Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins

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Highlights

  • Identification of several conopressin/conophysin sequences form diverse Conus species.

  • Two conophysin sequences are characterized by mass spectrometry in C.monile.

  • Four distinct conopressin sequences were detected in C.monile venom.

  • Detection of C-terminal Gly deleted conopressins suggests “messy processing”.

Abstract

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct Conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (∗ indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.

Introduction

The peptide hormones oxytocin and vasopressin have been extensively investigated in vertebrates [[1], [2], [3], [4], [5]]. The discovery of these peptide hormones in Hydra suggests a role for these peptides as neurotransmitters in primitive organisms [6]. The more recent discovery of insulin analogs in cone snail venom [[7], [8], [9]], emphasises the importance of hormone like peptides in Conus venom duct extracts [10]. Olivera and co-workers identified the invertebrate homolog of an oxytocin/vasopressin peptide in marine cone snails nearly thirty years ago [11].

The conopressins were originally characterised from the venom of the fish hunting marine cone snails, C.geographus and C.striatus [11]. Conopressins, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Arg/Lys-Gly-NH2, were shown to exercise behavioural effects in mice following intracerebral injection, which resembled those exhibited by Arg-vasopressin, suggesting specific interaction with a receptor in the central nervous system [11]. A noteworthy feature of the conopressins is the presence of a basic, Arg, residue in the macrocyclic disulfide loop, in contrast to the oxytocin/vasopressin class of peptides, which do not have charged residues within the disulfide ring [11].

In mammals, oxytocin and vasopressin are biosynthesized in the neurohypophysis as precursor proteins, which are proteolytically processed to yield the peptide hormones and the protein neurophysin [4,12]. Noncovalent complexes of the peptide hormone and neurophysins are stored as granules in the pituitary gland. The invertebrate analog of neurophysin was first identified from the venom of C.radiatus [13] All the fourteen Cys residues present in the mammalian neurophysins are conserved in conophysins. The high mutation rates for genes encoding conopeptide precursors suggests that sequence diversity may also be a feature of the conopressin class of peptides. Dutertre et al., have isolated conopressin-T from the venom of C.tulipa with the sequence Cys-Tyr-Ile-Gln-Asn-Cys-Leu-Arg-Val-NH2 [14]. Conopressin-T differs significantly from the known peptides of this class; two highly conserved residues at position 7 (Pro) and 9 (Gly) being replaced with Leu and Val, respectively. Conopressin-T binds to the oxytocin receptor (OTR) and the vasopressin V1a receptor [14]. The wide distribution of conopressins suggests a possible function as an endogenous hormone in gastropods [15,16]. Interestingly, the possibility that the conopressin may serve a role in prey capture has also been advanced [17].

In this report, we establish the presence of conopressins and conophysins in vermivorous and molluscivorous cone snails isolated from the southern coast of India. Complete characterization of precursor gene sequences has been obtained by mining the venom duct transcriptome and peptide characterization has been achieved by mass spectrometric studies of the total venom. Sequence diversity in conopressins is established with appreciable variations at position 8. C-terminus truncated peptides have also been detected in the venom samples. The presence of conophysins is experimentally established by mass spectrometry guided proteomic analysis.

Section snippets

Materials and methods

Details for the collection of Conus specimens have been described previously [18]. Briefly, Conus Specimen used in this study, C.amadis, C.araneosus C.loroisii and C.monile were collected from Gulf of Mannar, Tamil Nadu, India while C.ebraeus, C.frigidus, C.lividus, C.litteratus and C.miles were from Andaman and Nicobar Islands, India. The reagents used for protein isolation, electrophoresis reduction /alkylation and the reagents used for mass spectrometry were obtained from Sigma Aldrich.

Transcriptome derived sequences

Transcriptomic data for nine cone snail species collected off the south-eastern coast of India were mined for the conopressin/conophysin precursor sequences, using the previously annotated C.geographus conopressin/conophysin precursor sequence (Uniprot id:P05486) for the BLAST analysis. Fig. 1 shows a multiple sequence alignment of twenty precursor sequences along with the sequence of the crystallographically characterized noncovalent complex of vasopressin with the bovine neurophysin II. In

Author contributions

SK(proteomics); MV(transcriptomics); SP (mass spectrometry); MAV (peptide synthesis). SK, MV & PB (analysis of transcriptomics and proteomics data); SK and PB wrote the manuscript. All the authors read and approved the final manuscript.

Funding sources

This work was supported by a grant from Department of Biotechnology (BT/PR/11293/BRB/10/849/2008) and a DST/YOS Chair Professorship to PB and SERB National postdoctoral fellowship (PDF/2016/0020) to SK. The mass spectrometry facility is supported by an institutional program grant from the DBT at the Indian Institute of Science.

Declaration of Competing Interest

The authors declare no competing financial interest.

Acknowledgment

We are grateful to Prof. M. K. Mathew, NCBS for providing laboratory facilities.

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