Peptides, Vol. 13, pp. 1159-1163, 1992
0196-9781/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the USA.
Rainbow Trout (Oncorhynchusmykiss) Neuropeptide Y C. L. B A R T O N , * C. S H A W , *l D. W. H A L T O N *
A N D L. THIM~f
*Comparative Neuroendocrinology Research Group, Schools of Biology & Biochemistry and Clinical Medicine, The Queen's University of Belfast, Northern Ireland and ~'Novo Nordisk A/S, Bagsvaerd, Denmark
R e c e i v e d 12 J u n e 1992
BARTON, C. L., C. SHAW, D. W. HALTON AND L. THIM. Rainbowtrout(Oncorhynchusmykiss) neuropeptideY. PEPTIDES 13(6) 1159-1163, 1992.--Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pr•-Pr•-Lys-Pr•-G•u-Asn-Pr•-G•y-G•u-Asp-A•a-Pr•-Pr•-G•u-G•u-Leu-A•a-Lys-Tyr-Tyr-Thr-A•a-Leu-Arg-His-Tyr-••eAsn-Leu-lle-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchuskisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells. Rainbow trout Neuropeptide Y Coho salmon Pancreatic polypeptide Mass spectroscopy Primary structure Peptide phylogeny
NEUROPEPTIDE Y (NPY), a peptide of 36 amino acid residues, was originally isolated from porcine brain extracts by chemical methodology as a consequence of the presence of an amidated C-terminal tyrosyl residue (30). Sequence analysis of NPY indicated structural similarity to intestinal peptide tyrosine tyrosine (PYY), which was isolated by the same chemical methodology (31), and with pancreatic polypeptide (PP) (13,15). Due to the presence of many common structural features, these peptides have been grouped into the NPY/PP superfamily. Numerous immunohistochemical studies, using antisera raised against mammalian members of this peptide superfamily, have shown that immunoreactivity is present within the neuroendocrine tissues from a wide range of vertebrates (1 l) and invertebrates (12,21,29). The recent isolation of several invertebrate neuropeptides, designated neuropeptide F (NPF) (9,20,22), displaying structural similarity to vertebrate family members, has led to the speculation that these represent the phylogenetic precursors of vertebrate forms. If this is so, then the NPY/PP/NPF superfamily is of ancient evolutionary
Gas-phase sequencing
cells of the adrenal medulla (14). Pancreatic polypeptide is synthesized exclusively in pancreatic endocrine cells (18), and PYY is found within mucosal endocrine cells of the distal intestine (17). Within teleost fishes, the primary structures of analogous peptides have been determined from pancreatic extracts of the anglerfish (Lophiusamericanus) (l), coho salmon (Oncorhynchus kisutch) (16), daddy sculpin (Coitus scorpius) (5), and American eel (Anguilla rostrata) (7). All of these peptides exhibit greater structural similarity to mammalian NPY/PYY than to mammalian PP. To date, the primary structure of a teleost NPY or PYY has not been elucidated. In the rainbow trout (Oncorhynchus mykiss), NPY immunoreactivity has been chromatographically characterized in extracts of brain and pyloric ceca/intestine (3). Single NPYimmunoreactive peptides, with identical chromatographic characteristics, were resolved in each extract leading to the conclusion that, in both neuronal and endocrine tissues of this species, a single NPY-like peptide was expressed. As extracts of intestine/pyloric ceca from the trout might well contain NPY-like peptides from both neuronal and endocrine cell sources, the aim of the present study was to isolate and structurally characterize the NPY-immunoreactive peptide
origin.
Neuropeptide Y in mammals is synthesized in neurons of the central and peripheral nervous system and in chromaflin
Requests for reprints should be addressed to Dr. C. Shaw, Wellcome Research Laboratories, Mulhouse Building, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland.
1159
1160
BARTON ET AL. Vt
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FIG. 1. Sequential chromatographic purification of rainbow trout NPY on (a) Sephadex G-50 gel permeation column; (b) semi-preparative Partisil l00DS 3 column; (c) analytical Vydac C-8 column; and (d) analytical Vydac C-4 column. Profiles ofNPY immunoreactivity are show in (a)-(c) and the absorbance profile is shown in (d). For chromatographic conditions at each stage, see text.
from extracts of brain, where it is known to occur exclusively within neurons (3,10). METHOD
the remaining solution were concentrated by pumping through six Sep-Pak C-18 cartridges (Waters Associates, Milford', MA) arranged in series. Bound peptides were eluted with acetonitrile and the eluates were evaporated to dryness.
Tissue Collection and Preparation Specimens of both sexes of rainbow trout (Oncorhynchus mykiss) (2-3 years old; 35-50 cm in length; 900-1500 g in weight) were obtained from the Ministry of Agriculture fish farm at Movanagher, County Antrim, Northern Ireland and Seven Springs Fish Farm, Lame, County Antrim, Northern Ireland. Fish were killed by a blow to the head and the brains were dissected immediately, washed in fish saline, touch-dried, weighed, and frozen at - 2 0 ° C prior to peptide extraction.
Peptide Extraction Brain tissue from 60 trout (87 g wet weight) was homogenized in 435 ml of acidified ethanol (0.7 M HC1; 3:1; v/v). The homogenate was incubated at 4°C for 12 h and then centrifuged at 4000 x g for 30 min at 4°C. The supernatant was decanted and the tissue pellet was reextracted with 400 ml of acidified ethanol. The supernatants from both extracts were combined and ethanol was removed under reduced pressure. Peptides in
Peptide Isolation Peptide extract residues were dissolved in 3 ml of 2 M acetic acid and centrifuged for 30 min at 4000 X g to clarify. The supernatant was loaded directly onto a 90 x 1.6 cm column of Sephadex G50 (fine) (Pharmacia, Uppsala, Sweden), equilibrated in 2 Macetic acid, and eluted at a flow rate 12.5 ml/h. Fractions (2.5 ml) were collected at 12-min intervals. An aliquot (10 ul) of each fraction was removed and subjected to NPY radioimmunoassay employing an antiserum (code 8999) raised to a synthetic C-terminal NPY hexapeptide analog (25), which exhibits an almost absolute requirement for a C-terminal amide group. This antiserum was a kind gift from Dr. Thue Schwartz, Rigshospitalet, Copenhagen, Denmark. Fractions containing NPY immunoreactivity were chromatographed on a semipreparative (1 X 60 cm) Partisil 10 ODS 3 reverse-phase column (Whatman, Kent, UK) eluted with a linear gradient from trifluoroacetic acid (TFA)/water (0.1:99.9, v/v) to TFA/water/acetonitrile (0.1:29.9:
RAINBOW TROUT NPY
1161
TABLE 1 AUTOMATED EDMAN DEGRADATIONOF RAINBOW TROUT (Oncorhynchusmykiss) NEUROPEPTIDE Y CycleNo.
PTH-Amino Acid
Yield(pmol)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 !9 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Tyr(Y) Pro (P) Pro (P) Lys(K) Pro (P) Glu (E) Asn(N) Pro (P) GIy(G) Glu (E) Asp(D) Ala (A) Pro (P) Pro (P) Glu (E) Glu (E) Leu (L) Ala(A) Lys (K) Tyr (Y) Tyr (Y) Thr(T) Ala (A) Leu (L) Arg(R) His(H) Tyr(Y) lie (I) Asn(N) Leu (L) lie (I) Thr(T) Arg(R) GIn(Q) Arg(R) Tyr(Y)
263 222 249 207 215 196 197 188 166 168 158 134 127 129 80 109 109 104 51 82 109 46 45 72 70 28 56 53 42 38 48 32 40 26 44 20
Averagerepetitiveyield:93.5%. Singleletter notation for amino acids in parentheses.
70.0, v/v/v) over 70 min at a flow rate of 3 ml/min. Fractions (3 ml) were collected each rain and the elution of NPY immunoreactivity was determined by radioimmunoassay as before. Neuropeptide Y-immunoreactive fractions were then sequentially chromatographed on analytical Vydac C-8 and C-4 columns using gradients formed from TFA/water (0.1:99.9, v/v) to TFA/water/acetonitrile (0.1:74.9:25.0, v/v/v) in 5 min then to TFA/water/acetonitrile (0,1:49.9:50.0, v/v/v) over the next 60 min at a flow rate of 1.5 ml/min. Column effluents were monitored simultaneously at 214 n m and 280 nm, and in the final purification stage, peaks ofabsorbance were collected manually. The NPY-immunoreactive peptide was located by radioimmunoassay of fractions containing these absorbance peaks.
Structural Analyses Approximately 25 pmol of purified peptide was subjected to 252Cf-plasma desorption mass spectroscopy (PDMS) using a
Biolon 20K time-of-flight instrument. The sample was dissolved in ethanol/water/formic acid (50:40:10, v/v/v) and applied to a nitrocellulose-coveredtarget that was spin-dried and microrinsed. Spectra were recorded at 16 kV for 106 primary fission events. Approximately 280 pmol of purified peptide was subjected to automated Edman degradation using an Applied Biosystems 470A gas phase sequencer. The limit for detection of phenylthiohydantoin (PTH) amino acids was 0.5 pmol. RESULTS
Peptide Isolation The crude extract of trout brain contained the equivalent of 519 pmol/g wet weight of brain ofNPY immunoreactivity. The recovery of immunoreactive peptide after each chromatographic fractionation was of the order of 50-60% of that applied. Since only peak immunoreactive fractions were rechromatographed to facilitate the purification, 2.8 nmol of NPY-immunoreactive peptide was finally purified to apparent homogeneity. The elution profiles of each purification step are illustrated in Fig. la-d. A single immunoreactive peptide was resolved at each stage that was more hydrophilic than synthetic human NPY.
Structural Analyses The molecular mass of trout NPY was estimated by PDMS as 4303.9 Da. The entire primary structure of a 36 amino acid residue peptide was unequivocally established in a single gasphase sequencing run (Table 1). The computed molecular mass (4304 Da) from this sequence was consistent with that determined by mass spectroscopy. Due to the full molar cross-reactivity of trout NPY with the NPY antiserum employed, the peptide was deemed to be amidated. The primary structure of rainbow trout NPY was identical to that of PP previously isolated from the pancreas of the coho salmon (Oncorhynchus kisutch) (16). The primary structure of this neuropeptide is compared with frog and human NPY in Table 2. DISCUSSION The elucidation of the primary structure of rainbow trout NPY by isolation of the peptide from crude brain extracts represents the first determined from a teleost fish using this approach. Radioimmunoassay of chromatographic fractions throughout the purification procedure, with an antiserum directed against the C-terminus of mammalian NPY, consistently detected a single immunoreactive peptide. The sequence of this neuropeptide is identical to that of pancreatic polypeptide isolated from the endocrine pancreas of the coho salmon (Oncorhynchus kisutch) (16), and represents the first vertebrate NPY of identical primary structure to an analog isolated from extraneural tissues.
TABLE 2 COMPARISON OF THE PRIMARYSTRUCTURESOF TROUT, FROG, AND HUMAN NPY Trout NPY Frog NPY HumanNPY
YPPKPENPGEDAPPEELAKYYTALRHYINLITRQRY,NH
2
YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY,NH
2
YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY,NH2 * * * ** * *
* Sitesof aminoacid substitutions.
1162
BARTON ET AL.
The fishes are an interesting group with respect to the primary structural evolution of the NPY/PP superfamily. The sequences of peptides isolated from pancreatic extracts of elasmobranch (7,8,26), holostean (7,27), and teleost ( 1,5,7,16) fishes bear striking structural resemblances to mammalian NPY and PYY, all of which possess an N-terminal tyrosyl residue and terminate in the tetrapeptide amide sequence, -Arg-Gln-Arg-Tyr-NH2.In the pancreas of higher vertebrate groups, pancreatic polypeptides are more variable in these parameters, possessing a short-chain aliphatic residue at the N-terminus (Ala in amphibians, Thr in reptiles, Gly in birds, Ala or Ser in mammals) and terminating in -Arg-Pro-Arg-Phe-NH2 (amphibians and reptiles), -Arg-HisArg-Tyr-NH2 (birds), or -Arg-Pro-Arg-Tyr-NH2 (mammals) (13,23,28). As the fish pancreatic polypeptides differ little in primary structure from mammalian NPY, the aim of the present study was to elucidate the primary structure of a fish NPY by isolation from brain extracts. The primary structures of homed shark and goldfish NPY have been deduced from genomic DNA libraries using probes to the mammalian analog (19). While this approach is technically sound, it generates data that must be interpreted with caution. Due to the fact that fish possess NPYrelated peptides in pancreatic and intestinal tissues as well as within the brain, it may be difficult to identify the anatomical site of expression of peptides whose structures are determined by this approach. Isolation and sequencing of closely related peptides from discrete tissue extracts, the approach adopted in the present study, circumvents this problem of interpretation. As the primary structure of amphibian NPY, recently determined independently by two groups (4,24), differs in only a single site
(Lys for Arg at residue 19) from human NPY, the evolutionary constraints on primary structure of this neuropeptide are considerable. It was thus an unexpected finding that the primary structures of the neuropeptide and pancreatic peptide from two congeneric species of salmonid fish were identical. In a recent study that describes the immunocytochemical distribution and chromatographic characterisation of NPY immunoreactivity in the rainbow trout, immunoreactivity was found in central and peripheral neurons, intestinal endocrine cells, and pancreatic endocrine cells (3). In addition, immunoreactivity was attributed to single peptides of identical retention time when extracts of these tissues were subjected to chromatographic analysis. The possibility therefore exists that in the rainbow trout, and perhaps also in related Pacific salmonids, a single peptide is expressed in both central neurons and peripheral endocrine tissues. If so, this peptide would necessarily perform all of the regulatory functions of additional family members occurring in higher vertebrate peptide regulatory systems and would imply that these fishes have adopted a different evolutionary strategy to those of higher vertebrate groups. In terms of regulatory peptide evolution it is accepted that, as the nervous system predates the presence of an intestine that predates the presence of a pancreas, the neuropeptide is likely to be the protopeptide in cases where families of structurally related peptides are perceived as having arisen by gene duplication and mutation events (2). Within this scheme, one might speculate that in salmonid fishes, mutation has not occurred, leading to the expression of a single peptide in anatomically discrete tissues.
REFERENCES
1. Andrews, P. C.; Hawke, D.; Shively, J. E.; Dixon, J. E. A nonamidated peptide homologous to porcine peptide YY and neuropeptide YY. Endocrinology 116:2677-2681; 1985. 2. Barrington, E. J. W. The phylogeny of the endocrine system. Expefientia 42:775-781; 1986. 3. Barton, C. L.; Shaw, C.; Halton, D. W.; Johnston, C. F.; Buchanan, K. D. Distribution and characterisation of neuropeptide Y immunoreactivity in the brain and gastrointestinal tract of the rainbow trout, Oncorhynchus mykiss. Comp. Biochem. Physiol. (in press). 4. Chattel, N.; Conlon, J. M.; Danger, J.-M.; Fournier, A.; Tonon, M.-C.; Vaudry, H. Characterization of melanotropin-release-inhibiting factor (melanostatin) from frog brain: Homology with human neuropeptide Y. Proc. Natl. Acad. SCi.USA 88:3862-3866; 1991. 5. Conlon, J. M.; schmidt, W. E.; Gallwitz, B.; Falkmer, S.; Thim, L. Characterisationof an amidated form of pancreaticpolypeptidefrom daddy sculpin (Cottus scorpius). Regul. Pept. 16:261-268; 1986. 6. Conion, J. M.; Bjoruholm, B.; Jorgensen, F. S.; Youson, J. H.; Schwartz, T. W. Primary structure and conformational analysis of peptide methionine-tyrosine, a peptide related to neuropeptide Y and peptide YY isolated from lamprey intestine. Eur. J. Biochem. 199:293-298; 1991. 7. Conlon, J. M.; Bjenning, C.; Moon, T. W.; Youson, J. H.; Thim, L. NeuropeptideY-relatedpeptides from the pancreas of a teleostean (eel), holostean (bowfin) and elasmobranch (skate) fish. Peptides 12: 221-226; 1991. 8. Conlon, J. M.; Balasubramaniam, A.; Hazon, N. Structural characterisation and biological activity of a NPY-related peptide from the dogfish, Scyliorhinus canicula. Endocrinology 128:2273-2279; 1991. 9. Curry, W. J.; Shaw, C.; Johnston, C. F.; Thim, L.; Buchanan, K. D. Neuropeptide F: Primary structure from the turbellarian, Artioposthia triangulata. Comp. Biochem. Physiol. 101C:269-274; 1992. 10. Danger, J. M.; Breton, B.; Vallarino, M.; St-Pierre, S.; Pelletier,G.; Vaudry, H. Localisation,characterizationand neuroendocrineaction
of neuropeptide Y in the trout brain. Ann. NY Acad. SCi.611:504507; 1990. 11. EI-Salhy, M.; Grimelius, L.; Emson, P. C.; Falkmer, S. Polypeptide YY- and neuropeptide Y-immunoreactive cells and nerves in the endocrine pancreas of some vertebrates: An onto- and phylogenetic study. Histochem. J. 19:11I-117; 1987. 12. Endo, Y.; lwanaga, T.; Fujita, T.; Nishiitsutsuji-Uwo,J. Localisation of pancreatic polypeptide (PP)-likeimmunoreactivity in the central and visceralnervoussystemsof the cockroach Periplaneta. CellTissue Res. 227:1-9; 1982. 13. Glover, I. D.; Barlow, D. J.; Pitts, J. E.; Wood, S. P.; Tickle, 1. J.; Blundell, T. L.; Tatemoto, K.; Kimmel, J. R.; Wollmer, A.; Strassburger, W.; Zhang, Y. S. Conformational studies on the pancreatic polypeptide hormone family. Eur. J. Biochem. 142:379-385; 1985. 14. Gray, T. S.; Morley, J. E. Neuropeptide Y: Anatomical distribution and possible function in mammalian nervous system. Life SCi. 38: 389-401; 1986. 15. Kimmel, J. R.; Hayden, L. J.; Pollock, H. G. Isolation and characterizafionof a new pancreaticpolypepfidehormone. J. Biol.Chem. 250:9369-9376; 1975. 16. Kimmel, J. R.; Plisetskaya, E. M.; Pollock, H. G.; Hamilton, J. W.; Rouse, J. B.; Ebner K. E.; Rawitch, A. B. Structure of a paptide from coho salmon endocrine pancreas with homology to neuropeptide Y. Biochem. Biophys. Res. Commun. 141:1084-1091; 1986. 17. Kishimoto, S.; Kato, R.; Mukai, T.; Kanbara, A.; Okamoto, K.; Shimizu, S.; Daitoku, K.; Kajiyama, G. Distribution and endocrine morphology of polypcptide YY (PYY) containingcellsin the human gut. Hiroshima J. Med. SCi. 34:155-160; 1985. 18. Langslow,D. R.; Kimmel, J. R.; Pollock, H. G. Studies on the distribution of a new avian pancreatic polypcptide and insulin among birds, reptiles, amphibians and mammals. Endocrinology 93:558565; 1973. 19. Larhammer, D.; Blomqvist,A.; Lundell, I. Strong evolutionary conservation of neuropeptide Y between mammals, chicken, goldfish and horned shark. Neuroendocrinology 52(Suppl. 1):53; 1990.
RAINBOW T R O U T NPY 20. Leung, P. S.; Shaw, C.; Maule, A. G.; Johnston, C. F.; Irvine, G. B. The primary structure of neuropeptide F (NPF) from the garden snail, Helix aspersa. Regul. Pept. 41:71-81; 1992. 21. Maule, A. G.; Shaw, C.; Halton, D. W.; Johnston, C. F.; Fairweather, I. Localisation, quantification and characterisation of pancreatic polypeptide immunoreactivity in the parasitic flatworm Diclidophora merlangi and its fish host (Merlangius merlangus). Gen. Comp. Endocrinol. 74:50-56; 1989. 22. Maule, A. G.; Shaw, C.; Halton, D. W.; Thim, L.; Johnston, C. F.; Fairweather, I.; Buchanan, K. D. Neuropeptide F: A novel parasitic flatworm regulatory peptide from Moniezia expansa (Cestoda: Cyclophyllidea). Parasitology 102:309-316; 1991. 23. McKay, D. M.; Shaw, C.; Thim, L.; Johnston, C. F.; Halton, D. W.; Fairweather, I.; Buchanan, K. D. The complete primary structure of pancreatic polypeptide from the European common frog, Rana temporaria. Regul. Pept. 31:187-198; 1990. 24. McKay, D. M.; Shaw, C.; Halton, D. W.; Thim, L.; Buchanan, K. D. The primary structure and tissue distribution of an amphibian neuropeptide Y. Regul. Pept. 37:143-153; 1992. 25. O'Hare, M. M. T.; Schwartz, T. W. Expression and precursor processing of neuropeptide Y in human pheochromocytoma and neuroblastoma tumors. Cancer Res. 49:7010-70141 1989.
1163 26. Pan, J.-Z.; Shaw, C.; Halton, D. W.; Thim, L.; Johnston, C. F.; Buchanan, K. D. The primary slructure of peptide Y (PY) of the spiny dogfish, Squalus acanthiar. Immunocytochemical localisationand isolation fi'om the pancreas. Comp. Biochem. Physiol. 102B:1-5; 1992. 27. Pollock, H. G.; Kimmel, J. R.; Hamilton, J. W.; Rouse, J. B.; Ebner, K. E.; Lance, V.; Rawitch, A. B. Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide. Gen. Comp. Endocrinol. 61:375-3821 1987. 28. Pollock, H. G.; Hamilton, J. W.; Rouse, J. B.; Ebner, K. E.; Rawitch, A. B. Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin and two glucagon-like peptides. J. Biol. Chem. 263: 9746-97511 1988. 29. Sundler, F.; Hakanson, R.; Alumets, J.; VaUes, B. Neuronal localisation of pancreatic polypeptide (PP) and vasoactive intestinal polypeptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris). Brain Res. Bull. 2:61-651 1977. 30. Tatemoto, K. Neuropeptide Y: Complete amino acid sequence of the brain peptide. Proc. Natl. Acad. Sci. USA 79:5485-5489; 1982. 31. Tatemoto, K. Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion. Proc. Natl. Acad. Sci. USA 79:2514-2518; 1982.
0196-9781/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the USA.
Rainbow Trout (Oncorhynchusmykiss) Neuropeptide Y C. L. B A R T O N , * C. S H A W , *l D. W. H A L T O N *
A N D L. THIM~f
*Comparative Neuroendocrinology Research Group, Schools of Biology & Biochemistry and Clinical Medicine, The Queen's University of Belfast, Northern Ireland and ~'Novo Nordisk A/S, Bagsvaerd, Denmark
R e c e i v e d 12 J u n e 1992
BARTON, C. L., C. SHAW, D. W. HALTON AND L. THIM. Rainbowtrout(Oncorhynchusmykiss) neuropeptideY. PEPTIDES 13(6) 1159-1163, 1992.--Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pr•-Pr•-Lys-Pr•-G•u-Asn-Pr•-G•y-G•u-Asp-A•a-Pr•-Pr•-G•u-G•u-Leu-A•a-Lys-Tyr-Tyr-Thr-A•a-Leu-Arg-His-Tyr-••eAsn-Leu-lle-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchuskisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells. Rainbow trout Neuropeptide Y Coho salmon Pancreatic polypeptide Mass spectroscopy Primary structure Peptide phylogeny
NEUROPEPTIDE Y (NPY), a peptide of 36 amino acid residues, was originally isolated from porcine brain extracts by chemical methodology as a consequence of the presence of an amidated C-terminal tyrosyl residue (30). Sequence analysis of NPY indicated structural similarity to intestinal peptide tyrosine tyrosine (PYY), which was isolated by the same chemical methodology (31), and with pancreatic polypeptide (PP) (13,15). Due to the presence of many common structural features, these peptides have been grouped into the NPY/PP superfamily. Numerous immunohistochemical studies, using antisera raised against mammalian members of this peptide superfamily, have shown that immunoreactivity is present within the neuroendocrine tissues from a wide range of vertebrates (1 l) and invertebrates (12,21,29). The recent isolation of several invertebrate neuropeptides, designated neuropeptide F (NPF) (9,20,22), displaying structural similarity to vertebrate family members, has led to the speculation that these represent the phylogenetic precursors of vertebrate forms. If this is so, then the NPY/PP/NPF superfamily is of ancient evolutionary
Gas-phase sequencing
cells of the adrenal medulla (14). Pancreatic polypeptide is synthesized exclusively in pancreatic endocrine cells (18), and PYY is found within mucosal endocrine cells of the distal intestine (17). Within teleost fishes, the primary structures of analogous peptides have been determined from pancreatic extracts of the anglerfish (Lophiusamericanus) (l), coho salmon (Oncorhynchus kisutch) (16), daddy sculpin (Coitus scorpius) (5), and American eel (Anguilla rostrata) (7). All of these peptides exhibit greater structural similarity to mammalian NPY/PYY than to mammalian PP. To date, the primary structure of a teleost NPY or PYY has not been elucidated. In the rainbow trout (Oncorhynchus mykiss), NPY immunoreactivity has been chromatographically characterized in extracts of brain and pyloric ceca/intestine (3). Single NPYimmunoreactive peptides, with identical chromatographic characteristics, were resolved in each extract leading to the conclusion that, in both neuronal and endocrine tissues of this species, a single NPY-like peptide was expressed. As extracts of intestine/pyloric ceca from the trout might well contain NPY-like peptides from both neuronal and endocrine cell sources, the aim of the present study was to isolate and structurally characterize the NPY-immunoreactive peptide
origin.
Neuropeptide Y in mammals is synthesized in neurons of the central and peripheral nervous system and in chromaflin
Requests for reprints should be addressed to Dr. C. Shaw, Wellcome Research Laboratories, Mulhouse Building, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland.
1159
1160
BARTON ET AL. Vt
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60
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FIG. 1. Sequential chromatographic purification of rainbow trout NPY on (a) Sephadex G-50 gel permeation column; (b) semi-preparative Partisil l00DS 3 column; (c) analytical Vydac C-8 column; and (d) analytical Vydac C-4 column. Profiles ofNPY immunoreactivity are show in (a)-(c) and the absorbance profile is shown in (d). For chromatographic conditions at each stage, see text.
from extracts of brain, where it is known to occur exclusively within neurons (3,10). METHOD
the remaining solution were concentrated by pumping through six Sep-Pak C-18 cartridges (Waters Associates, Milford', MA) arranged in series. Bound peptides were eluted with acetonitrile and the eluates were evaporated to dryness.
Tissue Collection and Preparation Specimens of both sexes of rainbow trout (Oncorhynchus mykiss) (2-3 years old; 35-50 cm in length; 900-1500 g in weight) were obtained from the Ministry of Agriculture fish farm at Movanagher, County Antrim, Northern Ireland and Seven Springs Fish Farm, Lame, County Antrim, Northern Ireland. Fish were killed by a blow to the head and the brains were dissected immediately, washed in fish saline, touch-dried, weighed, and frozen at - 2 0 ° C prior to peptide extraction.
Peptide Extraction Brain tissue from 60 trout (87 g wet weight) was homogenized in 435 ml of acidified ethanol (0.7 M HC1; 3:1; v/v). The homogenate was incubated at 4°C for 12 h and then centrifuged at 4000 x g for 30 min at 4°C. The supernatant was decanted and the tissue pellet was reextracted with 400 ml of acidified ethanol. The supernatants from both extracts were combined and ethanol was removed under reduced pressure. Peptides in
Peptide Isolation Peptide extract residues were dissolved in 3 ml of 2 M acetic acid and centrifuged for 30 min at 4000 X g to clarify. The supernatant was loaded directly onto a 90 x 1.6 cm column of Sephadex G50 (fine) (Pharmacia, Uppsala, Sweden), equilibrated in 2 Macetic acid, and eluted at a flow rate 12.5 ml/h. Fractions (2.5 ml) were collected at 12-min intervals. An aliquot (10 ul) of each fraction was removed and subjected to NPY radioimmunoassay employing an antiserum (code 8999) raised to a synthetic C-terminal NPY hexapeptide analog (25), which exhibits an almost absolute requirement for a C-terminal amide group. This antiserum was a kind gift from Dr. Thue Schwartz, Rigshospitalet, Copenhagen, Denmark. Fractions containing NPY immunoreactivity were chromatographed on a semipreparative (1 X 60 cm) Partisil 10 ODS 3 reverse-phase column (Whatman, Kent, UK) eluted with a linear gradient from trifluoroacetic acid (TFA)/water (0.1:99.9, v/v) to TFA/water/acetonitrile (0.1:29.9:
RAINBOW TROUT NPY
1161
TABLE 1 AUTOMATED EDMAN DEGRADATIONOF RAINBOW TROUT (Oncorhynchusmykiss) NEUROPEPTIDE Y CycleNo.
PTH-Amino Acid
Yield(pmol)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 !9 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Tyr(Y) Pro (P) Pro (P) Lys(K) Pro (P) Glu (E) Asn(N) Pro (P) GIy(G) Glu (E) Asp(D) Ala (A) Pro (P) Pro (P) Glu (E) Glu (E) Leu (L) Ala(A) Lys (K) Tyr (Y) Tyr (Y) Thr(T) Ala (A) Leu (L) Arg(R) His(H) Tyr(Y) lie (I) Asn(N) Leu (L) lie (I) Thr(T) Arg(R) GIn(Q) Arg(R) Tyr(Y)
263 222 249 207 215 196 197 188 166 168 158 134 127 129 80 109 109 104 51 82 109 46 45 72 70 28 56 53 42 38 48 32 40 26 44 20
Averagerepetitiveyield:93.5%. Singleletter notation for amino acids in parentheses.
70.0, v/v/v) over 70 min at a flow rate of 3 ml/min. Fractions (3 ml) were collected each rain and the elution of NPY immunoreactivity was determined by radioimmunoassay as before. Neuropeptide Y-immunoreactive fractions were then sequentially chromatographed on analytical Vydac C-8 and C-4 columns using gradients formed from TFA/water (0.1:99.9, v/v) to TFA/water/acetonitrile (0.1:74.9:25.0, v/v/v) in 5 min then to TFA/water/acetonitrile (0,1:49.9:50.0, v/v/v) over the next 60 min at a flow rate of 1.5 ml/min. Column effluents were monitored simultaneously at 214 n m and 280 nm, and in the final purification stage, peaks ofabsorbance were collected manually. The NPY-immunoreactive peptide was located by radioimmunoassay of fractions containing these absorbance peaks.
Structural Analyses Approximately 25 pmol of purified peptide was subjected to 252Cf-plasma desorption mass spectroscopy (PDMS) using a
Biolon 20K time-of-flight instrument. The sample was dissolved in ethanol/water/formic acid (50:40:10, v/v/v) and applied to a nitrocellulose-coveredtarget that was spin-dried and microrinsed. Spectra were recorded at 16 kV for 106 primary fission events. Approximately 280 pmol of purified peptide was subjected to automated Edman degradation using an Applied Biosystems 470A gas phase sequencer. The limit for detection of phenylthiohydantoin (PTH) amino acids was 0.5 pmol. RESULTS
Peptide Isolation The crude extract of trout brain contained the equivalent of 519 pmol/g wet weight of brain ofNPY immunoreactivity. The recovery of immunoreactive peptide after each chromatographic fractionation was of the order of 50-60% of that applied. Since only peak immunoreactive fractions were rechromatographed to facilitate the purification, 2.8 nmol of NPY-immunoreactive peptide was finally purified to apparent homogeneity. The elution profiles of each purification step are illustrated in Fig. la-d. A single immunoreactive peptide was resolved at each stage that was more hydrophilic than synthetic human NPY.
Structural Analyses The molecular mass of trout NPY was estimated by PDMS as 4303.9 Da. The entire primary structure of a 36 amino acid residue peptide was unequivocally established in a single gasphase sequencing run (Table 1). The computed molecular mass (4304 Da) from this sequence was consistent with that determined by mass spectroscopy. Due to the full molar cross-reactivity of trout NPY with the NPY antiserum employed, the peptide was deemed to be amidated. The primary structure of rainbow trout NPY was identical to that of PP previously isolated from the pancreas of the coho salmon (Oncorhynchus kisutch) (16). The primary structure of this neuropeptide is compared with frog and human NPY in Table 2. DISCUSSION The elucidation of the primary structure of rainbow trout NPY by isolation of the peptide from crude brain extracts represents the first determined from a teleost fish using this approach. Radioimmunoassay of chromatographic fractions throughout the purification procedure, with an antiserum directed against the C-terminus of mammalian NPY, consistently detected a single immunoreactive peptide. The sequence of this neuropeptide is identical to that of pancreatic polypeptide isolated from the endocrine pancreas of the coho salmon (Oncorhynchus kisutch) (16), and represents the first vertebrate NPY of identical primary structure to an analog isolated from extraneural tissues.
TABLE 2 COMPARISON OF THE PRIMARYSTRUCTURESOF TROUT, FROG, AND HUMAN NPY Trout NPY Frog NPY HumanNPY
YPPKPENPGEDAPPEELAKYYTALRHYINLITRQRY,NH
2
YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY,NH
2
YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY,NH2 * * * ** * *
* Sitesof aminoacid substitutions.
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BARTON ET AL.
The fishes are an interesting group with respect to the primary structural evolution of the NPY/PP superfamily. The sequences of peptides isolated from pancreatic extracts of elasmobranch (7,8,26), holostean (7,27), and teleost ( 1,5,7,16) fishes bear striking structural resemblances to mammalian NPY and PYY, all of which possess an N-terminal tyrosyl residue and terminate in the tetrapeptide amide sequence, -Arg-Gln-Arg-Tyr-NH2.In the pancreas of higher vertebrate groups, pancreatic polypeptides are more variable in these parameters, possessing a short-chain aliphatic residue at the N-terminus (Ala in amphibians, Thr in reptiles, Gly in birds, Ala or Ser in mammals) and terminating in -Arg-Pro-Arg-Phe-NH2 (amphibians and reptiles), -Arg-HisArg-Tyr-NH2 (birds), or -Arg-Pro-Arg-Tyr-NH2 (mammals) (13,23,28). As the fish pancreatic polypeptides differ little in primary structure from mammalian NPY, the aim of the present study was to elucidate the primary structure of a fish NPY by isolation from brain extracts. The primary structures of homed shark and goldfish NPY have been deduced from genomic DNA libraries using probes to the mammalian analog (19). While this approach is technically sound, it generates data that must be interpreted with caution. Due to the fact that fish possess NPYrelated peptides in pancreatic and intestinal tissues as well as within the brain, it may be difficult to identify the anatomical site of expression of peptides whose structures are determined by this approach. Isolation and sequencing of closely related peptides from discrete tissue extracts, the approach adopted in the present study, circumvents this problem of interpretation. As the primary structure of amphibian NPY, recently determined independently by two groups (4,24), differs in only a single site
(Lys for Arg at residue 19) from human NPY, the evolutionary constraints on primary structure of this neuropeptide are considerable. It was thus an unexpected finding that the primary structures of the neuropeptide and pancreatic peptide from two congeneric species of salmonid fish were identical. In a recent study that describes the immunocytochemical distribution and chromatographic characterisation of NPY immunoreactivity in the rainbow trout, immunoreactivity was found in central and peripheral neurons, intestinal endocrine cells, and pancreatic endocrine cells (3). In addition, immunoreactivity was attributed to single peptides of identical retention time when extracts of these tissues were subjected to chromatographic analysis. The possibility therefore exists that in the rainbow trout, and perhaps also in related Pacific salmonids, a single peptide is expressed in both central neurons and peripheral endocrine tissues. If so, this peptide would necessarily perform all of the regulatory functions of additional family members occurring in higher vertebrate peptide regulatory systems and would imply that these fishes have adopted a different evolutionary strategy to those of higher vertebrate groups. In terms of regulatory peptide evolution it is accepted that, as the nervous system predates the presence of an intestine that predates the presence of a pancreas, the neuropeptide is likely to be the protopeptide in cases where families of structurally related peptides are perceived as having arisen by gene duplication and mutation events (2). Within this scheme, one might speculate that in salmonid fishes, mutation has not occurred, leading to the expression of a single peptide in anatomically discrete tissues.
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1163 26. Pan, J.-Z.; Shaw, C.; Halton, D. W.; Thim, L.; Johnston, C. F.; Buchanan, K. D. The primary slructure of peptide Y (PY) of the spiny dogfish, Squalus acanthiar. Immunocytochemical localisationand isolation fi'om the pancreas. Comp. Biochem. Physiol. 102B:1-5; 1992. 27. Pollock, H. G.; Kimmel, J. R.; Hamilton, J. W.; Rouse, J. B.; Ebner, K. E.; Lance, V.; Rawitch, A. B. Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide. Gen. Comp. Endocrinol. 61:375-3821 1987. 28. Pollock, H. G.; Hamilton, J. W.; Rouse, J. B.; Ebner, K. E.; Rawitch, A. B. Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin and two glucagon-like peptides. J. Biol. Chem. 263: 9746-97511 1988. 29. Sundler, F.; Hakanson, R.; Alumets, J.; VaUes, B. Neuronal localisation of pancreatic polypeptide (PP) and vasoactive intestinal polypeptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris). Brain Res. Bull. 2:61-651 1977. 30. Tatemoto, K. Neuropeptide Y: Complete amino acid sequence of the brain peptide. Proc. Natl. Acad. Sci. USA 79:5485-5489; 1982. 31. Tatemoto, K. Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion. Proc. Natl. Acad. Sci. USA 79:2514-2518; 1982.
