Structural characterization of neuropeptide Y from the brain of the dogfish, Scyliorhinus canicula

Peptides,Vol. 13, pp. 493--497,1992

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

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Structural Characterization of Neuropeptide Y From the Brain of the Dogfish,

Scyliorhinus canicula J. M I C H A E L C O N L O N , *l C H R I S T I N A B J E N N I N G t A N D N E l L HAZON:~

*Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University School of Medicine, Omaha NE 68178 tDepartment of Zoophysiology, University of Goteborg, S-400 31, Sweden and ~.Department of Preclinical Medicine, Gatty Marine Laboratory, St Andrews KY16 8LB, UK Received 16 D e c e m b e r 1991 CONLON, J. M., C. BJENNING AND N. HAZON. Structuralcharacterizationof neuropeptide Yfrom the brainof the doglish, Scyliorhinus canicula. PEPTIDES 13(3) 493-497, 1992.--A peptide of the pancreatic polypeptide (PP) family was isolated in pure form from the brain of an elasmobranch fish, Scyliorhinuscanicula(European common dogfish). The primary structure of the peptide was established as: Tyr-Pr~-Ser-Lys-Pr~-Asp-Asn-Pr~-G~y-G~u~43~y-A~a-Pr~-A~a-G~u-Asp-Leu-A~a-Lys-Tyr2°-TyrSer-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu3°-lle-Thr-Arg-Gln-Arg-Tyr-NH2.This sequence contains only two amino acid substitutions compared with pig neuropeptide Y (NPY) (Gly for AspH and Lys for Arglg), and two substitutions (Gly for Aspn and Leu for Met17)compared with frog NPY. The amino acid sequence of NPY from dogfishbrain is appreciablydifferent from the neuropeptide Y-related peptide previously isolated from dogfish pancreas (five amino acid substitutions). The data indicate that evolutionary pressure to conserve the complete primary structure of neuropeptide Y has been very strong. It is suggested that the NPY-related peptide present in the pancreas of elasmobranch and teleost fish represents the piscine equivalent of mammalian peptide tyrosine tyrosine (PYY). Pancreatic polypeptide family

Neuropeptide Y

Peptidetyrosine tyrosine

STRUCTURAL similarities between members of the pancreatic polypeptide family of regulatory peptides has led to the suggestion that they are homologous (16). In mammals, the PP family comprises NPY, synthesized in neurons of the central and peripheral nervous systems and in chromaflin cells of the adrenal medulla (18), PYY, localized primarily to the endocrine-like L cell in the mucosa of the lower intestinal tract (17), and PP produced in the pancreatic islets (9). Numerous immunohistochemical studies, using antisera raised against mammalian peptides, have shown that PP family peptides are found in a wide range of lower vertebrate species (1,2,8,13,19), but structural and biological characterization of such nonmammalian peptides is limited. Neuropeptide Y has been isolated from the brain of the European green frog, Rana ridibunda, and has been shown to inhibit a-MSH release from the neurointermediate lobe of the frog pituitary (3). Pancreatic polypeptide has been isolated from the pancreas of the frog Rana temporaria ( 1 l) and, more recently, PYY from the intestine of Rana ridibunda has been characterized structurally (7). The endocrine pancreas of certain teleost fish (aoglerfish, eel, salmon, sculpin) and the holostean fish, the alligator par [re-

Elasmobranch

Dogfish

Brain

viewed in (5)], produces peptides of unknown function that show greater structural similarity to mammalian NPY and PYY than to PP. The elasmobranch fish, represented in the present day by sharks, rays, and skates, diverged from the line of evolution leading to mammals approximately 350 million years ago. This class of vertebrate is believed to be the first to have developed a pancreatic islet containing the four cell types (A, B, D, and PP) found in the mammalian pancreas (8). Previous work has led to the isolation of NPY-related peptides from the pancreata of two species ofelasmobranch fish, the European common dogfish, Scyliorhinus canicula (4), and the skate, Raja rhina (5), but the relationship of these peptides to mammalian NPY is unclear. This study describes the purification and structural characterization of a second peptide of the PP family from an extract of the brain of the dogfish that is distinct from the pancreatic peptide but is structurally very similar to mammalian NPY. METHOD Synthetic peptides were supplied by Peninsula Laboratories, Inc., and other chemicals by Sigma Chemical Co. Reagents for

Requests for reprints should be addressed to J. M. Conion.

493

494

CONLON, BJENNING AND HAZON

peptide sequence analysis were supplied by Applied Biosystems Inc.

Radioimmunoassay Dogfish NPY was detected using antiserum 8995, which was raised against the synthetic cysteine-extended COOH-terminal hexapeptide of human NPY (Cys-Ile-Thr-Arg-Gln-Arg-TyrNH2) in a radioimmunoassay procedure that has been described previously (14). The antiserum shows full cross-reactivity with human NPY and PYY. Human NPY was used as standard and '25I-Bolton Hunter-labeled NPY (Amersham) was used as radiolabeled tracer.

Tissue Extraction Whole brains were removed from adult dogfish of both sexes (0.65-2.0 kg b.wt.; n = 55) immediately after sacrifice under tricaine methanosulfonate (MS-222; 1:10,000 w/v) anesthesia. Tissue (32.5 g) was frozen on dry ice and stored at - 2 0 °C until the time of extraction. The tissue was boiled in water (260 ml) for 10 min, the mixture was acidified to 3% (v/v) with glacial acetic acid, and stirred for 1 h at room temperature. After centrifugation (20,000 × g, 1 h at 4°C), peptides were isolated from the supematant by passage through eight Sep-Pak C18 cartridges (Waters Associates) connected in series at a flow rate of 1 ml/ min. Bound material was eluted with 70% (v/v) acetonitrile/ water and lyophilized.

Purification of Doghsh NPY The extract, after partial purification on Sep-Pak cartridges, was redissolved in 1% (v/v) trifluoroacetic acid (5 ml) and injected onto a (250 × l0 mm) Vydac 218TP510 (C18) reversed-phase HPLC column (Separations Group) equilibrated with trifluoroacetic acid (0. l%)/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min, held at this concentration for 30 min, and raised to 49% (v/v) using a linear gradient. Absorbance was measured at 214 nm and 280 nm, and individual peaks were collected by hand. Fractions were assayed for NPY-like immunoreactivity at a dilution of 1:300. The fraction containing NPY-like immunoreactivity was rechromatographed on a Vydac

214TP54 (C4) column (250 × 4.6 mm) equilibrated with acetonitrile:water:trifluoroacetic acid (21.0:78.9:0.1; v/v/v)) at a flow rate of 1.5 ml/min. The concentration ofacetonitrile in the eluting solvent was raised to 42% (v/v) over 40 min using a linear gradient. Dogfish NPY was purified to near homogeneity on a Vydac 219TP54 phenyl column (250 × 4.6 mm) equilibrated with acetonitrile:water:trifluoroacetic acid (21.0:78.9:0.1) at a flow rate of 1.5 ml/min. The concentration ofacetonitrile in the eluting solvent was raised to 42% (v/v) over 40 min.

Structural Analysis The amino acid composition of dogfish NPY was determined by precolumn derivatization with phenylisothiocyanate as previously described (4). Hydrolysis in 5.7 M HC1 (24 h at 110°C) of approximately 500 pmol peptide was carried out. Automated Edman degradation of approximately I nmol peptide was carried out using an Applied Biosystems model 471A sequenator modified for on-line detection of phenylthiohydantoin amino acids under gradient elution conditions (4). The method of Schmidt et al. (15) was used to show that the COOH-terminal residue in dogfish NPY is c~-amidated. Dogfish NPY (approx. 500 pmol) in 0.2 M ammonium bicarbonate buffer, pH 7.8 (100/~l), was incubated for 16 h at 37 ° C with 1-tosylamine-2-phenylethylchloromethyl ketone-treated trypsin at a substrate:enzyme ratio of 50: t. The reaction mixture was lyophilized and redissolved in 0.001 M EDTA (20 tzl). An aliquot (5 #l) was applied to the sintered glass disc of an Applied Biosystems model 120A derivatizer and phenylthioearbamyl derivative of the COOH-terminal amino acid was produced. The retention time on reversedphase HPLC of this derivative was compared with the retention times of the corresponding derivatives of L-tyrosine and L-tyrosinamide using an Applied Biosystems model 130A separation system. RESULTS

Purification of Dog/ish NPY The concentration of NPY-like immunoreactivity in the extract of dogfish brain was 95 pmol/g tissue wet weight. The immunoreactivity in serial dilutions of the extract diminished in parallel with the human NPY standard in radioimmunoassay.

0.6.

V -50 w -40

...i ~: I--

-30

<

-20 -10

10

ao

ab

4b

so ~o TIME (min)

rb

ab

9b

-

FIG. l. Reversed-phase HPLC on a semipreparative Vydac C18 column of an extract of dogfish brain after partial purification on Sep-Pak cartridges. The fraction denoted by the bar contained NPY-like immunoreactivity measured with an antiserum raised against the COOH-terminal hexapeptide of h u m a n NPY. The arrows indicate when peak collection began and ended. The dashed line shows the concentration of acetonitrile in the eluting solvent.

DOGFISH NPY

495

TABLE

0.6-

I

A U T O M A T E D E D M A N DEGRADATION OF i

~

~

-40

-30

P

an <

/

- 20

_i)/

NEUROPEPTIDE Y FROM DOGFISH BRAIN

.50

I,IJ ..J

I-w o <

,0

CycleNo.

Residue

Yield(pmol)

1 2 3 4 5 6 7 8 9

Tyr Pro Ser Lys Pro Asp Asn Pro Gly

394 381 69 245 254 102 181 225 281

10

Glu

135

11

253 261 175 234 94 52 163

FIG. 2. Purification of doglish NPY on an analytical Vydac C4 reversed-

15

phase HPLC column. The bar denotes the peak containing NPY-like immunoreactivity, which was purified further.

16 17

Giy Ala Pro Ala Glu Asp Leu

18

Ala

177

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Lys Tyr Tyr Ser Ala Leu Arg His Tyr lie Asn Leu lie Thr Arg Gin Arg Tyr

105 145 170 2! 125 94 51 43 91 68 56 32 52 16 31 37 27 11

o

2b

lb

12

~0

13 14

TIME (min)

The elution profile on a semipreparative Vydac C 18 column of the extract of dogfish brain, after partial purification on SepPal< cartridges, is shown in Fig. 1. Neuropeptide Y-like immunoreactivity was associated with the single peak denoted by the bar. Rechromatography of the fraction on an analytical Vydac C4 column (Fig. 2) showed that the peak was heterogeneous but NPY immunoreactivity was associated with the major component denoted by the bar. Dogfish NPY was purified to near homogeneity by chromatography on an analytical Vydac phenyl column (Fig. 3). The peptide was eluted as a sharp, symmetrical peak and the final yield of pure material was approximately 2 nmol.

Structural Characterization The primary structure of dogfish NPY was determined by automated Edman degradation and the results are shown in Ta-

The detection limit for amino acid phenylthiohydantoins was 0.5 pmol.

• 50

-4O ill _J w

I I

-3o

[fl <

z

-2o

<

-10

TIME (rain)

FIG. 3. Purification to near homogeneity of dngtish NPY on an analytical Vydac phenyl column. The peak containing the peptide (purity > 95%) is shown by the bar. The final yield of peptide was 2 nmol.

ble 1. Unambiguous assignment of amino acid phenylthiohydantoin derivatives was possible for 36 cycles of operation of the sequencer, and no trace of the derivative o f a glycine residue was seen during cycle 37. The sequence analysis data indicated that the peptide was at least 95% pure. The amino acid composition of the peptide was established as (residues/tool peptide): Asx 3.1 (4), Glx 2.4 (3), Ser 1.8 (2), Gly 2.4 (2), His 0.9 (1), Arg 2.6 (3), Thr 1.0 (1), Ala 4.1 (4), Pro 4.5 (4), Tyr 5.2 (5), Ile 2.1 (2), Leu 3.0 (3), Lys 1.8 (2). The values in parentheses show the number of residues predicted from the proposed structure. With the exception of slightly low values for the acidic residues, the agreement between the sequence analysis and amino acid composition data was good. The reduced yields of the phenylthiocarbamyl derivatives of Asx and Glx may be a consequence of contamination of the sample or the glass frits of the amino acid analyzer with metal ions (12). The presence of an a-amidated COOH-terminal tyrosine residue in dogfish NPY was demonstrated by chromatographic

496

CONLON, BJENNING AND HAZON TABLE 2 A COMPARISON OF THE PRIMARY STRUCTURES OF D(X~FISH NPY WITH OTHER PEPTIDES OF THE PP FAMILY

Pig NPY

5 i0 15 20 25 30 35 YPSKP DNPGE DAPAE DLARY YSALR HYINL ITRQR Y-NH 2

I~gPYY

--A-- E A . . . . . SP- E-S--

Frog NPY

................

Frog PYY

--P-- E. . . . . .

Dogfish NPY

..........

Dogfish PYY

- - P -- E . . . . . . .

-AS . . . . L--

V. . . . .

M-K. . . . . . . . . . . . . . . . . SP- EMTK- LT . . . . . . . . G. . . . . . .

V. . . . .

K. . . . . . . . . . . . . . . . .

P - ~.- - K. . . . . . . . . . . . . . . . .

(-) denotes residue identity. DogfishNPY and frog NPY were isolated from brain tissue, dogfish PYY was isolated from pancreas, and frog PYY from intestine.

analysis of the products of digestion with trypsin. The enzyme liberates the free COOH-terminal residue by cleavage at the COOH-terminal side of the Arg3s residue. The retention time of the phenylthiocarbamyl derivative of the Tyr 3~ residue was 15.25 rain, compared with retention times of 15.26 min and 11.67 min for the retention times of the corresponding derivatives of L-tyrosinamide and L-tyrosine, respectively. It is concluded that the primary structure of dogfish NPY is identical to pig NPY except for the substitutions Gly for Asp at position 11 and Lys for Arg at position 19. DISCUSSION This study has described the first isolation and structural charactenza'tion of a PP family polypeptide from central nervous tissue of a fish, the European common dogfish, and the work complements a previous study (4) in which a structurally related peptide was isolated from the pancreas of the same species. As shown in Table 2, the amino acid sequence of the peptide isolated from dogfish brain is very similar (two amino acid substitutions) compared with pig NPY. It is proposed, therefore, that this peptide, rather than the pancreatic NPY-related peptide, represents the authentic dogfish NPY. The data indicate that evolutionary pressure to conserve the full sequence of the peptide has been very strong. The amino acid substitution Lys for Arg at position 19 also occurs in the sequence of frog NPY (3) and in the pancreatic NPY-related peptide from the dogfish (4), skate (5), and from several teleost fish [reviewed in (5)]. The Asp residue at position 11 has been quite strongly conserved in PP family peptides, but the substitution Gly for Asp 1~ in dogfish NPY is also found in PP isolated from the alligator pancreas (10). Recent elucidation of the primary structure of PYY isolated from the intestine of the frog, Rana ridibunda (7), has demonstrated a close structural similarity with the NPY-related paptide isolated from d ~OLdktkpancreas (Table 2). Both peptides share sequence identities at positions 3 (Pro), 5 (Glu), 14 (Pro), and

16 (Glu) that are not found in NPY molecules. Despite the fact that the dogfish pancreatic NPY-related peptide contains an isoleucine residue at position 31, rather than the valine that is present in pig and frog PYY, it is proposed that the fish pancreatic PP family peptide is the piscine equivalent of mammalian and amphibian PYY. The present data s ~ r t the hypothesis (7) that duplication of an ancestral PP family gene has led to genes encoding a NPY molecule that has been strongly conserved during evolution, and a PYY molecule that is present in both the pancreas and gut offish. A second duplication takingplace after the time of divergence of the fish and amphibia has led to a separate PYY gene expressed in the gut and a PP gene expressed in the pancreas. Consistent with this hypothesis, a PP family peptide has been isolated from the intestine of the sea lamprey that is structurally quite distinct from NPY (6). The intestine of S. canicula contains NPY/PYY-Iike immunoreactivity measured with the antiserum used in the present study, and purification and characterization of this material are underway. The physiological role of the PP family of peptides in elasmobranchs is not understood. A previous study has shown that a synthetic replicate of the dogfish pancreatic NPY-related peptide produced a doseMependent increase in arterial blood pressure in the conscious dogfish and that the peptide was equipotent with human NPY (4). The close structural similarity between dogfish and human NPY suggests that the former peptide will probably also be hypertensive in the dogfish. The substitution at position 11 (Gly for Asp) is located in a putative//-turn region of the molecule (6,16), and the presence of the flexible glycine residue would not be expected to alter significantly the overall conformation in this region. ACKNOWLEDGEMENTS This work was supported by a NATO Collaborative Research Grant CRG 89094, by the Paul and Marie eerglums Foundation, and the Swedish-American Foundation. We thank Dr. M. M. Oq'lare, Queen's University of Belfast, N. Ireland for a gift of antiserum.

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2. Brodin, L.; Rawitch, A.; Taylor, T.; Ohta, Y.; Ring, I-L;Hokfelt, T.; GriHner, S.; Terenius, L, MultiOe forms ~ ~ tm4~ related compounds in the lamprey C'W3"~ characterization

DOGFISH NPY

3.

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7. 8.

9. 10.

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