Amino acid sequences of a hyperglycaemic hormone and its related peptides from the Kuruma prawn, Penaeus japonicus

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Aquaculture 135 (1995) 205-212

Amino acid sequences of a hyperglycaemic hormone and its related peptides from the Kuruma prawn, Penaeus japonicus Wei-jun Yang a, Katsumi Aida a, Hiromichi Nagasawa b,* aDepartment of Fisheries, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan b Ocean Research Institute. The University of Tokyo, Nakano-ku, Tokyo 164, Japan

Abstract

Major peptides have been extracted from sinus glands of the Kuruma prawn, Penaeus japonicus, and purified to homogeneity by reverse-phase HPLC. From the results of amino-terminal sequence and mass spectral analyses and bioassay of the peptides, one of the peptides was considered to be a hyperglycaemic hormone. The complete amino acid sequence of the peptide has been determined; it consists of 72 amino acid residues and has an amidated carboxyl-terminus. Sequence analyses of the other four peptides revealed that they all were structurally related to the hyperglycaemic hormone. All of these peptides were found to be produced by a single individual. Kqvwords:

Crustacea;Kuruma prawn; Penaeus japonicus; Hyperglycaemichormone; Amino acid sequence

1. Introduction

It has been shown that various kinds of neuropeptides are produced in the eyestalks of crustacea (for review: Keller, 1992). Most of them are synthesized in the X-organ and transferred to the sinus gland, from where they are released into the haemolymph. Among them, three physiologically important neuropeptide hormones, crustacean hyperglycaemic hormone (CHH), molt-inhibiting hormone (MM) and vitellogenesis-inhibiting hormone (VIH), have been recently characterized chemically. The amino acid sequences of these peptide hormones are similar to each other, forming a new peptide family referred to as the CHH family. They consist of 72-78 amino acid residues with six conserved Cys residues. It is believed that they have evolved from a common ancestral molecule. Recently, a CHH * Corresponding author at: Ocean Research Institute, The University of Tokyo, 1-15-1 Minarnidai, Nakano-ku, Tokyo 164, Japan, Tel: 81-3-5351-6489 Fax: 81-3-5351-6488. 0044-8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved .SsDrOO44-8486(95)01015-7

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related peptide has also been reported from an insect (Audsley et al., 1992), but no similar peptides have been discovered in animals other than in arthropods. CHH is by far the most abundant of the three peptides in all the crustacean species examined thus far, and the amino acid sequences of CHHs from various species have been determined in the shore crab, Curcinus maenas (Kegel et al., 1989), the lobster, Homarus americanus (Tensen et al., 1991), the crayfishes, Orconectes limosus (Kegel et al., 1991), Procumburus bouuieri (Huberman et al., 1993) and Procumburus clurkii (Yasuda et al., 1994)) and the terrestrial isopod, Armudillidium uulgure (Martin et al., 1993). It has also been reported that a peptide isolated from H. americanus has both MIH and CHH activities (Chang et al., 1990). However, in prawns, no sequence data for CHH-family peptides have been reported until now. In this paper we report the complete amino acid sequence of CHH from the Kuruma prawn, Penueusjuponicus. The production of at least five molecular species of CHH-family peptides by a single individual is also described.

2. Materials and methods The Kuruma prawns (Penueus juponicus) were obtained from a prawn farm in Oita, Japan. Sinus glands were removed from live prawns immediately after eyestalk ablation and kept in a OS-ml microtube with 0.9% NaCl in 30% aqueous acetonitrile (10 ~1 per sinus gland) on ice. They were stored at - 80 “C until extraction. The sinus glands were homogenized on ice in the above NaCl/acetonitrile solution using a Mixer Pellet Pestle (Kontes, NJ, USA), which was just fitted to a OS-ml microtube, and the homogenate was kept on ice for 30 min. After centrifugation at 15 000 g for 10 min, the pellet was extracted and centrifuged as above. The two supernatants were combined and concentrated under reduced pressure to remove acetonitrile. The resultant extract was then subjected to reverse-phase HPLC using an Asahi Pak ODP-50 column (4.6 X 150 mm, Asahi Chemical Industry, Tokyo). Fractionation was performed with a 50-min linear gradient of O-50% acetonitrile in 0.05% trifluoroacetic acid (TFA) at a flow rate of 1 ml min-‘. The elution was monitored by measuring the absorptions at 225 nm and 280 nm. All peak materials were collected manually and numbered in the order of elution. The amounts of proteins were estimated by the absorbance at 225 nm using bovine serum albumin as the standard (Beaven and Holiday, 1952). Amino-terminal amino acid sequences of both S-carboxamidomethylated peptides (60160 pmol) and fragment peptides were analyzed on an Applied Biosystems model 476A protein sequencer in the pulsed-liquid mode. Each intact peptide (0.4-1.0 nmol) was dissolved in 70 ~1 of 0.25 M Tris-HCl buffer (pH 8.0)) to which 20 ~1 of dithiothreitol solution ( 1 mg ml-’ in 0.1 M Tris-HCl buffer, pH 9.0, containing 2 M urea) was added. The mixture was incubated at 35 “C for 30 min. Subsequently, 20 ~1 of iodoacetamide solution (2 mg ml-’ in the same buffer as used in dithiothreitol solution) was added, and the incubation was continued under the same conditions as above. The reaction mixture was acidified by the addition of 20 ~1 of 1 M HCl and the resulting mixture was fractionated by reverse-phase HPLC using a Senshu Pak VP304 column (4.6 x 250 mm, Senshu Kagaku, Tokyo). A 40-min linear gradient elution of

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lO-50% acetonitrile in 0.05% heptafluorobutyric acid was performed, and monitored by the absorbances at 225 and 280 nm. Two proteolytic enzymes were used. Trypsin: the intact peptide (No. 42,500 pmol) was dissolved in 100 ~1 of 0.1 M Tris-HCl buffer (pH 8.0), to which TPCK-treated trypsin (Sigma) in 0.001 M HCl was added so that the enzyme to substrate ratio was 1: 10 (w/w). The mixture was maintained at 37 “C for 3 h with occasional shaking. The digestion was stopped by the addition of 20 ~1 of 1 M HCl. Endoproteinase Glu-C: the intact peptide (No. 42, 500 pmol) was dissolved in 100 ~1 of 0.1 M Tris-HCl buffer (pH 8.0) and incubated with endoproteinase Glu-C (Boehringer Mannheim) at an enzyme to substrate ratio of 1: 10 (w/w) at 35 “C for 10 h. The digestion was stopped by adding 20 ~1 of 1 M HCl. The digests were fractionated by reverse-phase HPLC using the same column as used in the fractionation of intact peptides with a 65-min linear gradient elution of O-65% acetonitrile in 0.05% TFA at a flow rate of 1 ml mitt- ‘. The fragment peptides were collected manually by monitoring the absorbance at 225 nm. Mass spectral analyses were performed on two different types of mass spectrometers. TOF-MS: TOF mass spectra were measured on a Kompact MALDI-III (Shimadzu, Kyoto) time-of-flight mass spectrometer with 2-(4-hydroxyphenylazo)benzoic acid as the matrix in the positive ion mode. FAB-MS: High resolution FAB mass spectrum of El, an endoproteinase Glu-C fragment, was measured on a JMS-SX102 mass spectrometer (JEOL, Tokyo) with a mixture of glycerol and thioglycerol ( l:l, v/v) as the matrix. Calibration was done using the two characteristic peaks derived from glycerol, m/z 829.46 and 921.47. The target was bombarded with xenon atoms and the spectrum was measured in the positive ion mode. The bioassay for hyperglycaemic activity was done essentially according to the methods reported previously (Leuven et al., 1982). Two sinus gland equivalents of the No. 42 peptide were injected into a Kuruma prawn (ca. 20 g), which had been eyestalk-ablated one day before. Hemolymph (ca. 100 ~1 each) was taken just before ablation, just before injection, and 2 h after injection. Glucose in each hemolymph sample was measured by the glucose-oxidase peroxidase method (Papadopoulas and Hess, 1960). Before and during the experiment, the prawns were kept at 18 f 2°C. Data were analyzed by analysis of variance (ANOVA) using the Duncan’s multiple range test, with P < 0.05 taken as significant.

3. Results Peptides were extracted in four batches from a total of 1500 sinus glands and fractionated by a single run of reverse-phase HPLC. Fig. 1 shows a typical chromatogram from 300 sinus glands. By measuring TOF mass spectra of all the peak materials recovered from HPLC, five major peak materials from peak Nos. 34,40,42,48 and 50 were thought to be CHH-family peptides, whose protonated molecular ion peaks were observed at m/z 8,368, 8,487, 8,353, 8,328 and 8,314, respectively. Reductive carboxamidomethylation of these five peak materials followed by mass determination resulted in an increase in mass of 312-362. Theoretically, this reaction brings about an increase in mass of 348 by the addition of six carboxamidomethyl groups to six Cys residues, provided a molecule has three cystine residues. Notwithstanding the low

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0.5

50

f e

a 0.25

?5

6 r.9

2 e

E

,o ::

0 1,

20

Retention

4"

3,

30

25

time

45

50

(mln)

Fig. 1. Reverse-phase HPLC elution profile of extracts from 300 sinus glands of the Kuruma prawn, Peaueus juponicus. Column: Asahi Pak ODP-50 (4.6X 150 mm). Solvent: O-50% acetonitrile in 0.05% TFA. Flow rate:

1 ml min-‘. Detection: absorbance at 225 nm. Temperature: 40 “C. The concentration of acetonitrile is indicated by the dotted line.

accuracy of TOF-MS, the results indicated the presence of three intramolecular disulfide bonds in each molecule and therefore it was highly probable that they all were CHH-family peptides. This was confirmed by amino-terminal amino acid sequence analysis of each Scarboxamidomethylated derivative. More than 43 residues could be identified (Fig. 2) and the sequences were highly similar to each other and also similar, but somewhat less, to the known CHHs from other crustaceans. The yields of the five peptides isolated from 300 sinus glands were 1.5,0.6,2.8, 1.8 and 0.9 nmol for Nos. 34,40,42,48 and 50, respectively. Since the No. 42 peptide was the most abundant of the five peptides, further structural analysis of this peptide was performed. Trypsin digestion of the intact No. 42 peptide afforded a new peptide named T3, whose protonated molecular mass was 74 19. This peptide consisted of four peptide chains, among which three sequences (T3- 1, -2 and -3) were easily assigned based on the amino-terminal sequence. The fourth peptide (T3-4) starting from Ser41 was assigned by subtracting the above three sequences from the total. T3-4 was considered to be a carboxyl-terminal peptide, but the residues could be identified up to the 70th residue and, judging from the mass spectral data, two more unidentified residues probably remained at the carboxyl-terminus.

Fig. 2. Amino-terminal amino acid sequences of the five major eyestalk peptides from the Kumma prawn and Penaeus japonicus. Identical residues are boxed.

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10

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209

30

20

40

SLFDPACTGIYDBPLLRKLGRLCDDCYNVFREPKVATGCR +

t-----

T3-1 ___( 50 I FLDCLEYLI

SNCYHNL

T3-2 -------I+

60

T3-3 _(

IO

PSHLPEEHMAAMQTV-NH2

r^l

________ 1

T3-4 +E2-i+El-------_(

Fig. 3. Summary of sequence analyses of the No. 42 peptide. Wavy lines: residues identified by amino-terminal sequencing. T3-1, -2, -3 and -4: tryptic peptides connected together by three disulfide bonds. El and E2: endoproteinase Glu-C peptides.

ti ----

/

100

P 50

_________----L

I

I

15

20

25

I

30

Retention

35

time

40

45

50

E 3 u

0

55

(min)

Fig. 4. Elution patterns of the sinus gland extracts from each of three in Fig. 1 except that the gradient program was slightly different.

prawns.HPLC conditions were the same as

In order to determine the carboxyl-terminal sequence, the intact No. 42 peptide was digested with endoproteinase Glu-C. Four major fragment peptides named El-E4 were obtained (data not shown). El was found by sequence analysis to be a carboxyl-terminal fragment, and one more residue, Thr7’, was identifiable. Thus, there remained only a carboxyl-terminal unidentified residue. The high resolution FAB mass spectrum of El showed a protonated molecular ion peak at m/z 887.43. The only possibility for the carboxylterminal residue which could satisfy this value was Val with an amidated structure (calculated value: m/z 887.42). Thus, the complete amino acid sequence of the No. 42 peptide was unambiguously determined (Fig. 3). Since the amounts of the five peptides from an individual prawn, or a pair of sinus glands, were sufficient for detection on HPLC chromatograms, we examined whether all these peptides were produced by a single individual. A pair of sinus glands from 10 prawns were

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separately extracted and subjected to reverse-phase HPLC. The elution patterns were quite similar to one another (Fig. 4)) and all the five peaks could be observed in each chromatogram, indicating that they all were produced in a single prawn. Hyperglycaemic activity of the No. 42 peptide was assessed. Glucose level in the haemolymph before eyestalk ablation was 209 f 23 pg ml-’ hemolymph (mean f S.E., n = 7). This level decreased considerably to 23 + 9 pg ml- I one day after eyestalk ablation, which was likely caused by stopping the supply of CHH from the eyestalks. This level reached 40 f 15 pg ml-’ (significant, P < 0.05, compared with the value before injection) after injection of two sinus gland equivalents of the No. 42 peptide. The sequence similarity of the No. 42 peptide to the known CHHs, together with the evidence for its hyperglycaemic activity, though the data were still preliminary, lead us to conclude that this peptide was Kuruma prawn CHH.

4. Discussion In this investigation, neuropeptides of the Kuruma prawn were isolated from the sinus glands and the complete amino acid sequence was determined for the most abundant species. This is the first characterization of CHH from prawns. Since the sinus gland extracts consisted mainly of neuropeptides, a single HPLC purification afforded sufficiently pure peptides. The peptides were characterized by a combination of automated microsequencing and TOF mass measurement. In particular, the latter equipment was quite efficient in this type of experimentation, because each TOF mass spectrum could be obtained in a short time with a small amount of sample (sub-picomole), though accuracy was somewhat poor ( f 0.1% from the manufacturer’s specification). The amino-terminus of the No. 42 peptide was free, which contrasted with the fact that all the CHHs obtained thus far from various decapods are blocked by pyroglutamate. We experienced difficulty in determining the sequence beyond the 44th residue, because any fragments corresponding to that part could not be recovered after trypsin digestion of the S-carboxamidomethylated peptide. Thus, to obtain the sequence information for that part, the intact peptide was digested with trypsin. As expected, the carboxyl-terminal peptide was obtained as a peptide chain of a bigger molecule consisting of four peptide chains connected together by three disulfide bonds, whose sequences could be analyzed on the basis of the amino-terminal sequence. Regarding the determination of the carboxyl-terminus, since the only possibility was Val-amide from the high resolution FAB mass spectrum, analysis was very simple. The arrangement of three disulfide bonds was also determined, and the results showed that they were connected in the same manner as in the Cam-CHH (Kegel et al., 1989) and Arv-CHH (Martin et al., 1993). The details will be reported elsewhere. The amino acid sequence of the No. 42 peptide thus determined is compared with the known CHHs. The total number of amino acid residues (except for Arv-CHH), the distribution of six Cys residues and the carboxyl-terminal amide are identical. The similarity is higher in the amino-terminal part, suggesting that this part is important for expressing hyperglycaemic activity. The similarities with known CHHs are 40-50%; 42% with CamCHH, 50% with Orl-CHH, 50% with Hoa-CHH-A, 49% with Hoa-CHH-B, 40% with ArvCHH, 50% with Prb-CHH, and 50% with Pm-CHH. In contrast, the similarities with

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Cam-MIH (29%) and Hoa-VII-I (26%) are much lower than those with CHHs. In addition, there was a molecule (the No. 46 peptide in Fig. 1) whose sequence was more similar to Cam-MIH or Hoa-VIH than to the known CHHs, as far as the forty amino-terminal residues are concerned. Thus, we consider that this molecule would be MIH or VIH of this species, though the complete amino acid sequence has not been determined nor has the activity been assessed. These data together with the preliminary assay data showing hyperglycaemic activity strongly indicate that the No. 42 peptide is a hyperglycaemic hormone of this species. The presence of more than two molecular species of CHH has been reported in H. americanus (Soyez et al., 1991) and P. bouuieri (Huberman et al., 1993)) but it is not clear whether they can be derived from a single individual or not. Our present experiment clearly showed that five molecular species of CHH-family peptides were produced in a single prawn as their production was high enough for detecting W peaks on the HPLC chromatogram. This indicates the presence of at least five genes encoding respective CHH-family peptides in the genome. It appears to be of great interest to analyze the distribution of these genes, because it might provide insight into the process of gene multiplication regarding the CHH family.

Acknowledgements We are grateful to J. Nakayama of the Department of Agricultural Chemistry, The University of Tokyo, for high resolution FAB mass spectrometty. We also thank Prof. C. Ooka of the Department of Biology, Tokyo Metropolitan University for teaching us how to dissect sinus glands, and Dr. Marcy N. Wilder of the Department of Fisheries, The University of Tokyo for critical reading of the manuscript. This work was partly supported by a Grantin-Aid for Scientific Research (No. 04660132) from the Ministry of Education, Science and Culture of Japan, and by funds from the Cooperative Program (No. 18,1994) provided by The Ocean Research Institute, University of Tokyo.

References Audsley, N., McIntosh, C. and Phillips, J. E., 1992. Isolation of a neuropeptide from locust corpus cardiacum which influences ileal transport. J. Exp. Biol., 173: 261-274. Beaven, G. H. and Holiday, E. R., 1952. Ultraviolet absorption spectra of proteins and amino acids. Advances in Protein Chemistry, 7: 319-386. Chang, E. S., Prestwich, G. D. and Bruce, M. J., 1990. Amino acid sequence of a peptide with both molt-inhibiting and hyperglycaemic activities in the lobster, Homarus americanus. Biochem. Biophys. Res. Commun., 171, 818-826. Huberman, A., Aguilar, M. B., Brew, K., Shabanowitz, I. and Hunt, D. F., 1993, Primary structure of the major isomorph of the crustacean hyperglycaemic hormone (CHH-I) from the sinus gland of the Mexican crayfish Procambarus bouuieri (Ortmann): Interspecies comparison. Peptides, 14: 7-16. Kegel, G., Reichwein, B., Tensen, C. P. and Keller, R., 1991. Amino acid sequence of crustacean hyperglycaemic hormone (CHH) from the crayfish, Orconectes limosus: Emergence of a novel neuropaptide family. Peptides, 12: 909-913.

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Kegel, G., Reichwein, B., Weese, S., Gaus, G., Peter-Katalinic, J. and Keller, R., 1989. Amino acid sequence of the crustacean hyperglycaemic hormone (CHH) from the shore crab, Carcinus maenas. FEBS Len., 225: lO14. Keller, R., 1992. Crustacean neuropeptides: Structures, functions and comparative aspects. Experientia, 48: 439448. Leuven, R. S. E. W., Jaros, P. P., Van Herp, F. and Keller, R., 1982. Species or group specificity in biological and immunological studies of crustacean hyperglycaemic hormone. Gen. Comp. Endocrinol., 46: 288-296. Martin, G., Sorokine, 0. and Van Dorsselaer, A., 1993. Isolation and molecular characterization of a hyperglycaemic neuropeptide from the sinus gland of the terrestrial isopod Armadillidium uulgare (Crustacea) . Eur. J. Biochem., 211: 601-607. Papadopoulas, N. M. and Hess, W. C., 1960. Determination of neuraminic (siahc) acid, glucose and fructose in spinal fluid. Arch. Biochem. Biophys., 88: 167-171. Soyez, D., LeCaer, J. P., Noel, P. Y. and Rossier, J., 1991, Primary structure of two isoforms of the vitellogenesis inhibiting hormone from the lobster Homarus americanus. Neuropeptides, 20: 25-32. Tensen, C. P., DeKleijn, D. P. V. and Van Herp, F., 1991, Cloning and sequence analysis of cDNA encoding two crustacean hyperglycaemic hormones from the lobster Homarus ameticanus. Eur. J. Biochem., 200: 103-106. Yasuda, A., Yasuda, Y., Fuji& T. and Naya, Y., 1994, Characterization of crustacean hyperglycaemic hormone from the crayfish (Procambarus da&i): multiplicity of molecular forms by stereoinversion and diverse functions. Gen. Comp. Endocrinol., 95: 387-398.