THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 261, No. 11, Issue of April 15, pp. 48124819, 1986 Printed in U.S.A.
(Received for publication, September 12, 1985)
Nancy M. SherwoodS, Stacia A. Sowers, Daniel R. Marshakli, Blair A. Fraserll ,and Michael J. Brownstein
From the $Biology Department, University of Victoria, Victoria, British Columbia, Canada V 8 W 2Y2, the §Zoology Department, University of New Hampshire, Durham, New Hampshire 03824, the TLaboratory of Cell Biology, National Institute of Mental Health, and the 1) Division of Biochemistry and Biophysics, Center for Drugs and Biologics, Food and Drug Administration, Bethesda, Maryland 20892
Theprimarystructureofgonadotropin-releasing hormone (GnRH) isolated from whole brains of lam-prey is pGlu-His-Tyr-Ser-Leu-Glu-Trp-Lys-Pro-Gly-NH2. This unique decapeptide was isolated and purified from brain extracts by reverse-phase high perform-ance liquid chromatography. The structure of the pep-tide was established from chymotryptic fragments that were identified by protein sequence analysis and fast atombombardmentmassspectrometry.Thepeptide reacts with anantiserumraisedagainstmammalian GnRH andis structurally identified as a member of the GnRH family by the amino and carboxyl termini of pG1u1-His2 and P ~ O ~ - G ~ ~ ~ ~ N H ~ , theconservation of Ser4 in the internal segment of the molecule and its length of 10 amino acids. For the first time, amino acid substitutions are found in positions 3 and 6, critical for biological potency and conformation, respectively. Ad-ditionally, a second form of GnRH (lamprey I1 GnRH), representing about 10% ofthetotal GnRHimmuno-reactive material in the brain, was isolated; its amino acid compositiondiffers by 3 residues from lamprey IGnRH. Synthetic lamprey I GnRH elevates plasma es-tradiol in adult female lampreys.
The pituitary gonadotropins are released in most verte-brates by a factor, gonadotropin-releasing hormone (GnRH’), synthesized in the brain. This factor, isolated in mammals from hypothalami of pigs (Matsuo et al., 1971) and sheep (Burgus et al., 1972) and from placentas of humans (Tan and Rousseau, 1982), is a decapeptide (Fig. 12). Mammalian GnRH injected into submammalian species induces various reproductive events including gonadotropin release (Crim et al., 1978; Peter, 1983; Sherwood, 1986a). However, distinct forms of GnRH are clearly present in submammalian verte-brates as shown by recent immunological, chromatographic (King and Millar, 1980; Sherwood, 1986b; Sherwood et al., 1986), and structural studies. Two forms of GnRH exist in chicken brains (King and Millar 1982a 1982b; Miyamoto et al., 1982,1983, 1984) andadistinct form in salmon brain (Sherwood et al., 1983). The major form of GnRH in amphib-*This workwas supported by the Canadian Medical Research Council, the International Development Research Centre of Canada, the Great Lakes Fisheries Commission, the National Institute of Mental Health, and theFood and Drug Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: GnRH, gonadotropin-releasing hor-mone; HPLC, high performance liquid chromatography. ian brain has the same amino acid composition as mammalian GnRH (Rivier et al., 1981a), but two minor GnRH forms also exist, which are chromatographically and immunologically similar to salmon GnRH (Sherwood et al., 1986). It is of considerable interest to knowif members of the most ancient class of vertebrates, Agnatha, also contain GnRH material. The only living members of this class are the hagfish and lamprey. Injections of a synthetic mammalian GnRH analogue advance ovulation and induce steroidogenesis in lampreys (Sower et al., 1983, 1985). And yet these agna-thans lack the hypothalmo-hypophysial pathways present in other vertebratesfor GnRH. Theyhave neither a portal blood system as in tetrapods and certain primitive fish nor GnRH nerve fibers terminating in the pituitary as in most teleosts (Ball, 1981). The GnRH-staining fibers appear to terminate on a layer of connective tissue above the pars distalis or in the neurohypophysis (Crim et al., 1979a, 1979b; Nozaki and Kobayashi, 1979; Nozaki et al., 1984). Lamprey GnRH appar-ently diffuses toward the pituitary gonadotropes or reaches them by an unknown path (Gorbman, 1980,Nozaki et al., 1984). Earlier we confirmed that lamprey, but not hagfish, brains contain aGnRH-like molecule that can be detected by some, but not all, antisera raised against mammalian GnRH. This immunoreactive material eluted on HPLC in the same position as mammalian GnRH (Sherwood and Sower, 1985). However, it was clear that structural analysis was needed to determine if the GnRH-like peptide in this primitive verte-brate was similar to the amphibian-mammalian GnRH mol-ecule or, if not, couldoffer clues about the nature of an ancestralGnRH molecule. Our currentstructural studies show that lamprey braincontains two distinct forms of GnRH (Table 2). The structure of the major form shows the molecule is a unique peptide (Fig. 5) with only 50% homology to mammalian GnRH. However, it clearly contains a frame-work which is common to all vertebrate GnRH family mem-bers.
The decapeptide reported here is the first peptide from the lamprey brain to be structurally analyzed. The structure of Portions of this paper (“Materialsand Methods,” “Results,” Figs. 1-4 and 6-11, Footnote 3, and Tables 1 and 3)are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be-thesda, MD20814. Request Document No.85M-3082, cite the au-thors, and include a check or money order for $10.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
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this lamprey peptide has a unique internal amino acid se-quence; only the amino and carboxyl termini of pGlu1-His2 and P ~ O ' - G ~ ~ ~ ~ N H ~ identify this molecule as a possible mem-ber of the GnRH family (Fig. 12). The molecule has 50% homology with mammalian and chicken I GnRH and 60% homology with salmon and chicken I1 GnRH. The amino acid sequence of lamprey GnRH shows that the length, termini, and probably amino acids 1-3, thought to be required for biological effect, are the important parts of the GnRHframe-work. The length of the molecule has remained stable for at least 500 million years, the approximate time in evolution when the ancestors to jawed vertebrates separated from the jawless agnathans. Considering the essential role GnRH plays in reproduction, it is surprising that the molecule has not been more highly conserved. The blocked amino terminus of the molecule prevented microsequencing of the intact peptide. The amino terminus was determined from the tripeptide pG1u1-His2-Tyr3after chymotryptic digestion (Fig. 5). The presence of pGlul was then deduced by finding Glu in the amino acid analysis; the mass spectra of the tripeptide and intact peptide were con-sistent with the proposed structure, The amino acid substitutions which have occurred between lamprey GnRH and other vertebrateGnRHpeptides may offer clues about function. Studies of GnRH analogs have shown that changes in single residues may lead to considerable potency and conformational changes. Folding is thought to occur at a 6-7' ,i? turn in mammalian GnRH (Kopple, 1981; Struthers et al., 1985) and presumably also in salmon and
pGlu-His-Tyr-Ser-Leu-Glu-Trp-Lys-Pro-Gly-NH2
CH-2 CH-4
chicken I and I1 GnRH. In mammalian GnRH, certain sub-stitutions in the 6-position produce enhanced biological po-tency (Rivier et d . , 1981b). These substitutions may stabilize a preferred backbone conformation. It remains to be deter-mine if the negatively charged Glu6 in lamprey GnRH would alter the biological potency and folding of mammalian GnRH. This could be tested with synthetic Gly'-lamprey GnRH in lamprey and with Glu'-mammalian GnRH in mammals. The conservation of Ser4 in all 5 GnRH peptides implies an important role because Ser hasthe largest mutation prob-ability of the amino acid residues (Schulz and Schirmer, 1979). Hydrophilic residues such as Ser are usually on the protein surface and changed more frequently. The stability of Ser in the evolution of GnRH may be due to theimportance of the hydrogen bonds which Ser4 is believed to make with Pro9 for maintaining the folded molecule (Struthers et al., 1985). The first report of a substitution in the functional region of the GnRH molecule is Tyr3 in lamprey GnRH; the other four GnRH peptides have Trp3. The functional region, which is considered to be residues 2-3 or 1-3, is necessary for release of luteinizing hormone and follicle-stimulating hormone from the pituitary in mammals (Schally and Coy, 1977; Rivier et al., 1981b). The Tyr3 to Trp3 change may reflect the high mutation probability (Schulz and Schirmer, 1979). A dramatic increase in potency is produced by the T y r to Trp interchange in mammalian (m) GnRH; Tyr3-m GnRH has only 0.1-0.4% potency compared to native Trp3-m GnRH in a mammalian assay (Schally and Coy, 1977). This implies that lamprey and mammalian GnRH receptors have different requirements be-cause native Tyr3-lamprey GnRHbindsandhasintrinsic activity in its own species as shown by the release of steroids after treatment with synthetic lamprey GnRH. Whether the evolutionary change to Trp3 was dueto potency enhancement or receptor requirements might be partially answered by com-paring Trp3- andTyr3-lamprey GnRH in lampreys. The amino acid substitutions in position 7 give some indi-cation of the order in which the GnRH peptides may have evolved. Trp7 appearsin lamprey, salmon, and chicken I1 GnRH; Leu7 is in chicken I and mammalian GnRH. It seems likely that lamprey, salmon, and chicken I1 evolved first or from an ancestral molecule with Trp7. The substitution to Leu7 must have been coincident with or prior to the appear-ance of chicken I and mammalian GnRH. This substitution
Lamprey Salmon Chicken IChicken II Mammal 123
45678910 FIG. 12. Comparison of primary structures of the five known vertebrate GnRH molecules. The boxes show the residues which lamprey shares with all other family members.Lamprey GnRH shows 50% homology with mammal and chicken I GnRH, it shows 60% homology with salmon and chicken I1 GnRH because of the shared Trp residue in position 7. conserves the bulky nonpolar side chain. In position 5 it is difficult to tell if either His5 (chicken 11) or Tyr5 (salmon, chicken I, and mammal) evolved first from Leu' in lamprey or if each evolved separately from Leu5. Since multiple forms of GnRH exist in many submammalian spe-cies, both the Tyr' and His5 form can exist simultaneously as in chicken. More primary structures are needed to determine the speciation of the 5-position in GnRH. The most variable position is 8; each of the GnRHpeptides has a different amino acid. The difference in neutral or basic amino acids in position 8 is the basis of a functional differ-entiation in the oxytocin (neutral)/vasopressin (basic; Arg/ Lys)-family. There is little evidence for such a division in GnRH. It is known that the salmon (Leu') GnRH-like mole-cule appears to act as a neurotransmitter in frog sympathetic ganglia (Eiden and Eskay, 1980; Jan et al., 1983; Jones et al., 1984) or in fish retina (Stell et d., 1984). However, mamma-lian (ArgS) GnRH may act as a local hormone in placenta and both salmon and mammalian GnRH have gonadotropin-re-leasing properties (Peter et al., 1985). Rather, the modifica-tions in position 8 may reflect changes in receptor molecules and binding sitesin the respective species. For example, lamprey has Lys', but the substitution of Lys into mammalian GnRH produces a molecule with only 7.6% luteinizing hor-mone releasing hormone activity in a mammalian bioassay (Schally and Coy, 1977). The amphiphilic secondary structure of lamprey, chicken I, and mammalian GnRH illustrates another common feature of the family. The @-pleatedsheet conformation of mamma-lianGnRH shows thatthe amino acids have alternating hydrophobic and hydrophilic side chains (Kaiser and KCzdy, 1984; Struthers et al., 1985) as does chicken I1 GnRH. Like-wise, lamprey GnRH has hydrophobic residues pG1u1-Tyr3-Leu5-Trp7-Prog alternating with His2-Ser4-G1~6-Lyss-Gly10NH2.The other 2 GnRH molecules do not fit the alter-natingpatternas well. In solution the folded molecule is thought to have a hydrophobic core with H bonding between Trp3 and Gly", His' and Gly", and Ser4 and Prog; this bonds amino and carboxyl termini together. However, when GnRH is exposed to a water/lipid inteface such as a membrane, it may become extended with the hydrophobic side chains on one side and thehydrophilic on the other. The second form of lamprey (lamprey 11) GnRH was more hydrophobic on a Cls HPLC column compared with the first form (lamprey I)
