tissue there was an initial decrease in contractility, followed by an increase to peak concentration-dependent contraction, and then a period of oscillation until the peptide was washed from the organ bath (Fig. 1, Fig. 2). When tested on the invertebrate hindgut assay, all three peptides elicited increases in contraction (Fig. 3), with similar EC50 values (Table 2).
Discussion
In this study, we examined the differential effects of the octopus venom peptides Oct-TK-I, Oct-TK-II and Oct-TK-III on vertebrate and invertebrate tissue-specific contractile activity using the rat ileum (Rattus norvegicus) and crayfish hindgut (P. clarkia) assays. Our findings indicate that OCT-TK-I, OCT-TK-II and OCT-TK-III are differentially active when assayed for invertebrate- and vertebrate-specific effects. For all three peptides, the C-terminal amide was essential for any activity,
Acknowledgements
BGF was funded by an Australian Research Council Future Fellowship and by the University of Queensland. SAA was the recipient of postdoctoral fellowship (PDRF Phase II Batch-V) from Higher Education Commission (HEC Islamabad) Pakistan. TNWJ was funded by an Australian Postgraduate Award. EABU acknowledges funding from the University of Queensland (International Postgraduate Research Scholarship, UQ Centennial Scholarship, and UQ Advantage Top-Up Scholarship) and the Norwegian State Education
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Joint first-authorship.