Structural and Functional Evolution of the Vasopressin/Oxytocin Superfamily
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The Journal of Neuroscience, September 1995, 15(g): 5989-5998 Structural and Functional Evolution of the Vasopressin/Oxytocin Superfamily: Vasopressin-Related Conopressin Is the Only Member Present in Lymnaea, and Is Involved in the Control of Sexual Behavior R. E. Van Kesteren,’ A. B. Smit,’ R. P. J. De Lange,’ K. S. Kits,’ F. A. Van Golen,’ R. C. Van Der Schors,’ N. D. De With,’ J. F. Burke,* and W. P. M. Geraertsl ‘Graduate School Neurosciences Amsterdam, Institute of Neurosciences, Faculty of Biology, Department of Experimental Zoology, Vrije Universiteit, Amsterdam, The Netherlands and *Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton, United Kingdom It has been suggested that the gene duplication that led to Vasopressin and oxytocin are nonapeptides that are present in the formation of the vasopressin/oxytocin two-gene family all placental mammals. Although similar in structure, they serve occurred early during vertebrate evolution. However, the different functions (Ramsay, 1983). Vasopressin has anti-diuretic existence of both vasopressin- and oxytocin-related pep- activities and is involved in hydromineral regulation, whereas tides in invertebrates suggests that this duplication may oxytocin has uterotonic and milk-ejection activities and regulates have occurred much earlier, although there is no evidence aspects of reproductive behavior. Peptides related to both vaso- for the co-occurrence of vasopressin- and oxytocin-related pressin and oxytocin are present in all vertebrate classes, except peptides in the same invertebrate species. We report here the cyclostomes, which have only the vasopressin-related pep- that in Lymnaea only the vasopressin-related peptide Lys- tide vasotocin (reviewed by Acher, 1993). Peptides of the va- conopressin, but not an oxytocin-related peptide, is pres- sopressin family have a basic amino acid residue at position 8 ent. Moreover, it is very likely that an oxytocin-like cDNA in common, whereas oxytocin and related peptides have a neu- or gene is absent. The conopressin gene is expressed in tral amino acid residue at this position. The chemical nature of neurons that control male sexual behavior, and its gene this amino acid residue is thought to be of critical importance products are present in the penis nerve and the vas defer- in the interactions of the peptides with their respective receptors. ens. Conopressin induces muscular contractions of the The structure of the protein precursors of the vasopressin/ vas deferens and inhibits central neurons that control fe- oxytocin superfamily are very similar, with a signal peptide do- male reproductive behavior. Thus, although structurally re- main followed by the nonapeptide, neurophysin, and copeptin lated to vasopressin, conopressin has functional and be- domains (reviewed by Acher, 1993), although the copeptin do- havioral characteristics typical for oxytocin. Physiological main is lacking in the oxytocin and mesotocin precursors. The and receptor binding data suggest that conopressin and precursor proteins are encoded by different genes that have an [Ile*]-conopressin, a synthetic oxytocin-like analog of cono- identical exon-intron organization in mammals (Schmale et al., pressin, are functionally equivalent in Lymnaea, and that 1983; Ivell and Richter, 1984; Ruppert et al., 1984; Sausville et the chemical nature of the amino acid residue at position al., 1985; Hara et al., 1990), bony fish (Morley et al., I990), and 8 does not result in a functional difference. Therefore, we cyclostomes (Heierhorst et al., 1992). Together with the phylo- suggest that invertebrates contain only a single member of genetic distribution of the peptides, these data suggest that the the vasopressin/oxytocin gene family and that the amino superfamily evolved from an ancestral gene by the mechanism acid change that distinguishes vasopressin from oxytocin of gene duplication. Because lampreys and hagfish have only vasotocin (Lane et al., 1988; Heierhorst et al., 1992), it has been is functionally neutral in invertebrates. hypothesized that the vasotocin gene is the present-day repre- [Key words: conopressin, vasopressin/oxytocin gene sentative of the ancestral gene, and that the gene duplication family, evolution, male sexual behavior, peptide sequenc- occurred between the radiation of cyclostomes and cartilaginous ing, gene cloning, receptor interaction, Lymnaea stagnalis] fish, about 450 million years ago (Acher, 1980). Vasopressin-related peptides are not restricted to the verte- brates, but have been identified in several invertebrate phyla as Recetved Oct. 3, 1994; revised Apr. 20, 1995; accepted Apr. 24, 1995. well, including insects (Proux et al., 1987), molluscs (Cruz et This work was supported by the Foundation for Life Sciences (SLW: Grant SOS-26.203), which is subsidized by the Netherlands Organization for Scien- al., 1987; McMaster et al., 1992) and annelids (Salzet et al., tific Research (NWO). Support was also received from the EC (BIO2CT- 1993). Oxytocin-related peptides have similarly been found in CT930169). We thank Dr. P. Van Veelen for the mass determination, Dr. G. Reich for performing the anti-oxytocin RIA, Mrs. E. R. Van Kesteren and Mr. molluscs (Reich, 1992) and annelids (Oumi et al., 1994). The J. C. Lodder for technical assistance, and Mrs. T. Laan for secretarial assistance. presence of both vasopressin- and oxytocin-related peptides in Correspondence should be addressed to Dr. R. E. Van Kesteren, Department invertebrates leads to an alternative hypothesis stating that du- of Zoology, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam. plication of the ancestral gene occurred prior to the divergence Copyright 0 1995 Society for Neuroscience 0270.6474/95/155989-10$05.00/O of vertebrates and invertebrates, over 600 million years ago (Van 5990 Van Kesteren et al. - Conopressin Controls Sexual Behavior in Lymnaea Kesteren et al., 1992). However, this hypothesis has not been Li et al. (1989). In short, 1% of each fraction was dot blotted on nitro- verified by experimental data showing co-occurrence of vaso- cellulose paper, heat fixed at 106°C for 1.5 min, and then stained with antisera W 1E and 121. Corresponding immunoreactive fractions were pressin- and oxytocin-related peptides in a single invertebrate pooled and applied to a Nucleosil 120 C-l 8 column (3 urn, 4.6 X 150 species, or by demonstrating functional divergence of the two mm) equilibrated with solvent A. Elution was achieved by a linear types of peptide in invertebrates. Thus, species variation instead gradient- of 20-35% solvent B in 60 min. Fractions were Ivoohilized.d I of gene duplication may explain the appearance of either vaso- redissolved in bidistilled water, and screened in the DIA. The immu- noreactive fraction was applied to the same C-18 column equilibrated pressin- or oxytocin-related peptides in invertebrates. with 25 mM NH,Ac for the CNS material. Elution was achieved by a In this article, we attempt to distinguish between these pos- linear gradient of IO-40% of 25 mM NH,Ac in 60% acetonitrile in 60 sibilities by studying vasopressin- and oxytocin-related peptides min. The vas deferens and penis complex material was applied to the in the mollusc Lymnuea stugnalis. Previous cDNA cloning stud- same column equilibrated with 0.05% HCl, and elution was achieved ies (Van Kesteren et al., 1992) have identified a vasopressin-like by applying 0.05% HCl/60% acetonitrile in a linear gradient of O-5% in 5 min. and 5-35% in 60 min. FmCtiOnS were lvoohilized. redissolved precursor, pro-Lys-conopressin, in Lymnaea. To demonstrate that in bidistilled water, and screened using the DIA: L conopressin has the typical tertiary structure of vasopressin-re- Sequencing, co-elution, and muss determination. The immunoreac- lated peptides and is transported to peripheral target tissues, we tive fraction of the final HPLC step was subjected to automated Edman have now identified Lys-conopressin from both the CNS and the degradation using a model 473A pulse liquid protein sequencer (Ap- male copulatory organs. At the same time, we show evidence plied Biosystems). To verify the sequence and complete the structural characterization, synthetic Lys-conopressin was used in co-elution ex- for the absence of an oxytocin-related peptide or cDNA. More- periments employing the same HPLC system, solvents, and gradients over, genomic analysis strongly suggests that the conopressin as in the first and the third rpHPLC step described above. In addition, gene is a single copy gene in Lymnuea, and that related genes the purification procedure was repeated as described above, and the are absent. Although structurally related to vasopressin, cono- immunoreactive fraction of the final HPLC step was subjected to mass pressin has functions in the control of sexual behavior that in spectrometry using a Bio-ion 20/30 plasma desorption mass spectro- meter (Applied Biosystems). mammals usually are associated with oxytocin. The implications Anti-oxytocin radioimmunoussuy. CNS of 200 snails were homoge- of these findings for theories of the molecular and functional nized, prepurified, and subjected to HPGPC as described above. All evolution of the vasopressin/oxytocin superfamily are discussed. fractions were lyophilized, redissolved in bidistilled water, and tested in an anti-oxytocin radioimmunoassay as described (Reich, 1992). Materials and Methods Polymerase chain reaction (PCR) umplijicution of putative oxytocin encoding cDNAs. Two degenerate oligonucleotides, OTI and OT2, were Animals, peptides, and antibodies. Adult specimens of L. stugnalis (shell height 28-34 mm), bred in the laboratory under standard condi- synthesized, based on the putative oxytocin-like sequences Cys-(Phe/ tions, were used. Synthetic Lys-conopressin G was obtained from Bach- Tyr)-(Phe/Ile)-Arg-Asn-Cys-Pro-(Leu/Ile/Val)-Gly and the following em Feinchemikalien AG (Budendorf, Switzerland). [Ilex]-conopressin amidation and processing sequence Gly-Lys-Arg [OTl : 5’.GGAAGC- (Cys-Phe-Be-Arg-Asn-Cys-Pro-Be-Gly-amide) was synthesized using a TTG(TC)T(AT)(TC)(AT)T(ATC)(AC)G(GATC)AA(TC)TG(TC)CC- peptide synthesizer from Applied Biosystems (Foster City, CA).