Chapter 4 Molecular evolution of neurohypophysial hormone precursors

J. Joosse, R . M . Buijs and F.J.H. Tilders (Eds.)

Progress i n Brain Research, Vol. 92

@ 1992 Elsevier Science Publishers B.V. All rights reserved

39 CHAPTER 4

Molecular evolution of neurohypophysial hormone precursors Akihisa Uranol, Susumu Hyodo2 and Masakuzu Suzuki' I

Laboratory of Molecular Biology, Ocean Research Institute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan and 2Department of Biology, College of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan

Introduction Comparative approaches to the study of neurosecretory systems have widened our understanding of the regulation of various aspects of physiology in both vertebrates and invertebrates. Approaches using morphological, physiological, pharmacological, immunological and biochemical methods have revealed unequivocally that peptidergic neurohormones are important agents for intercellular communication. Surprisingly many identical or similar peptides, including neurohypophysial hormones, occur in the nervous system of a variety of animals, from coelenterates to mammals. Neurohypophysial hormones are peptides produced mainly by hypothalamic magnocellular neurons. At least ten distinct nonz.peptide principles characterized in a wide variety of vertebrates can be separated into two groups: the vasopressin (VP) family and the oxytocin (OT) family (see Table I). The neurohypophysis of a given vertebrate species, except for the cyclostomes, generally contains at least one VP-like peptide and one OT-like peptide. Additional sequences were obtained from invertebrate species, conopressin I and I1 (Cruz et al., 1987), locust diuretic hormone (Proux et al., 1987) and tunicate oxytocin-like peptide (Iwakiri et al., 1990) (Table I). The molecular evolution of these peptides has attracted comparative endocrinolo-

gists, and several schemes were proposed for an evolutionary pathway based on amino acid sequences and phyletic distributions (Gorbman et al., 1983; Acher, 1985). It is generally accepted that VPlike and OT-like families emerged from a common ancestral molecule by gene duplication about 450 million years ago and then evolved independently. Amphibian mesotocin (MT) has been supposed to be derived from fish isotocin (IT) and to have been replaced by oxytocin in mammals, while vasotocin (VT) was replaced by vasopressin. The primary structures of the precursors of mammalian neurohypophysial hormones, [Arg'lvasopressin and oxytocin, were first determined by sequence analysis of cDNAs from bovine hypothalami (Land et al., 1982, 1983). The gene organization for vasopressin and oxytocin was then clarified in bovine (Ruppert et al., 1984), rat (Ivell and Richter, 1984) and human (Sausville et al., 1985). Thereafter, the primary structure of neurohypophysial hormone precursors was deduced from the nucleotide sequences of cDNAs encoding them in lower vertebrates such as the toad (Nojiri et al., 1987), white sucker (Heierhorst et al., 1989), chum salmon (Heierhorst et al., 1990; Hyodo et al., 1991) and masu salmon (Suzuki et al., 1992). This molecular information has enabled us to reconsider the evolution of neurohypophysial hormones based on the primary structure of their precursors.

40 TABLE I Phyletic distribution of vasopressin-like and oxytocin-like peptides The loci with the same amino acid in both vasopressin and oxytocin are indicated by *. Name of peptide

Amino acid sequence

Distribution

Vasopressin family Arg-vasopressin Lys-vasopressin Phenypressin Vasotocin Arg-conopressina Lys-conopressina Diuretic hormoneb

Cys - Tyr - Phe - Gln - Asn - Cys -Pro - Arg - Gly - NH, * a * * c * L y s * * P h e * t * * * t e * * I l e * * * t t * t Ile lle Arg t c * t * * Phe Ile Arg * * * Lys * a Leu Ile The t * * * e

Mammals Mammals Metatherians Non-mammalian vertebrates Mollusc (Conus) Mollusc (Conus) Insect (Locust)

Oxytocin family Oxytocin Mesotocin

Cys - Tyr - Ile - Gln - Asn - Cys -Pro - Leu - Gly - NH, * * * I l e e * * *

Mammals Metatherians Non-mammalian tetrapods Bony fish Ray Shark Shark Tunicate

Isotocin Glumitocin Valitocin Aspargtocin Oxytocin-like peptide'

* * * * *

* * * * t

* * * *

*

Ser Ser

*

t

*

A s n * Ser Asp

* * * t

*

Ile Glu * V a l

* *

*

Asn

* e

* t

Ser-RFWST

'Cruz et al., (1987); bProux et al., (1987); 'Iwakiri et al., (1990).

Structure of neurohypophysial hormone precursors

Comparisons of the overall structure of neurohypophysial hormone precursors are important to elucidate their evolutionary pathway. As is shown in Fig. 1, mammalian studies on gene organization (e.g. Ruppert et al., 1984) showed that the [Arg8]-vasopressin (AVP) precursor is composed of ternary segments for AVP preceded directly by a signal peptide, AVP-neurophysin (a carrier protein specific to AVP), and a glycoprotein named copeptin. The oxytocin precursor consists of binary segments for oxytocin and OT-specific neurohysin but lacks a glycoprotein domain. Vasotocin and mesotocin are amphibian counterparts of mammalian vasopressin and oxytocin, respectively. The vasotocin and mesotocin precurin the toad contain a peptide followed directly by the hormones, which in turn are connected to the specific neurophysins by

/yR\ fryH Signal Peptide VP

VP Precursor

VP Gene

Copeptin

~~~~ EXON A

O T Gene

Neurophysln

N H ~

intron I

EXON B

d r + , H - + H T -

OT P r e c u r s o r

V[

COOH

NH2

Signal OT Peptide

EXON C

intron I1

Neurophysin

Fig. 1. Structure of vasopressin (VP) and oxytocin (OT) precursors and their genes. Vasopressin precursor is composed of ternary segments for vasopressin, neurophysin and the glycoprotein copeptin, whereas oxytocin precursor consists of binary segments for oxytocin and OT-specific neurophysin. These segments are encoded by separate exons in the precursor genes. Based on Ruppert et al. (1984) and Ivell and Richter (1984).

41

h-provasopressin (h-proVP) t-provasotocin

(t-proVT)

cs-provasotocin-I (cs-proVT-I) ms-provasotocin-I (ms-proVT-I) ms-proisotocin-I (ms-proIT-I) cs-proisotocin-I (cs-proIT-I) t-promesotocin

(t-proMT)

h-prooxytocin

(h-proOT)

h-proVP t -proVT cs-proVT-I ms -proVT-I

ms-proIT-I cs-proIT-I t -proMT h-proOT

h -proVP

, -I Copeptin 1 SCVTEPECREGFHRRA R ASD-RSNATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY

t -proVT

TCVVDSSCLDEDSERR R VTP-EQNMTQMDGSASDLLLRLMHMANRQQQSKHQFY

J:;?.

,.>.,. .,. , ,.,.

&.- *L

cs -proVT-I

J-"

y A .

*

-L I.

4 *J:

.L .I

.j:;*:;*<

I .

a ;k

* - P A

,* ;; 1; :. 1, , -L. \

;L\

* :>'

Je;*:

;+?$d:;*: *f J:;*:;*:;kw:;~:

3k ;:'

.,,. * .....

-'--'-

9-J-J-

SCLLDSDCLD-DSF--R QPPSEQYSSLMEGLAGDLLQWMLH-ATRRERPQ ,\ k; 3r"JJ; Jr >': 9: GCSIDQSCTEEDE---A EYISQSVSS-SHG--HDLLMKLLNMISHTPPHRVHK .,I

Jr ;':;'eJ:;\;l:;':~:;':;?.

J:;':JrJrJ:;?.JrJ:~tr;tc;~c;':;~:

cs-proIT-I

; L

J-

J-"

ms-proIT-I

*

** * 9r+:9:** ;\ *, . ,.,.

SCVLDPDCL-EDSK--R QSPSEQNAALMGGLAGDLL-RILH-ATSRGRPQ :v; : , ;

ms-proVT-I

-L ,I

J-

,I

;'ci':J:

,.

-1. I .

;'c;~':;':;':;':;':Jc;':;~c;~~;~'cJr~'c;':;'c;~Jc;\;~:

GCSIDQSCTEEDE---A EYISQSVSS-SHG--HDLLMKLLNMISHTPPHRVHK ;\

fr

Jr :2'

9r

t-proMT

SCTMDPAC-EQDSVFS

h-pro0T

GCHADPAC-DAEATFSQR

;'

.:,.,.,.

J J.-L.L

AJc

Fig. 2. Comparison of amino acid sequences among neurohypophysial hormone precursors. Precursors of vasopressin-like and oxytocin-like peptides were aligned so as to optimize homology. Identical amino acid residues are indicated by asterisks. The conserved regions of neurophysins are indicated by the frame. From Suzuki et al. (1992).

42

Gly-Lys-Arg, a processing and carboxyl-terminal amidation signal (Nojiri et al., 1987). The vasotocin precursor further includes a glycoprotein of 36 amino acids following the VT-neurophysin, although the occurrence of processing between neurophysin and a glycoprotein is uncertain (Michel et al., 1987; Chauvet et al., 1988). The structural organizationof the vasotocin and mesotocin precursors is thus highly homologous to that of the vasopressin and oxytocin precursors, respectively (Fig. 2). The neurohypophysial principles in teleostean fish are vasotocin and isotocin. The primary structures of vasotocin and isotocin precursors were determined in tetraploid fish such as the white sucker (Heierhorst et al., 1989), chum salmon (Heierhorst et al., 1990; Hyodo et al., 1991) and m a w salmon (Suzuki et al., 1991). Two different precursors were obtained for vasotocin and for isotocin in the chum salmon and the white sucker, and were designated proVT-I and proVT-11, and proIT-I and proIT-11. The masu salmon has only proVT-I and proIT-I mRNAs. All these precursors were found to contain a signal peptide and a hormone connected to a specific neurophysin by GlyLys-Arg. The carboxyl termini of VT- and ITneurophysins are about 30 amino acids longer than in the neurophysins of toad and mammalian neurohypophysial hormone precursors. These extended regions, even of vasotocin, do not contain a glycosylation site. Nonetheless, they show marked similarity in their leucine-rich core segments with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. Since a single nucleotide mutation in salmonid vasotocin genes would generate a potential glycosylation site (Am-X-Thr/Ser) in the comparable position, the amphibian and mammalian copeptins may be derived from the “extended” carboxyl terminal of an ancestral neurophysin. The central portions of AVP- and OTneurophysins, often referred to as the conserved regions, contain similar amino acid sequences, regardless of the mammalian species from which they were obtained (Acher, 1985). Our cloning and

sequencing studies have confirmed that this is also true for lower vertebrates (Fig. 2). When compared in the same species, the homology of the conserved region between VP-like and OT-like peptides is higher than those of other corresponding regions in salmonid fish (Hyodo et al., 1991) as well as in mammals (Ivell et al., 1984; Ruppert et al., 1984), although it is not so high in the toad (Nojiri et al., 1987).

Synonymous and nonsynonymous substitutions Evolutionary relationships among the neurohypophysial hormone precursors were estimated by statistical calculation of nucleotide substitution rates of coding regions according to the method of Miyata et al. (1986), in which nucleotide sites and substitutions are classified as synonymous and nonsynonymous. The structures of mammalian (Ruppert et al., 1984) and white sucker (Morley et al., 1990) genes encoding neurohypophysial hormone precursors, in particular the presence of three exons (Fig. l), were considered in the calculation. The nucleotide sequences of precursors were thus divided into three regions: region A, the moiety which encodes the signal peptide, the hormone and the amino-terminal portion of neurophysin; region B, the segment that encodes the central conserved portion of neurophysin; and region C, the segment that encodes the carboxyl-terminal portion of neurophysin and the copeptin. Following calculation of nucleotide substitution rates, both synonymous (K,) and nonsynonymous ( K J , the values were corrected for the effect of multiple hits at a single site. Evolutionary distances among neurohypophysial hormone precursors were estimated by the formula: T = cKs/2v, where T = evolutionary distance between two sequences; cKs, a mean value of corrected synonymous nucleotide substitution rates; v = 3.1 x 10-9/locus/year, the mutation rate calculated from mammalian genomic data (Miyata et al., 1986). When the evolutionary distances of neurohypophysial hormone precursors were estimated among

43

teleost fish, we adopted a hypothesis based on the fossil record (Harland et al., 1967) and on an isozyme study (Lim et al., 1975) indicating that the divergence of the masu salmon and the white sucker occurred 100 million years ago. Since the corrected rates of synonymous substitutions (cKs)are almost the same for vasotocin and isotocin precursor genes, the mean mutation rate (v) of genes encoding neurohypophysial hormone precursors was estimated as 8.4 x 10-9/locus/year in teleosts. The calculated values of synonymous nucleotide substitutions among the salmon, toad and human are shown in Tabel 11. Between VP-like and OT-like peptides in the same species, the rate of nucleotide substitution for region B, which encompasses the exon that encodes the central conserved segment of neurohypophysial hormone precursor, was considerably lower than those for the other two segments, regions A and C, in the chum salmon and the human. Similarly, the rates of nonsynonymous nucleotide substitution for region B are markedly lower than for the other regions (data not shown). The same result was also found in the masu salmon (CKs:region A, 0.829; region B, 0.331; region C , 0.720). These results suggest the occurrence of gene conversion encompassing the exons encoding the

central portions of neurophysins in salmonid fish similar to that found for the human equivalents (Sausville et al., 1985). The exon that encodes the conserved central portion of neurophysin, thus, seems to be susceptible to a gene conversion event irrespective of species, although the rate of nucleotide substitution for the conserved regions of the neurophysins of vasotocin and of mesotocin in the toad were less differed than those characterizing the other two segments. It is well known that estimated values of the evolutionary distances among phylogenically distant animals usually contain considerable variation and hence are not fully reliable. However, there are no other good indicators which enable intuitive understanding of evolutionary relationships among neurohypophysial hormone precursors; hence, we estimated the evolutionary distances of genes encoding them from the synonymous nucleotide substitution rates (Table 11). Possible evolutionary pathway Based on the evolutionary distances and the structural organization of precursors, we have proposed a model which may describe the evolutionary

TABLE 11 Rates of synonymous substitution and evolutionary distances for coding regions of cDNAs encoding neurophypophysial hormone precursors The rates were separately calculated for Region A, region B and region C in Fig. 2. Region A , the signal peptide, hormone and Nterminal portion of neurophysin; region B, the central conserved portion of neurophysin; and region C , the C-terminal portion of neurophysin. T, evolutionary distance in million years ~-

--

Sequences compared ~~

Salmon VT-I vs. salmon VT-II Salmon IT-I vs. salmon IT-11 Salmon VT-I vs. salmon IT-I Salmon VT-I1 vs. salmon IT-I1 Salmon VT-I1 vs. toad VT Toad VT vs. toad MT Toad VT vs. human AVP Human AVP vs. human O T ~~~

Region A

T __

Region B

Region C Total

0.826 1.131 1.263 0.917 2.062 1.510 1.281 0.461

0.634 0.774 1 .663a 1 .425a 2.073 2.292 1.456 0.672a

-~

~

0.498 0.442 0.286 (46 million years) 0.276 (45 million years) 2.503 2.631 2.645 0.142 (23 million years)

0.663 1.110 1.795 1.615 1.600 2.260 0.823 0.776 -

aTotal value was calculated after eliminating region B. (Data from Hyodo et al., 1991)

102 125 268 229 334 370 234 108

44

<@%-

DUPLICATION

BI-

satnon IT-I satnon VT-I

CONVERSION

toad VT arn~h*lianV T

- human OT +CONVERSION

~~~

350

. . .

250

200

100

50

(-Myr)

Fig. 3 . A plausible evolutionary relationship among neurohypophysial hormone precursors of chum salmon, toad, and human. See text for further explanation. Based on Hyodo et al., (1991).

pathway of neurohypophysial hormone precursors (Hyodo et al., 1991) (Fig. 3). Since extant cyclostomes, the most primitive vertebrates, possess only vasotocin, we adopted the hypothesis that the VP-like and OT-like families of neurohypophysial hormones emerged from an ancestral vasotocin by gene duplication (Acher, 1985). Teleost vasotocin and isotocin precursors might be derived from an ancestral fish vasotocin precursor by gene duplication about 230 - 270 million years ago. A second gene duplication then induced 2 different genes for salmonid vasotocin precursors and isotocin precursors about 100 - 125 million years ago. Tetraploidization of salmonid fish (Ohno et al., 1968) which might be responsible for the second gene duplication thus preceded the gene conversion event around 45 million years ago. The evolutionary distance of vasotocin precursors estimated between the chum salmon and the masu salmon is about 21 million years, whereas that of isotocin precursors is only 3.5 million years (substitution rates not shown in Table 11). The divergence of chum salmon and masu salmon suggested by an isozyme study is about 3.0 million years ago (Numachi, 1984). This value for divergence time of species is consistent with that for isotocin precursors, but not with that for vasotocin precursors. It seems unjustified to assume that, in salmonid fish,

the nucleotide substitution rate of vasotocin precursor genes is unusually rapid, because the evolutionary distance of about 100 million years for vasotocin precursor genes between masu salmon and white sucker is comparable to that for isotocin precursor genes. A possible explanation for the above discrepancy is a duplication of VT-I precursor gene in a common ancestor of chum salmon and masu salmon, since Southern blot analysis showed the presence of two types of VT-I precursor genes in the genome of m a w salmon (Suzuki et al., 1992). The divergence time of 370 million years was estimated for the distance between vasotocin and mesotocin in the toad. In the models presented for the evolution of neurohypophysial hormones on the basis of the amino acid sequences and the phyletic distribution, amphibian mesotocin is considered to be derived from teleost isotocin and replaced by oxytocin in mammals. However, as mentioned above, teleost vasotocin and isotocin precursors might be derived from an ancestral fish vasotocin precursor by gene duplication about 230 - 270 million years ago. It would therefore be reasonable to consider that the teleost isotocin and amphibian mesotocin precursors are derived directly from the corresponding ancestral vasotocin precursors by gene duplication. This idea is supported by the comparisons of overall structural organization of neurohypophysial hormone precursors. In the toad, the vasotocin precursor has the ternary organization which consists of vasotocin, VT-specific neurophysin and copeptin, and the mesotocin precursor composed of binary segments for mesotocin and MTneurophysin lacking a glycoprotein domain. By contrast, in the salmonid and the white sucker, both the vasotocin and isotocin precursors contain binary segments, hormone and hormone-specific neurophysin the carboxyl-terminal of which is extended by about 30 amino acids. Mammalian vasopressin and oxytocin precursors are probably derived from ancestral vasotocin and mesotocin precursors of amphibians about 230 million years ago. The structural organizations and the amino acid sequences of the vasotocin and mesotocin precursors are highly homologous to

45

those of the vasopressin and oxytocin precursors, respectively (Nojiri et al., 1987). These facts strongly support the hypothesis that vasotocin is the molecule ancestral to [Arg8]-vasopressin, whereas mesotocin is the molecule ancestral to oxytocin. A problem arising here is the direction of the recent gene conversion which occurred to encompass the exons encoding the conserved regions of neurophysins. We tentatively hypothesize that an exon encoding the conserved region of OTneurophysin was converted into the corresponding exon for AVP-neurophysin, because the rate of synonymous nucleotide substitution between toad mesotocin and rat oxytocin cDNAs (cKs = 1.238) is smaller than that between vasotocin and vasopressin cDNAs (cKs = 1.457).

Problems arising from fish studies Southern and Northern blot analyses of genomic DNA and mRNA from single masu salmon showed the presence of 2 different genes for each of vasotocin and isotocin pecursors (proVT-I, proVT11, proIT-I and proIT-11); however, only proVT-I and proIT-I areexpressed (Suzuki et al., 1992). One plausible explanation is an occurrence of mutation in the 5 ’-upstream region and/or the coding region. In chum salmon, the expression level of proVT-I1 is considerably lower than that of proVT-I, probably because of a change of the codon for cysteine to a stop codon (Hyodo et al., 1991). Such a decrease in expression of neurohypophysial hormone precursor was also found in the Brattleboro rat (Majzoub et al., 1984; Ivell et al., 1986). These findings indicate the possibility that regulatory differentiation of hormonal genes may take place rather frequently. Morley et al. (1990) suggested the presence of 4 vasotocin genes, 2 proVT-I and 2 proVT-11, in the white sucker. Based on the 45% amino acid heterogeneity between proVT-I and proVT-11, they assume that duplication of the ancient vasotocin precursor gene occurred about 450 million years ago. These genes may have duplicated again at the time of tetraploidization of the white sucker 100 million years ago. Meanwhile, masu salmon seems

to have at least 3 vasotocin precursor genes, 2 proVT-I and 1 proVT-11, although the divergence time among them could not be estimated. These results imply that the evolutionary pathway of VPlike peptides is more complicated than that of OTlike peptides.

Conclusions Based on the evolutionary distances and the structural organization of cDNAs encoding neurohypophysial hormone precursors, we have proposed a possible evolutionary pathway of neurohypophysial hormone precursors (Fig. 3). The evolutionary pathway of vasopressin-like peptides in fish seems to be more complicated than that of oxytocin-like peptides. Teleost isotocin and amphibian mesotocin may have been derived directly from vasotocin by gene duplication. Vasotocin appears to be the ancestral molecule of mammalian vasopressin, whereas mesotocin may be ancestral to oxytocin.

Acknowledgements We are grateful to Professor Howard A. Bern, University of California at Berkeley for critical reading of the manuscript. A part of our study referred to in this article was supported by research grants from the Ministry of Education, Science and Culture, and the Fisheries Agency, Japan.

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Majzoub, J.A., Pappey, A , , Burg, R. and Habener, J.F. (1984) Vasopressin gene is expressed at low levels in the hypothalamus of the Brattleboro rat. Proc. Natl. Acad. Sci. USA, 81: 5296-5299. Michel, G., Chauvet, J., Chauvet, M.T. and Acher, R. (1987) One-step processing of the amphibian vasotocin precursor: structure of a frog (Rana esculenta) “big” neurophysin. Biochem. Biophys. Res. Commun., 149: 538 544. Miyata, T., Hayashida, H., Kikuno, R. and Yasunaga, T. (1985) Computer analysis of homology between genes. In M. Takanami, S. Nishimura and M. Matsumura (Eds.), Methods f o r Gene Research, Tokyo Kagaku Dojin, Tokyo, pp. 381 - 425, (in Japanese). Morley, S.D., Schonrock, C., Heierhorst, J., Figueroa, J., Lederis, K. and Richter, D. (1990) Vasotocin genes of the teleost fish Catostomus commersoni: gene structure, exonintron boundary, and hormone precursor organization. Biochemistry, 29: 2506 - 25 1 1. Nojiri, H., Ishida, I., Miyashita, E., Sato, M., Urano, A. and Deguchi, T. (1987) Cloning and sequence analysis of cDNAs for neurohypophysial hormones vasotocin and mesotocin for the hypothalamus of toad, Bufo japonicus. Proc. Natl. Acad. Sci. USA, 84: 3043 - 3046. Numachi, K. (1984) A study o n the divergence and phylogeny of salmonids by isozymes. The Heredity (Japan), 38: 4 - 11. Ohno, S., Wolf, U. and Atkin, N.B. (1968) Evolution from fish t o mammals by gene duplication. Hereditas, 59: 169- 187. Proux, J.P., Miller, C.A., Li, J.P., Carney, R.L., Girardie, A , , Delaage, M. and Schooley, D.A. (1987) Identification of an arginine vasopressin-like diuretic hormone from Locusta Migratori. Biochem. Biophys. Res. Commun., 149: 180 - 186. Ruppert, S., Scherer, G. and Schutz, G. (1984) Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence. Nature, 308: 554 - 557. Sausville, E., Carney, D. and Battey, J . (1985) The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. J. Biol. Chem., 260: 10236- 10241. Suzuki, M., Hyodo, S. and Urano, A. (1992) Cloning and sequence analyses of vasotocin and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou. Zool. Sci., 9, in press. ~