Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 4593-4597, May 1992 Neurobiology Evolution of the vasopressin/oxytocin superfamily: Characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis (gastropod mollusc/neuropeptide/conopressin/neurophysln/prohormone organization) RONALD E. VAN KESTEREN*t, AUGUST B. SMIT*, ROELAND W. DIRKSI, Nico D. DE WITH*, WUNAND P. M. GERAERTS*, AND JOOS JOOSSE* *Faculty of Biology, Unit Molecular Neurobiology/Endocrinology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands; and tDepartment of Cytochemistry and Cytometry, University of Leiden, Wassenaarseweg 72, 2333 AC Leiden, The Netherlands Communicated by Berta Scharrer, December 27, 1991 ABSTRACT Although the nonapeptide hormones vaso- thought that these genes evolved from an ancestral gene about pressin, oxytocin, and related peptides from vertebrates and 400 million years ago (6, 7). The prohormones contain, in some nonapeptides from invertebrates share smilities in addition to the nominal peptide domain, a highly conserved amino acid sequence, their evolutionary relationships are not cysteine-rich neurophysin domain, which probably is involved dear. To investigate this issue, we doned a cDNA encoding a in binding ofthe hormones during axonal transport. The mam- vasopressin-related peptide, Lys-conopressin, produced in the malian vasopressin precursor further includes a copeptin do- central nervous system of the gastropod mollusc Lymnaea main that is cleaved off and may play a role as a prolactin stagnalis. The predicted preproconopressin has the overall releasing factor (8). This copeptin domain is entirely absent in architecture of vertebrate preprovasopressins, with a signal the oxytocin precursor and is represented as a C-terminal highly peptide, Lys-conopressin, that is flanked at the C terminus by divergent extension of the neurophysin domain in the vaso- an amidation signal and a pair of basic residues, followed by a pressin-related precursors of lower vertebrates. neurophysin domain. The Lymnaea neurophysin and the ver- The high identity in amino acid sequence of the vertebrate tebrate neurophysins share high sequence identity, which and invertebrate peptides has led to the hypothesis that the includes the conservation of all 14 cysteine residues. In addi- vasopressin/oxytocin superfamily may have evolved from a tion, the Lymnaea neurophysin possesses unique structural common ancestral form, ofwhich the nature ofthe precursor characteristics. It contains a putative N-linked glycosylation and gene is unknown. To further investigate the evolutionary site at a position in the vertebrate neurophysins where a strictly origin ofthe vasopressin/oxytocin superfamily, we have now conserved tyrosine residue, which plays an essential role in cloned and sequenced the cDNA encoding conopressin from binding ofthe nonapeptide hormones, is found. The C-terminal Lymnaea.§ Our analysis ofthe deduced amino acid sequence copeptin homologous extension of the Lymnaea neurophysin for Lymnaea preproconopressin indicates that it is organized has low sequence identity with the vertebrate counterparts and much like the vasopressin (related) precursors of the verte- is probably not cleaved from the prohormone, as are the brates, with a signal sequence followed by conopressin and mammalin copeptins. The conopressin gene is expressed in a remarkably conserved neurophysin domain having a diver- only a few neurons in both pedal ganglia of the central nervous gent copeptin-homologous C-terminal sequence. Thus, we system. The conopressin transcript is present in two sizes, due could unequivocally demonstrate that the typical architec- to alternative use of polyadenylylation signals. The data pre- ture of the precursors of the vasopressin/oxytocin hormone sented here demonstrate that the typical organization of the superfamily must have been present in the Archaemetazoa, prohormones of the vasopressin/oxytocin superfamily must a stem group from which both the vertebrates and inverte- have been present in the common ancestors of vertebrates and brates diverged about 600 million years ago. invertebrates. The vasopressin/oxytocin hormone superfamily includes va- MATERIALS AND METHODS sopressin, oxytocin, and related peptides in vertebrates and Animals. Adult Lymnaea stagnalis (shell height, 28-34 in invertebrates (ref. 1; Fig. 1). Invertebrate members include mm), bred in the laboratory under standard conditions (9), the vasopressin-like diuretic hormone from the locust Lo- were used. custa migratoria (2), the conopressins from the gastropod PCR. Two degenerate oligonucleotides, oligo 1 and oligo 2, molluscs Conus geographus, Conus striatus (3), and Lym- were synthesized, based on the amino acid sequence of naea stagnalis (unpublished observations) and cephalotocin Lymnaea conopressin [oligo 1, 5'-CCAAGCTTTG(CT)TT- from the cephalopod mollusc Octopus vulgaris (4). All of (CT)AT(ACT)(AC)G(GATC)AA(CT)TG(CT)CC-3'; oligo 2, these hormones are nonapoptides that share close similarities 5'-CCAAGCTTAA(CT)TG(CT)CC(GATC)AA(GA)GG- in primary and tertiary structure. Biological activity may (GATC)GG-3']. Both oligonucleotides were provided with differ, however, mainly due to the amino acid residue in HindIII restriction site extensions on the 5' ends and were position 8, which is a basic amino acid in vasopressin and used as primers in a PCR, with cDNA isolated from a AgtlO vasopressin-related peptides and a neutral amino acid in cDNA library of the central nervous system (CNS) (10) as oxytocin and oxytocin-related peptides. template. As reverse primer, the A oligo was used that is In vertebrates, vasopressin, oxytocin, and related peptides complementary to a sequence in the left arm ofAgtlO (A oligo, are encoded by two types of structurally related genes and 5'-AGCAAGTTCAGCCTGGTTAAGTCC-3'). In the first synthesized as part of larger precursor molecules (5). It is Abbreviations: CNS, central nervous system; nt, nucleotide(s). The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M86610). 4593 . Downloaded by guest on September 26, 2021 4594 Neurobiology: van Kesteren et al. Proc. Natl. Acad Sci. USA 89 (1992) vasopressin(-related) peptides Cys Phe Ile Arg Asn Cya Pro Lys Gly-NH2 Lys-conopressin Lymnaea stagnalis, Conus geographus Cys Ile Ile Arg Ann Cys Pro Arg Gly-NH2 Arg-conopressin Conus striatus Cys Leu Ile Thr Ann Cys Pro Arg Gly-N82 diuretic hormone Locustanmgratoria Cys Tyr Ile Gln Aan Cys Pro Arg Gly-N12 vasotocin non-mammalian vertebrates Cys Tyr Phe Gln A n Cys Pro Arg Gly-N32 Arg-vasopressin mammals Cys Tyr Phe Gln Ann Cys Pro Lys Gly-NE2 Lys-vasopressin mammals Cys Phe Phe Gln Ann Cys Pro Arg Gly-NR2 phenypressin mammals oxytocin(-related) peptides Cys Tyr Phe Arg An Cys Pro Ile Gly-NH2 Cephalotocin Octopus vulgaris Cys Tyr Ile Asn An Cya Pro Leu Gly-NB2 Aspargtocin cartilaginous fishes (sharks) Cya Tyr Ile Gln An Cys Pro Val Gly-NE2 Valitocin cartilaginous fishes (sharks) Cys Tyr Ile Ser An Cya Pro Gln Gly-NH2 Glumitocin cartilaginous fishes (rays) Cys Tyr Ile Ser An Cya Pro Ile Gly-NH2 Isotocin bony fishes Cys Tyr Ile Gln An Cy. Pro Ile Gly-NH2 Mesotocin mammals, birds, reptiles, amphibians, lungfishes Cys Tyr Ile Gln An Cya Pro Leu Gly-NH2 Oxytocin mammals 1 2 3 4 5 6 7 8 9 FIG. 1. Compilation of the primary sequences of various peptides of the vasopressin/oxytocin superfamily. Residues at positions 1, 5, 6, 7, and 9 that are shared by all members ofthe superfamily are indicated in boldface type. Each peptide is indicated by name and by species/group in which it has been identified. PCR, cDNA was amplified between oligo 1 and the A oligo by 3' part ofthe mRNA encoding the conopressin region and the using 30 cycles: 940C for 1 min, 450C for 1 min, and 720C for hypothetical neurophysin region of the preprohormone. A 2 min. After amplification, 5 uml of the PCR mixture were degenerate oligonucleotide primer that corresponds to the reamplified under the same conditions, but using oligo 2 and N-terminal part ofconopressin and an oligonucleotide primer the A oligo. Amplified cDNA was digested with EcoRI and that is complementary to a sequence in the left arm of AgtlO HindIll, subcloned, and sequenced. were used to amplify cDNA from a AgtlO cDNA library ofthe Labeling of the Conopressin cDNAs. Radiolabeled cDNA CNS of Lymnaea stagnalis. Analysis of the PCR mixture by was synthesized by primer extension on a single-stranded electrophoresis on an agarose gel revealed many products. M13 clone containing a conopressin cDNA derived either by To increase the specificity of the PCR, a degenerate oligo- PCR or from AgtlO. An M13-specific primer was used, and part of [a-32P~dATP was incorporated during the reaction. The spe- nucleotide primer that corresponds to the C-terminal cific activity of the probes was >2 x 108 dpm/pug. conopressin and the same A oligonucleotide primer were used Screening of the cDNA Library. Approximately 2 x 10l to amplify the cDNA mixture of the first round of amplifi- recombinant AgtlO phages from a CNS-specific cDNA library cation. Analysis of the second PCR mixture on agarose gel (10) were plated at 7 x 104 plaques per 245 x 245 mm dish and revealed a major product of700 base pairs, which was cloned absorbed to Hybond-N filters (Amersham). After prehybrid- in M13mpl8 and sequenced. It appeared to contain the PCR ization, the filters were hybridized with the radiolabeled primer followed by an open reading frame encoding the conopressin cDNA (derived by the PCR) for 16 h, washed in endoproteolytic processing site Lys-Arg and a large cysteine- 2x SSC (lx SSC = 150 mM NaCl/15 mM sodium citrate, pH rich peptide. The first 9 amino acids of this peptide had only 7.4)/0.1% SDS at 650C for 30 min, and autoradiographed.