Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius)

Regulatory Peptides, 16 (1986) 261-268

261

Elsevier RPT 00541

Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin

(Cottus scorpius) J.M. Conlon a, W.E. Schmidt a, B. Gallwitz a, S. Falkmer b and L. Thim c ~Clinical Research Groupfor Gastrointestinal Endocrinology of the Max-Planck-Society, Department of Medicine, University of Gdttingen, Gofllerstrafie lOd, D-3400 Gdttingen, F.R.G., bDepartment of Tumor Pathology, Karolinska Institute, Stockholm, Sweden, and cNOVO Research Institute, Bagsvaerd, Denmark

(Received4 September 1986;revisedmanuscript receivedand accepted22 October 1986)

Summary The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as:

YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally ct-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals. teleost pancreatic islet; HPLC; primary structure

Introduction The primary structure of pancreatic polypeptide (PP) has been established for several mammalian (cat, cow, dog, human, pig, sheep, rat), avian (chicken, goose, Address for correspondence." Dr. J.M. Conlon, Klinische Arbeitsgruppe der MPG, GoBlerstraBe 10d,

D-3400 G6ttingen, F.R.G. Tel. (0551/396067). 0167-0115/86/$03.50 © 1986ElsevierSciencePublishers B.V. (BiomedicalDivision)

262 turkey), and reptilian (alligator) species (reviewed in Ref. 1). In the lower vertebrates, however, structural information is confined to the sequence of the peptide from the anglerfish (Lophius americanus) [2]. This 37-amino acid residue peptide shows greater homology towards porcine peptide YY [3] and porcine neuropeptide Y [4] than towards porcine PP and differs from mammalian/avian PPs in that the COOH-terminus ends in tyrosine-glycine rather than tyrosine-amide [2]. In this study, pancreatic polypeptide has been isolated from the 'principal islets' or Brockmann bodies of a second teleostean fish, the daddy sculpin (Cottus scorpius) [5]. The primary structure of the peptide was determined by automated Edman degradation and the presence of a COOH-terminal ~-amidated amino acid was established using a recently developed HPLC method [6].

Materials and Methods

Isolation procedures Daddy sculpin (n = 30) were collected at the Kristineberg Marine Biological Station, Fiskeb/ickskil, Sweden. Both pyloric and splenic islets (233 mg) were taken and immediately frozen on solid CO2. The tissue was extracted at 4°C with ethanol/0.7 M HC1 (3:1 v/v; 10 ml) using a Teflon-glass homogenizer. The homogenate was stirred overnight at 4°C, centrifuged (20 000 x g for 1 h) and ethanol removed from the supernatant under reduced pressure at 30"C. Peptides were isolated from the solution using Sep-pak C18 cartridges (Waters Associates) as previously described [7].

High-performance liquid chromatography The eluate from the Sep-pak cartridges was lyophilized and redissolved in trifluoroacetic acid (0.1% v/v; 1 ml). The solution was injected onto a Supelcosil LC-18DB column (250 x 10 mm) eluted at 30"C and at a flow rate of 2 ml/min with a linear gradient (120 ml) formed from acetonitrile/water/trifluoroacetic acid (21.0:78.9:0.1) and acetonitrile/water/trifluoroacetic acid (49.0:50.9:0.1). UV-absorbance was monitored at 214 nm. The peak designated by the star (Fig. 1) was lyophilized and redissolved in trifluoroacetic acid (0.1% v/v; 100 #1). The solution was injected onto a Supelcosil LC-3DP column (250 x 4.6 mm) eluted at 30°C and at a flow rate of 1.5 ml/min under isocratic conditions using acetonitrile/water/trifluoroacetic acid (24.5:75.4:0.1).

Structural analysis Amino acid composition was determined using approx. 1 nmol peptide as described [8]. The primary structure of the peptide was determined by automated Edman degradation using an Applied Biosystems model 470A gas-phase sequencer [9]. The detection limit for PTH-amino acids was 0.5 pmol. The presence of a COOH-terminal tyrosine amide in the peptide was established using a recently developed HPLC method [6] which is a modification of the thin-layer chromatography procedure of Tatemoto and Mutt [10]. Briefly, sculpin pancreatic polypeptide (approx. 2 nmol)

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was incubated with TPCK-treated trypsin (0.2 nmol; E. Merck, Darmstadt, F.R.G.) at 37°C for 24 h. Control incubations were carried out in parallel using synthetic human pancreatic polypeptide (2 nmol) and porcine PYY (2 nmol). The reaction mixtures were lyophilized and degradation fragments were derivatized with phenylisothiocyanate according to [11]. Phenylthiocarbamyl (PTC)-derivatives of COOHterminal amino acids were selectively extracted into an organic phase using triethylamine-saturated butyl acetate. The PTC-derivative of tyrosine amide was identified by reverse-phase HPLC using a Spherisorb ODS-2 column (4.6 x 250 mm; Latek Co., Heidelberg, FRG) eluted under the condition described in [6]. The column was calibrated with the PTC-derivatives of tyrosine amide, tyrosine-glycine and norleucine.

Results

Purification of the peptide The elution profile of an extract of sculpin islet tissue on a semi-preparative octadecyldimethylsilylsilica reverse-phase HPLC column is shown in Fig. 1. A preliminary screening of the major peaks of uv-absorbance by determination of their amino acid composition indicated that the peak designated by the star contained a high relative amount of proline. This is a characteristic feature of mammalian [1] and anglerfish [2] pancreatic polypeptides that is not found with the other islet hormones.

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TIME ( rain} Fig. 1. Reverse-phase HPLC on a semi-preparative C18 column of an extract of Brockmann bodies from the daddy sculpin. Details of the elution conditions are given in the text and the dashed line shows the concentration of acetonitrile in the eluting solvent. Pancreatic polypeptide is shown by the asterisk, peak 1 represents (TyrT,Glyt0)somatostatin-28, peak 2 glucagon, peak 3 somatostatin-14 and peak 4 glucagonlike peptide (GLP).

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Sculpin pancreatic polypeptide was purified to apparent homogeneity by chromatography under isocratic elution conditions on a diphenylmethylsilylsilica column (chromatogram not shown). The yield of peptide was approx. 5 nmol.

Structural analysis The amino acid composition of sculpin pancreatic polypeptide is shown in Table I. Unambiguous assignation of residues (1-36) of the peptide was possible by automated Edman degradation (Table II). Agreement between the amino acid composition and the sequence analysis was good except that the composition data suggested that an additional glycyl residue might be present in the molecule. No trace of PTH-glycine was detected in cycle 37 of the Edman degradation but the possibility that a COOH-terminal glycyl residue had been washed out of the glass fibre disc of the sequencer must be considered. In order to differentiate between a COOH-terminal tyrosine amide and tyrosine-glycine COOH, the Arg35Tyr 3~ bond of sculpin pancreatic polypeptide was cleaved with trypsin and the reaction mixture incubated with phenylisothiocyanate. The resulting PTC-derivative was extracted into an organic phase using alkaline butylacetate and subjected to reverse-phase HPLC (Fig. 2). The retention time of the PTC-derivative of tyrosine amide from the sculpin peptide (25.98 min) was identical (within the limit of an interassay variation of 1.5%) to that of a tyrosine amide standard (25.96 min) and to tyrosine amide derived from human pancreatic polypeptide (25.79 min) and porcine PYY (25.82 min). The peak of UVabsorbance of the PTC-tyrosine derived from sculpin PP, human PP and porcine PYY was asymmetric in all cases suggesting that the derivative may undergo partial

TABLE I Amino acid composition of sculpin pancreatic polypeptide Residue

Relative amount

Asx Thr Ser Glx Pro Gly Ala Val lie Leu Tyr His Lys Arg Trp

3.08 (3) 1.04 (1) 2.52 (2) 4.31 (4) 4.85 (5) 2.90 (2) 3.88 (4) 1.84 (2) 1.03 (1) 1.32 (1) 3.51 (4) 1.85 (2) 1.21 (1) 2.71 (3) ND (1)

The values in parentheses represent the relative amounts predicted from the amino acid sequence. ND: not determined.

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TABLE II

Automated Edman degradation of sculpin pancreatic polypeptide Cycle No.

PTH-amino acid

Yield (pmol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Tyr Pro Pro Gln Pro Glu Ser Pro Gly Gly Asn Ala Ser Pro Glu Asp Trp Ala Lys Tyr His Ala Ala Val Arg His Tyr Val Asn Leu lie Thr Arg Gin Arg Tyr

1072 905 820 690 764 401 210 504 683 778 639 575 144 315 218 157 224 273 255 252 87 301 350 197 133 68 194 155 241 157 123 63 98 100 101 99

The average repetitive yield was 93.6%.

rearrangement during the extraction procedure. The PTC-derivative of synthetic tyrosine-glycine was not extracted into the organic phase and the derivative was not detected in the aqueous phase following trypsinization of sculpin PP. It is concluded, therefore, that the COOH-terminal residue of sculpin pancreatic polypeptide is tyrosine amide.

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Fig. 2. Identification by HPLC of the C-terminal ct-amidated amino acid residue of sculpin pancreatic polypeptide. Digestion of the peptide with TPCK-trypsin, coupling to phenylisothiocyanate, selective extraction of the PTC-derivative of the amino acid a-amide and conditions of chromatography are described in the text and in [6]. (A) Procedure carried out in the absence of peptide. The arrow indicates the retention time of PTC-tyrosine amide and the norleucine internal standard is designated NL. (B) PTCderivative of tyrosine amide (designated by the asterisk) released from sculpin pancreatic polypeptide. TABLE III A comparison of the sequence of sculpin pancreatic polypeptide with peptides from the anglerfish and pig.

Sculpin

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Discussion A previous immunohistochemical study [12], using an antiserum to bovine pancreatic polypeptide, has shown that the 'principal islet', or Brockmann body situated close to the pylorus of the daddy sculpin contains cells producing pancreatic polypeptide-like immunoreactivity but such ceils are absent from the islet situated close to the spleen. The primary structure of sculpin pancreatic polypeptidc is compared with the corresponding peptide from the anglerfish [2] and with pancreatic polypep-

267

tide, neuropeptide Y and peptide YY from the pig (Table III). Although the anglerfish (superorder Paracanthopterygii) and the sculpin (superorder Acanthopterygii) may not be closely related phylogenetically, the Brockmann bodies are histologically very similar. Unlike the principal islets of most bony fishes, the islets of the anglerfish and sculpin are completely free from traversing strands of exocrine parenchyma [5]. The sequence of pancreatic polypeptide has been strongly conserved between the species (86% homology). The biological role of PP in fishes is unknown but the fact that evolutionary pressure has acted to conserve the structure is suggestive of a physiological function. Sculpin PP, in common with all mammalian and avian peptides yet characterized, contains an amidated COOH-terminal residue. Evidence from fast-atom bombardment/mass spectrometry, however, has indicated that anglerfish PP terminated with a glycyl residue [2]. The tyrosine amide of sculpin PP is probably derived from a COOH-terminal tyrosine-glycine by the action of a specific ascorbic acid-dependent enzyme [13] and the possibility exists that the sculpin islet contains a mixture of amidated and non-amidated forms but only the amidated form was isolated in this study. In agreement with the observations of Andrews et al. [2], sculpin PP shows stronger homology with porcine PYY (61%) and porcine NPY (61%) than with porcine PP (47%). The degree of homology with bovine PP is slightly higher (56%) but decreases when compared with chicken PP (42%). Nevertheless, the Pro-5, Pro-8, Gly-9, Ala-12, Glu-15, Tyr-27, Arg-33 and Arg-35 residues that have been conserved in PPs from all species yet studied [1] are also conserved in the sculpin and anglerfish peptides. The structural similarities between PP, NPY and PYY have been interpreted as evidence for their evolution from a common ancestral peptide by successive gene duplications. The strong homology of fish PPs to mammalian NPY and PYY suggests that these gene duplications took place after the time of divergence of the lines of evolution leading to fish and mammals (approximately 350 million years ago). This study complements previous work in which the primary structures of glucagon, glucagon-like peptide (GLP), somatostatin-14 and [Tyr7Gly~°]somatostatin-28 from the daddy sculpin have been determined (submitted for publication). Recently, a 36 residue amidated form of pancreatic polypeptide has been isolated from the principal islets of the coho salmon [14].

Acknowledgements The authors thank Professor N. Hilschmann, Max-Planck-Institut fiir Experimentelle Medizin, G6ttingen, for providing facilities for amino acid analysis and the staff of the Kristineberg Marine Biological Station for their cooperation. This work was supported by the Stiftung Volkswagenwerk, the Swedish Medical Research Council and Boehringer Ingelheim Foundation.

268

References 1 Kimmel, J.R., Pollock, H.G., Chance, R.E., Johnson, M.G., Reeve, J.R., Taylor, I.L., Miller, C. and Shively, J.E., Pancreatic polypeptide from rat pancreas, Endocrinology, 114 (1984) 1725-1731. 2 Andrews, P.C., Hawke, D., Shively, J.E. and Dixon, J.E., A non-amidated peptide homologous to porcine peptide YY(PYY) and neuropeptide YY(NPK), Endocrinology, 116 (1985) 2677-2681. 3 Tatemoto, K., Isolation and characterization of peptide YY(PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion, Proc. Natl. Acad. Sci. USA, 79 (1982) 2514-2518. 4 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 657-660. 5 Falkmer, S., Experimental diabetes research in fish. On the morphology and physiology of the endocrine pancreatic tissue of the marine teleost Cottus scorpius with special reference to the role of glutathione in the mechanism of alloxan diabetes using a modified nitroprusside method, Acta Endocrinol., 37 (Suppl. 59) (1961) 1-122. 6 Schmidt, W.E., Conlon, J.M., Mutt, V., Carlquist, M., Gallwitz, B. and Creutzfeldt, W., Identification of the C-terminally ~t-amidated amino acid in peptides by high performance liquid chromatography, Eur. J. Biochem., (1987) In press. 7 Conlon, J.M., Deacon, C.F., O'Toole, L. and Thim, L., Scyliorhinin I and II: two novel tachykinins from dogfish gut, FEBS Lett., 200 (1986) 11 I-116. 8 Conlon, J.M. and McCarthy, D.M., Fragments of prosomatostatin isolated from a human pancreatic tumour, Mol. Cell. Endocrinol., 38 (1984) 81-86. 9 Moody, A.J., Thim, L. and Valverc}e, I., The isolation and sequencing of human gastric inhibitory peptide (GIP), FEBS Lett., 172 (1984) 142-148. 10 Tatemoto, K. and Mutt, V., Chemical determination of polypeptide hormones, Proc. Natl. Acad. Sci. USA, 75 (1978)4115-4119. 11 Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L., Rapid analysis of amino acids using precolumn derivatization, J. Chromatogr., 336 (1984) 93-104. 12 Stefan, Y. and Falkmer, S., Identification of four endocrine celt types in the pancreas of Cottus scorpius (Teleostei) by immunofluorescence and electron microscopy, Gen. Comp. Endocrinol., 42 (1980) 171-178. 13 Bradbury, A.F., Finnie, M.D.A. and Smyth, D.G., Mechanism of C-terminal amide formation by pituitary enzymes, Nature, 298 (1982) 68~688. 14 Plisetskaya, E.M., Pollock, H.G. and Kimmel, J.R., Primary structures and biological activities of salmon pancreatic islet hormones, Can. J. Physiol. Pharmacol. Gastrointest. Horm., Suppl. (1986) 29.