Isolation and primary structure of gastrin-releasing peptide from a teleost fish, the trout (Oncorhynchus mykiss)

Peptides.Vol. 13, pp. 995-999, 1992

0196-9781/92 $5.00 + .00 Copyright © 1992PergamonPress Ltd.

Printed in the USA.

Isolation and Primary Structure of GastrinReleasing Peptide From a Teleost Fish, the Trout

(Oncorhynchusmykiss) JORGEN

JENSEN

A N D J. M I C H A E L

CONLON l

Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, N E 681 78 R e c e i v e d 4 M a y 1992 JENSEN, J. AND J. M. CONLON. Isolation and primary structure of gastrin-releasing peptidefrom a teleostfish, the trout (Oncorhynchus rnykis,s). PEPT1DES 13(5) 995-999, 1992.--lmmunohistochemical studies have established that fish gastrointestinal tissues contain peptides with gastrin-releasing peptide (GRP)/bombesin-like immunoreactivity, but the molecular nature of this material is unclear. In this study, the most abundant peptide that was immunoreactive towards an antiserum raised against pig GRP was isolated in pure form from an extract of the stomach of the rainbow trout (Oncorhynchusmykiss). The primary structure of the peptide was established as: Ser-G~u-Asn-Thr-G~y-A~a-~e-G~y-Lys-Va~°-Phe-Pr~-Arg-G~y-Asn-His-Trp-A~a-Va~-G~y2°-HisLeu-Met-NH2. Although this amino acid sequence is shorter than those of mammalian GRPs by four residues, the COOHterminal dodecapeptide is identical to the corresponding region in pig GRP. The data indicate, therefore, that the predominant molecular form of GRP in the stomach ofa teleost fish is structurally more similar to mammalian GRP than to the amphibian skin peptide, bombesin. Gastrin-releasing peptide

Bombesin

Trout

Stomach

THE stomach and intestine of m a m m a l s are richly innervated by intrinsic nerve fibers originating in ganglia of the myenteric and submucous plexi that contain gastrin-releasing peptide (GRP) (29). Gastrin-releasing peptide exercises complex effects on motility, feeding behavior, and neuroendocrine secretion [reviewed in (28)]. The primary structure of G R P has been established for the pig (19), h u m a n (25), dog (22), guinea pig (24), and chicken (18). In these species, G R P comprises 27 amino acid residues and shares a region of structural similarity at the COOH-terminal region with bombesin, a peptide isolated from the skin of the frog Bombina ( 1). However, nucleotide sequence analysis of cloned cDNAs encoding G R P (25) and bombesin (23) has shown that the two peptides are not homologous, and it has recently been shown that amphibian nervous and gastrointestinal tissues contain a mammalian-type G R P rather than bombesin (9,21). Several immunohistochemical and radioimmunoassay studies, using antisera that reacted with both G R P and bombesin, have shown that gastrointestinal tissues of teleost (3,14,17,26), holostean (12), elasmobranch (2-5,10,13), and holocephalan (27,30) fish contain GRP/bombesin-like peptides that are localized to both nerves and mucosal endocrine cells. The molecular nature of this material is a matter of controversy. Partial elucidation of the primary structure of a GRP/bombesin-like

Evolution

peptide from the gut of the dogfish, S~Tliorhinus canicula (6), indicated that this component was structurally more similar to m a m m a l i a n G R P than to bombesin. On the other hand, it has been proposed the GRP/bombesin-like material in the intestine of the longnose skate, Raja rhina (2), the dogfish, Scyliorhinus slellaris (5), and the ratfish, Hydrolagus colliei (27), resembles bombesin more closely than G R P in terms of its i m m u n o c h e m ical properties. In this study, we have determined the complete primary structure of the GRP/bombesin-like peptide from a teleost fish, the rainbow trout, and we propose that this peptide is more closely related to m a m m a l i a n GRP. METHOD Synthetic peptides were supplied by Peninsula Laboratories Inc. 3-[~25I][IodotyrosyPS]GRP (74 T b q / m m o l ) was supplied by Amersham Corporation.

Radioimmunoassay Trout G R P was detected using antiserum R354, which was raised against pig G R P in a radioimmunoassay procedure that has been described previously (24). Pig G R P was used as standard and 3-[~25I][iodotyrosyPS]GRP was used as radiolabeled tracer. The antiserum is directed against the COOH-terminal region of

Requests for reprints should be addressed to Dr. J. M. Conlon.

995

996

JENSEN AND CONLON

G R P and shows full molar reactivity with bombesin and neuromedin C [GRP(18-27)-peptide] (20) and 1% reactivity with neuromedin B. The antiserum requires the presence of a COOHterminal a-amidated methionine residue for binding.

TABLE 1 AUTOMATED EDMAN DEGRADATION OF TROUT GRP

Cycle No.

Tissue Extraction Adult trout (300-400 g) were obtained from Robinson Trout Farm, Grand Haven, MI. Stomach tissue (1090 g) was taken from 325 specimens and immediately frozen on dry ice. The tissue was homogenized with 6 volumes of ethanol/0.7 M HCI (3:1 v/v) using a Waring blender and stirred for 2 h at 0°C as previously described (16). After centrifugation (4000 X g for 30 min), ethanol was removed from the supernatant under reduced pressure. After a further centrifugation (4000 × g for 30 min), the extract was pumped at a flow rate of 2 m l / m i n through 10 Sep-Pak C-18 cartridges (Waters Associates) connected in series. Bound material was eluted with 70% (v/v) acetonitrile/water and lyophilized.

Pur([ication oJ Trout GRP The extract was redissolved in 1% (v/v) trifluoroacetic acid/ water (20 ml) and chromatographed on a (90 × 5 cm) Biogel P10 (fine) column (Bio-Rad) equilibrated with 1 M acetic acid at a flow rate of 72 ml/h. Fractions (12 ml) were collected and the presence of GRP-like immunoreactivity was determined by radioimmunoassay at appropriate dilution. The fractions denoted by the bar in Fig. 1 were pooled (total volume = 84 ml) and pumped at a flow rate of 2 m l / m i n onto a (250 X 10 mm) Vydac 218TP510 (C- 18) column (Separations Group) equilibrated with 0.1% (v/v) trifluoroacetic acid/water. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 rain, held at this concentration for 30 min, and then raised to 49% (v/v) over 60 min using a linear gradient. Absorbance was measured at 214 and 280 nm, and 2-ml fractions were collected. Four consecutive fractions denoted by the bar Fig. 2A were rechromatographed on a (250 × 4.6 mm) Vydac 214TP54 (C-4) column equilibrated with acetonitrile/water/trifluoroacetic acid (14.0:85.9:0.1; v/v/v) at a flow rate of 1.5 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 32%

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20 21 22 23

Residue

Yield (pmol)

Ser Glu Asn Thr Gly Ala lie Gly Lys Val Phe Pro Arg Gly ASh His Trp Ala Val Gly His Leu Met

62 1 l0 92 54 133 149 I 18 121 102 95 107 69 34 61 52 17 13 27 17 31 9 12 Trace

The detection limit for amino acid phenylthiohydantoins was 0.5 pmol.

(v/v) over 40 min using a linear gradient. The fraction denoted by the bar (Fig. 2B) was rechromatographed on a (250 × 4.6 mm) Vydac 219TP54 phenyl column under the same conditions of chromatography used with the C-4 column. Trout G R P was purified to apparent homogeneity by chromatography on a (250 × 4.6 mm) Vydac 218TP54 (C-18) column using the same elution conditions as previously.

Structural Characterization 30

-0.6

E

~2.5

0.5

>_ 2~.

-o.4

""

0 Z

1.0

O2

~0..5

0.1

1

0

o

~

4o

,

eo

,

eo FRACTION

loo

,

~2o

1~

0

~eo

NO.

FIG. 1. Gel permeation chromatography on a Biogel P-10 column of an extract of trout stomach after partial purification on Sep-Pak cartridges. The GRP-like immunoreactivity, measured at a dilution of 1:100, with an antiserum directed against the COOH-terminus of pig GRP, is shown by the histogram. Fractions denoted by the bar were pooled and subjected to reversed-phase HPLC. The arrows show the elution volume of neuropeptide 7 (1) and neurokinin A (2).

The primary structure of trout G R P was determined by automated Edman degradation using approximately 400 pmol of peptide. Its amino acid composition was determined by precolumn derivatization with phenylisothiocyanate using approximately 200 pmol of peptide. Tryptophan and cysteine residues were not determined. Full details of the methods and instrumentation have been provided previously (8,16). RESULTS

Purftcation (?/ Trout GRP The original extract of trout stomach contained GRP-like immunoreactivity equivalent to 5.8 pmol of pig G R P / g wet weight of tissue. In radioimmunoassay, the immunoreactivity in serial dilutions of the extract diminished in parallel with the synthetic pig G R P standard curve. The elution profile on a Biogel P-10 gel permeation column of the extract of trout stomach, after partial purification on SepPak cartridges, is shown in Fig. 1. Immunoreactive material was eluted from the column as a major peak with approximately the same elution volume as neuropeptide 3" (Mr 2321) and as a minor

TROUT GRP

997

peak with the same elution volume as neurokinin A (M~ 1134). The recovery of immunoreactivity from the column was approximately 60%. The fractions containing maximum immunoreactivity (denoted by the bar in Fig. 1) were pooled and chromatographed on a semipreparative C- 18 column (Fig. 2A). High concentrations of GRP-like immunoreactivity were associated with four fractions, denoted by the bar. Chromatography of this material on an analytical C-4 column (Fig. 2B) showed that GRP-like immunoreactivity was associated with the leading edge of the major peak in the chromatogram, as delineated by the arrows. Chromatography of this fraction on an analytical Vydac phenyl column (Fig. 2C) showed that the material was still markedly heterogeneous, but GRP-like immunoreactivity was associated with a distinct peak shown by the bar. Trout GRP was purified to apparent homogeneity on an analytical Vydac C-18 column (Fig. 2D). The peptide was eluted from the column as a sharp, symmetrical peak and the final yield of pure material was approximately 600 pmol.

Peptide Characterization The primary structure of trout GRP was determined by automated Edman degradation (Table 1). Unambiguous assignment of amino acid phenylthiohydantoin derivatives was possible for 22 cycles of operation of the sequencer and the derivative

of methionine was observed during cycle 23. The amino acid composition of the peptide was established as: Asx 2.0 (2), Glx 1.4 (1), Ser 1.2 (1), Gly 3.6 (4), His 1.4 (2), Arg 1.3 (1), Thr 1.3 (1), Ala 2.0 (2), Pro 1.2 (1), Val 1.7 (2), Met 0.3 (1) lie 1.0 (1), Leu 1.1 (1), Phe 1. l (1), Lys 1.4 (1) residues/mol peptide. The values in parentheses show the values predicted from the proposed structure. With the exception of the low value for the amount of methionine in the peptide, the agreement between the sequence analysis and the amino acid composition data was good, demonstrating that the full sequence of the peptide had been obtained. The composition and sequence analysis data indicated that the peptide was >95% pure. By analogy with other members of the GRP family of peptides, the COOH-terminal residue of trout is probably c~amidated, but there was insufficient pure material to demonstrate this chemically. However, the strong reactivity of the peptide with an antiserum directed against the a-amidated COOH-terminus of pig GRP (24) suggests that trout GRP also contains an a-amidated COOH-terminal methionine residue. The low value for the amount of methionine in the peptide is consistent with the observation that peptides with a COOH-terminal a-amidated methionine residue are susceptible to oxidation. The goldfish tachykinin, carassin (8), and guinea pig GRP (24) were also isolated in the metfiionine sulfoxide form using the same methodology as in the present study.

2.2. 40

0.28-

A

30

J

W I--

! t#)

8

W 0 F

.

.

.

.

iO

i ill/ /

I0

TIME (rnin)

TIME (min) 0.06-

0.4

0

40

C RI

3O 30

LU

.u

~r I 03 ,<

i

I

1 1

I

ni-

1 1

I

20

/j/tl W 0

1 1

..J

-er Z

/ 20

,<

O

,<

8

-m 8

o

lb

~) TIME (rain)

~)

o TIME (min.)

FIG. 2. Successive reversed-phase chromatographies on (A) a semipreparative Vydac C-18 column, (B) an analytical Vydac C-4 column, (C) an analytical Vydac phenyl column, and (D) an analytical Vydac C-18 column of an extract of trout stomach after partial purification by gel permeation chromatography. The fractions containing GRP are denoted by the bars and the arrows indicate when peak collection began and ended. The dashed line shows the concentration of acetonitrile in the eluting solvent.

998

JENSEN AND CONLON

TABLE 2 COMPARISON OF T H E PRIMARY S T R U C T U R E S OF PEPTIDES RELATED

TO GASTR1N-RELEASINGPEPTIDE Human Pig Guinea pig Dog Chicken Trout Dogfish Frog GRP- 10 Bombesin

V P L P AG G G T V L T KM Y P RGNHWA A-

V S V

VGH

LM

A

A -- V S V A A- V --GQ A A----Q P---- S P A -----I S E N T G A 1 -- , , • • -- V F A-- V E N Q-- S F P * * ---- F -----

S --

S ---


(--) denotes residue identity. In order to maximize the degree of structural similarity in the peptides, gaps (denoted by *) have been introduced into the trout and dogfish sequences but the position of these gaps is arbitrary. The amino acid sequence at the COOH-terminus of the dogfish peptide is only tentatively assigned.

DISCUSSION

This study presents the first complete primary structure of a member of the G R P / b o m b e s i n family of peptides to be isolated from the tissues of a fish. The amino acid sequence of the trout peptide is compared with that of some other members of this family in Table 2. Like the GRP-related peptide from the dogfish (6), trout G R P is smaller than its m a m m a l i a n and avian counterparts, but recent data suggest that evolutionary pressure to conserve the length of the peptide has not been strong. Alligator G R P (Y. Wang and J. M. Conlon, unpublished results) and G R P from the frog, Rana ridibunda (9), for example, contain 28 amino acid residues and G R P from the frog Bombina orientalis contains 29 residues (21). Nevertheless, the 12 amino acid region at the COOH-terminus of the trout peptide is the same as the corresponding region in m a m m a l i a n G R P s but shows three substitutions compared with bombesin. The lysine residue in position 13 of h u m a n G R P has also been conserved in the trout peptide. Structure-activity relationships have indicated that the COOH-terminal decapeptide of G R P is equipotent with GRP-27 in most test systems (29). It is not surprising, therefore, that evolutionary pressure to conserve the NH2-terminal region of the peptide has been very weak. At the present time, bombesin has been unambiguously identified only in the skin of the frogs Bombina bornbina (l) and Bombina variegata (23). The present study does not, however, entirely exclude the possibility that bombesin is present in the trout stomach. As shown in Fig. 1, the extract contained a low concentration of a smaller component that reacts with the antiserum to pig GRP. Trout GRP, in c o m m o n with other members of the G R P family, contains a single arginine residue that functions as a recognition site for processing enzyme(s).

Thus, pig spinal cord (20), guinea pig brain (24), dog small intestine (22), frog brain (9) and intestine (21), and human neuroendocrine tumors (7) contain the COOH-terminal decapeptide of G R P (GRP- 10), which has also been termed neuromedin C. There was insufficient material to characterize chemically the small molecular form of trout GRP, but the component was eluted from a C-18 reversed-phase HPLC column with the retention time that was similar to that of synthetic GRP-10 and distinct from that of synthetic bombesin. Thus, the data support the hypothesis (9) that gene-encoding G R P is phylogenetically ancient and that structural similarity between G R P and bombesin may be a result of convergent evolution. The physiological role of G R P in teleost fish is unknown and virtually all biological studies have been carried out using bombesin. Infusions o f b o m besin stimulate gastric acid secretion in the cod (15) and inhibit intestinal motility in this species (17). Studies with isolated muscle strips have shown that trout gastric smooth muscle contracts in response to low concentrations of bombesin (11), and the peptide has been shown to potentiate the contractile effect of acetylcholine on trout and cod gastric smooth muscle (26). A role as a satiety factor in bony fish has been suggested by the observation that intraperitoneal injections of bombesin into carp significantly delayed the onset of feeding (28). ACKNOWLEDGEMENTS We wish to thank the research team of Dr. K. Olson, University of Notre Dame, for help in collection of the trout tissues and Drs. C. Shaw and K. D. Buchanan, Queen's University of Belfast, N. Ireland, for a gift of antiserum. This work was supported in part by grants from the Erna and Victor Hasselblad Foundation, the Hierta-Retzius Foundation, and the Swedish Council for Forestry and Agricultural Research.

REFERENCES

1. Anasastasi, A.; Erspamer, V.; Bucci, M. Isolation and structure of bombesin and alytensin, two analogous active peptides from the skin of the European amphibians, Bombina and Alytes. Experientia 27:166-167; 1971. 2. Bjenning, C.; Farrell, A. P.; Holmgren, S. Bombesin-like immunoreactivity in skates and the in vitro effect of bombesin on coronary vessels from the longnose skate, Raja rhina. Regul. Pept. 35:207219; 1991. 3. Bjenning, C.; Holmgren, S. Neuropeptides in the fish gut. An immunohistochemical study of evolutionary patterns. Histochemistry 88:155-163; 1988.

4. Bjenning, C.; Jonsson, A. C.; Holmgren, S. Bombesin-like immunoreactive material in the gut and the effect of bombesin on the stomach circulatory system of an elasmobranch fish, Squalus ancanthias. Regul. Pept. 28:57-69; 1990. 5. Cimini, V.; Van Noorden, S.; Giordano-Lanza, G.; Nardini, V.; McGregor, G. P.; Bloom, S. R.; Polak, J. M. Neuropeptides and 5HT immunoreactivity in the gastric nerves of the dogfish (S¢Tliorhinus stellaris). Peptides 6(Suppl. 3):373-377; 1985. 6. Conlon, J. M.; Henderson, !. W.; Thim, L. Gastrin-releasing peptide from the intestine of the elasmobranch fish, Scyliorhinus canicula (common dogfish). Gen. Comp. Endocrinol. 68:415-420; 1987.

TROUT

999

GRP

I. ConIon, J. M.; McGregor, G. P.; Wallin, G.; Grimelius, L.; Thim,

8.

9.

IO.

II.

12.

13.

t4.

15.

16. 17.

18.

L. Molecular forms of katacalcin, calcitonin gene-related peptide and gas&in-releasing peptide in a human medullary thyroid carcinoma. Cancer Res. 48:2412-2416; 1988. Conlon, J. M.; O’Harte, F.; Peter, R. E.; Kah, 0. Carassin: A tachykinin that is structurally related to neuropeptide-y from the brain of the goldfish. J. Neurochem. 561432-1436; 199 I. Conlon, J. M.; O’Harte, F.: Vaudry, H. Primary structures of the bombesin-like neuropeptides in frog brain show that bombesin is not the amphibian gastrin-releasing peptide. Biochem. Biophys. Res. Commun. 178526-530; 1991. El-Salhy, M. Immunocytochemical investigation of the gastro-enter~pancreatic (GEP) neurohormonal peptides in the pancreas and gastrointestinal tract of the dogfish, Squalus acanthias. Histochemistry 80: 193-205; 1984. Holmgren. S. The effects of putative non-adrenergic, non-cholinetgic autonomic transmitters on isolated strips from the stomach of the rainbow trout, .%&no gairdwri’. Comp. Biochem. Physiol. 74C:229238; 1983. Holmgren, S.: Nilsson. S. VIP-. bombesin-, and neurotensin-like immunoreactivity in neurons of the gut of the holostean fish. Lepismftw pla~~r~iizez~~.Acta 2001. 64~25-32; 1983. Holmgren, S.; Nilsson, S. Bombesin-, gastrin, CCK-, %-hydroxytryptamine-, neurotensin-, somatostatin-, and VIP-like immunoreactivity and catecholamine fluorescence in the gut of the elasmobranch. Suz~alusacanrhias. Cell Tissue Res. 234:595-6 IS: 1983. Holmgren, S.: Vaillant, C.; Dimaline, R. VIP-, substance P-, gastrin/ CCK-, bombesin-, somatostatin- and glucagon-like immunoreactivities in the gut of the rainbow trout, Salmo gairdneri. Cell Tissue Res. 223:141-153; 1982. Holstein B.; Humphrey. C. S. Stimulation of gastric acid secretion and suppression of VIP-like immunoreactivity by bombesin in the Atlantic codfish, Gadus morhua. Acta Physiol. Stand. 109:2 17-223; 1980. Jensen. J.: ConIon, J. M. Substance-P-related and neurokinin-Arelated peptides from the brain of the cod and trout. Eur. J. B&hem. 206:659-664: 1992. Jensen, J.: Holmgren. S. Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua, Comp. Biochem. Physiol. 82C:81-89; 1985. McDonald, T. J.: Jornvall, H.: Ghatei, M.; Bloom, S. R.; Mutt. V. Characterization of an avian gastric (proventricular) peptide having

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

sequence homology with the porcine gastrin-releasing peptide and the amphibian Dentides bombesin and alvtensin. FEBS Lett. 122: 45-48;‘1980. . . McDonald, T. J.; Jornvall, H.; Nilsson, G.; Vagne, M.; Ghatei, M.; Bloom, S. R.; Mutt, V. Characterization of a gastrin-releasing peptide from porcine non-antral gastric tissue. Biochem. Biophys. Res. Commun. 90~227-233; 1979. Minamino, N.; Kanagawa, K.; Matsuo, H. Neuromedin C: A bombesin-like peptide identified in porcine spinal cord. B&hem. Biophys. Res. Commun. 119: 14-20; 1984. Nagalla, S. R.; Gibson. B. W.; Tang, D.; Reeve, J. R.; Spindel, E. R. Gastrin-releasing peptide (GRP) is not mammalian bombesin. J. Biol. Chem. 267:69 16-6922; 1992. Reeve, J. R.: Walsh, J. H.; Chew, P.: Clark, 8.; Hawke, D.; Shively, J. E. Amino acid sequences of three bombesin-like peptides from canine extracts. J. Biol. Chem. 258:5582-5588; 1983. Richter. K.; Eager, R.; Kreil, G. Molecular cloning of a cDNA encoding the bombesin precursor in skin of Bo~~j~~ vnrirgata. FEBS Lett. 262:353-355; 1990. Shaw, C.; Thim. L.; Conlon, J. M. Primary structure and tissue distribution of guinea pig gas&in-releasing peptide. J. Neurochem. 49:1348-1354: 1987. Spindel, E. R.: Chin, W. W.: Price, J.: Rees, L. H.: Besser, G. M.; Habener, J. F. Cloning and characterization of cDNAs encoding human gastrin-releasing peptide. Proc. Nat]. Acad. Sci. USA 81: 5699-5703: 1984. Thorndyke, M.; Holmgren, S. Bombesin potentiates the effect of acetylcholine on isolated strips of fish stomach. Regul. Pept. 30: 125-135: 1990. Thorndyke. M. C.: Reeve, J. R.; Vigna, S. R. Biological activity of a bom~sin-like peptide extracted from the intestine of the ratfish, Hydrolagus cuiiiei. Comp. Biochem. Physiol. 96C: 135- 140: 1990. Vigna, S. R.; Thorndyke, M. C. Bombesin. In: Holmgren, S., ed. The comparative physiology of regulatory peptides. London: Chapman and Hall; 1989:34-60. Walsh. J. H. Bom~sin-like peptides. In: Krieger, D.; Brownstein, M.: Martin. J.. eds. Brain .oentides. New York: John Wilev &Sons: . 1983:941-960. Yui, R.: Shimada, M.: Fujita. T. Immunohistochemical studies of peptide- and amine-containing endocrine cells and nerves in the gut and the recta1 gland of the ratfish. Chimaera mu~.~~ro.~a.Cell Tissue Res. 260:193-201; 1990.