Molecular cloning and sequencing of trypsin cDNAs from Penaeus vannamei (Crustacea, Decapoda): Use in assessing gene expression during the moult cycle

tnt. J. Biochem.

1357-2725(95)00169-7

Vol. 28, NO. 5, pp. 551-563, 1996 Copyright 0 1996 Ekevier ScienceLtd Printed in Great Britain. All rights reserved 1357-2725/96$15.00+ 0.00 Cell Bid.

in A B. KLEIN,* G. LE MOULLAC, Marine Biology Laboratory, Cedex, France

D. SELLOS, A. VAN WORNfWOUDT

URM IFREMER-14-Collige

de France, BP 225, F-29182 Concarneau

protease in Crastaeea. This Trypsinkthl?rwaPta #zmd of Pemens vQnmmzez,revealing three major isoforms sewed mizzorcompzments.Five cDNAs encoding five isoforms of try@a were detected by two saccessivettcmhgs of an ampIilkd cDNA library from the digestive$and of P. vanmu&. Tbe 0fWaminoacidscontaihga 1orgestisolatedaudseqencedcDNAeacodedapreprwmymae ~~~~ pepI& of 14 reaidaes and a bigbly bydropb@bictisspI sequenceof 14 amino . al@tnMsreveakdabigbdegreeofidentitybetweentbetrypsinfRrom P. vBIuullAeiand tbt from crayfkb (74%) aad an eqaa1kvel of 40). Dot bkt bybridkation and reveakd that znRNA expression tberegulationoftrypsinb f trypsin cDNA from P. patative 2s sequence in a crastacean specks, medmnbm of trypin synthesisin tbese important marine organisms. Copyrigbt 0 1996 Elsevier Science Ltd KeyworC: Seriae proteases Trypsin cDNA Precursor seqaence Penaeus vunnamei Crastacea EMBL accessionnumber X86369

mRNA expression

ht. J. Biochem. CelI Biol. (1996) 28, 551-563

and Moriarty, 1983; Kimoto et al., 1983). Shrimp trypsin was first detected in the digestive gland of Penaeus set$ms (Gates and Travis, 1969) and later isolated and characterized from other penaeid species (Lee and Lawrence, 1982; Galgani et al., 1985; Tsai et al., 1986; Honjo et al., 1990). In the digestive gland of P. japoniCUS,trypsin contributes about 6% of the total soluble protein, making it one of the most important proteases in this organ (Galgani et al., 1985). The activation mechanism for digestive enzymes by specific proteolytic cleavage has been extensively studied in mammalian species; however, no data is available about zymogens of crustacean trypsins. Whether a proenzyme of trypsin is synthesized in Penaeidae is still an open question (Titani et al., 1983; Sellos and Van Wormhoudt, 1992).

INTRODUCTION

The digestive endopeptidase trypsin is a member of the family of serine proteases. This important family is characterized by a common catalytic mechanism involving the presence of a catalytic triad composed of specific residues: serine, histidine and aspartic acid. The importance of trypsin and trypsin-like enzymes for many biological processes and industrial applications, such as digestion, dietary adaptation, evolution and food-processing, have made them the subject of much study in vertebrates (Dayhoff, 1972; Hermodson et al., 1973) and invertebrate species (Gibson and Barker, 1979; Zwilling and Neurath, 1981; Lee and Lawrence, 1982; Dal1 -.*To whom all correspondence should be addressed. Received 2 June 1995; accepted 6 November 1995. 551

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B. Klein

The serine protease family, including trypsin, chymotrypsin, elastase and thrombin, have evolved from a common ancestor (Neurath, 1984) and the sequences of trypsin seem to be highly conserved (Zwilling et al., 1975). Therefore, the comparison of mRNA coding sequences can assist our understanding of the evolutionary pathway of trypsin and other serine proteases. The characterization and the study of specific proteases in shrimp is an essential first step towards identifying their role in the development and the growth of these marine species. Crustaceans exhibit pronounced biochemical changes associated with specific moult stages. The synthesis and secretion of digestive enzymes, and their regulation by ecdysteroids and neuropeptides have been adequately demonstrated (Sedlmeier, 1988; Sedlmeier and Resch, 1989; Favrel et al., 1991). Protein synthesis is stimulated by ecdysteroids in premoult (Gorrel and Gilbert, 1969; Van Wormhoudt et al., 1978) while an inhibitory factor is found in the eyestalk (McWhinnie and Mohrherr, 1970). The variation of the activity of several digestive enzymes and the amount of the mRNA are known to be stage-specific (Bauchau and Mengeot, 1965; Van Wormhoudt, 1983; Van Wormhoudt and Favrel, 1988; Van Wormhoudt et al., 1995). The present paper describes the purification of shrimp trypsin, its characterization and the molecular cloning of five different trypsin cDNAs. Through the characterization of putative zymogen and signal sequences and from the analysis of the trypsin gene family, the relationship between the trypsin of P. vannumei and that of other species is discussed. Quantitative stagespecific differences in trypsin mRNA expression and trypsin specific activity are used to describe the regulation of the trypsin synthesis during the moult cycle.

MATERIALS

AND METHODS

et a/

kfeasurement of spectjic activity and puriJication of trypsin

The tissue of the digestive gland (2 g) was homogenized in 50 ml phosphate buffer (lOmM, pH 7.5 at 4°C) with an Ultraturrax. This suspension was centrifuged for 20 min at 55,OOOg at 4°C and the trypsin activity of the supernatant was measured by the method of Erlanger et al. (1961) with benzoyl-arginineparanitroanilide (BAPNA) in a 0.1 M Tris buffer (pH 8 at 25°C). Protein content was estimated according to Lowry et al. (1951) with bovine serum albumin (BSA) as the standard. The crude extract was lyophilized, adjusted to 10 ml and centrifuged again. The clarified extract was then filtered through an ACA54 (IBF) column (140 x 2.7 cm) and eluted with ammonium acetate buffer (10 mM, pH 7.5) at a flow rate of 35 ml/hr. Fractions showing trypsin activity were collected and subjected to affinity chromatography. They were loaded on a paraaminobenzamidine column and eluted with 0.15 M benzamidine in Tris buffer 50 mM containing 0.5 M NaCl, pH 7.5 (Galgani et al., 1985). Trypsin was separated from benzamidine by filtration on a G25 Sephadex column with ammonium acetate 10 mM, pH 7.5 as buffer, lyophilized and specific activity measured. One unit of trypsin activity corresponded to 1 pmole of p-nitro-anilide liberated in 1 min. Electrophoretic

analysis

Polyacrylamide gel electrophoresis was carried out as described by Davis (1964). Proteins were stained using the silver staining method (Morrisey, 1981). The specific activity of trypsin was measured with N-benzoyl-L-arginine CInaphthylamide (BANA) as a substrate (Zwilling et al., 1969). PAGE electrophoresis of the trypsin isoforms under denaturating conditions was realized according to Weber and Osborne (1969) on 12% polyacrylamide gels containing 1% sodium dodecyl sulphate. The standard proteases for molecular weight determination were purchased from LKB (low molecular weight standards).

Biological materials

Shrimps (Penaeus vannamei) were obtained from IFREMER (Tahiti). The digestive glands were dissected from specimens and were immediately frozen in liquid nitrogen and stored at - 80°C. Moult stages were determined according to Drach and Tchernigovtzeff (1967).

Construction and screening of the P. vannamei digestive gland cDNA library

A lambda-ZAP-cDNA library for shrimp digestive gland was constructed following the instructions of the lambda-ZAP-cDNA synthesis kit (Stratagene). Starting with 1 pg of poly A+ RNA, the obtained primary cDNA

553

Sequencingtrypsin cDNAs from Penaeus vannamei library contained 6 x lo6 independent phages. The amplified cDNA library containing lOi pfu/ml was screened with an anti-sense degenerate oligonucleotide probe based on a consensus sequence of trypsin. This highly conserved sequence was discovered by alignment of known amino acid sequences of trypsins from mammals, insects and crustaceans. Information about nucleotide composition of this region provided the NBRF-Bisance data library. The synthesized oligonucleotide was composed of 26 bases (Try C: 5’ GCA/G CA1 CCA/G TAT CCC CAG C/GT/AC/G ACA/T/G AT 3’), anti-sense to the sequence coding for residues 212-219 in the established chymotrypsin numbering system. Plaques were transferred to Hybond-N+ membranes (Amersham) and screened with the probe end-labelled with 50 PCi [y3’P]ATP. Prehybridization of the replica nylon filters was performed for 2 hr at 80°C and for 16 hr at 40°C in a solution containing NaCl (0.6 M), sodium citrate sodium pyrophosphate (0.06 M), (O.OS%), EDTA (10 mM), Sarkosyl (OS%), Fico11 (O.l%), PVP (O.l%), BSA (0.1%) and sonicated salmon sperm DNA (240 pg/ml). Hybridization was carried out at 80°C for 1 hr and at 40°C for 48 hr in the above solution containing the denatured labelled probe ( lo6 c.p.m./ml). Following hybridization, the filters were washed with 4 x SSCO.l% N-Law rylsarcosine (Na-salt) for 2 x 15 min and 1 x 30 min at room temperature and then for 15 min at 50°C. Filters were exposed for 48 hr to an X-ray film (Amersham) with an enhancer screen. Digestion of clone HP1 3 (longest cDNA obtained after the first screening performed with the consensus oligonucleotide probe) with Aua I provided a 5’-0.5 kb cDNA-fragment, which was purified by electrophoresis (1.2% agarose), extracted with the Geneclean kit (Bio 101, Inc.) and labelled with the random priming kit from New England Biolabs using [LZ~~P]~ATP. Prehybridization and hybridization were carried out in a 50% formamide solution containing 1M NaCl, 1% SDS and 100 rig/ml yeast RNA. The membranes were washed twice in 2 x SSC at room temperature for 5 min and twice in 2 x SSCjO.S”/, SDS at 50°C for 30 min. A supplementary screening was carried out using a 23-mer degenerate sense oligonucleotide probe based on a 5’-terminal region (nucleotides 41-76) of identified cDNAs from the shrimp library: CCC ACC TTC CGT/C CGC GGT BC2Xi5--D

CTC AA. Hybridization conditions same as described above. RNA

extraction

were the

and Northern blot analysis

Total RNA of the digestive gland at different stages of P. vannamei adults were extracted following the instructions of the MicroS&e Total RNA Separator Kit (Clontech, U.S.A.) and stored at -70°C. The yield of purified RNA was measured by spectrophotometry. Concentration was adjusted to approx. 3.6 pg/pl and the samples were then subjected to Northern blot analysis. Total RNA was denatured by deionized glyoxal and separated on agarose gels (1.5%) in 10 mM phosphate buffer (pH 6.4) following the procedure of McMasters and Carmichael (1977). The gel was placed in contact with a Hybond N+ membrane (Amersham, U.K.) and blotted essentially as described by Thomas (1980). A I Hind III digest was used as size marker. The fixation of RNA was obtained by baking the filters at 80°C for 2 hr. moult

Filter hybridization

An EcoRI-ClaI-fragment of 425 bp of a trypsin encoding cDNA was used as a probe for hybridization of trypsin mRNA. Prehybridization was performed for 4 hr, hybridization for 16 hr in presence of the [o!~~P] dATP-labelled fragment at 42°C in a 50% formamide solution as mentioned above. The filters were subjected to two 5-min washings in 2 x SSC at room temperature, two 30-min washings in 2 x SSC/O.S% SDS at 65°C and two final 30min washings in 0.1 x SSC at room temperature. Filter-bound radioactivity was determined by counting in a scintillation spectrometer. For each sample the relative mRNA level (c.p.m./pg RNA) was calculated and expressed as the mean + SD. The differences between the moult stages were calculated using the Student’s f-test. Dot blot hybridization

The relative level of trypsin encoding mRNA at different moult stages was measured by dot blot hybridization. Samples containing 3.6 and 7.2 pg of total RNA from each shrimp were dotted on sheets of Nylon (Hybond N+, Amersham) and baked for 2 hr at 80°C. Three to six animals were tested for each moult stage. The membranes were subjected to filter hybridization and filter-bound radioactivity was determined as previously described.

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B. Klein et (II

cDNA sequencing

Hybridization-positive recombinant pBluescript SK-phagemids were rescued from the bacteriophage (ZZAP) clones by in vivo excision, according to the instructions of the manufacturer’s protocol (Stratagene). The size of the inserts was determined by electrophoresis after digestion with EcoRI and XhoI. The longest inserts were selected. Sequencing was performed with singlestranded DNA using the dideoxynucleotide method @anger et al., 1977) and a reverse SK(-) primer. The entire determination of the nucleotide order on both strands was performed by the sequencing of double-stranded DNA following the manufacturer’s instructions of the USB Plasmid Sequencing Kit and using a reverse primer (S-TTGTGAGCGGATAACAATTTC-3’), a forward primer (5’-GTTTTCCCAGTCACGACGTTGTA-3’) and three synthetic specific oligo primers: Try A (sense strand to amino acid positions 96-102, following the established chymotrypsin numbering system), Try B (anti-sense strand to amino acid positions 97-103) and Try C (anti-sense strand to amino acid positions 212-219).

was dissolved in TE buffer (N 1 mg/ml) and stored at 4°C. Precipitated DNA (12 pg) was single and double digested with restriction enzymes for 2 hr and loaded on to a 0.6% agarose gel. Following electrophoresis, the gel was denatured for 30min in a solution containing 0.4 M NaOH and 0.6 M NaCl. The transfer was carried out using the same solution. After neutralization of the membrane in 1 M NaCl and 0.5 M Tris-HCI at pH 7, the DNA was fixed for 2 hr at 80°C then hybridized with the entire 854 pb probe as described above. RESULTS

AND

DISCUSSION

Biochemical characterization

of shrimp trypsin

After affinity chromatography, the trypsin specific activity was 3 1.5 Units/mg protein (Table 1) and 3.5 mg of pure trypsin was obtained from 2 g of digestive gland. Trypsin was composed of three major isoforms and three minor components (Fig. 1, lanes A and B). One of the smallest components in the crude extract, highly active on BANA, was also present in low quantity after purification (Fig. 1,

Try A

5’ GGC TTC ACC ATC AGC AAC GAC A 3’

Try B

5’ GAT GTC GTT GCT GAT GGT GAA

Try C

5’ GCA CA1 CCA TAI CCC CAG CTC ACA AT 3’ G G GAG T G

Electrophoresis was carried out with two or three successive loadings on a 5% acrylamide/bisacrylamide (29 : 1) denaturing gel. Southern blot analysis of trypsin genes

Genomic DNA was isolated from P. vannamei sperm cells. Powdered tissue (200 mg) was suspended in 2.4 ml digestion buffer (100 mM NaCl; 10 mM Tris-Cl, pH 8; 25 mM EDTA, pH 8; 0.5% SDS; and 0.1 mg/ml proteinase K). The samples were incubated for 5 hr at 50°C with shaking, and nucleic acids were extracted with phenol/chloroform/isoamyl alcohol. After centrifugation at 1700g for 10 min, the supernatant containing the DNA was precipitated with 0.5 vol of 7.5 M ammonium acetate and 2 vol of 100% ethanol. DNA was recovered by centrifugation at 1700 g for 2 min and washed with 70% ethanol. Final material

lane C). In P. juponicus, six isoforms have been reported (Galgani et al., 1985) while in P. indicus two variants have been found (Honjo et al., 1990). Molecular weight determined on denaturing SDS gel electrophoresis ranged between 3 1 and 32 kDa depending on the isoforms (Fig. 1, lane E). Analysis of N-terminal amino acids was determined on these different isoforms and gave the sequence: I V x G T D x K x G x L P Y Q L corresponding to the N-terminus of trypsin isoforms so far purified. Four residues were not determined. A part of this difficulty might be due to the polymorphism of the different isoforms. Library screening and shrimp characterization

trypsin

cDNA

The hybridization conditions used for screening with the synthetic degenerate oligonucle-

555

Sequencing trypsin cDNAs from Penaew vannamei Table 1. Purification

Crude extract Filtration step Affinity + G25

Proteins (mg) 120 37 3.5

of Penaeus vannamei trypsin

Total activity (units) 156 85 110

otide were highly stringent. About 70 hybridization-positive clones out of a total of about 6 x lo4 were detected in the cDNA library of the digestive gland. The sequencing of the five longest inserts revealed nucleotide sequences encoding for two of the mature protein isoforms of P. uannamei trypsin. The longest of these cDNAs (HP1 3) was composed of 736 bp. Rescreening the library with a 532 bp fragment of the S-region of the previously isolated cDNA yielded about 42 positive clones out of a total of 6 x 104 independent clones from the shrimp library. The 14 longest inserts were selected and characterized revealing the nucleotide sequences of three isoforms of trypsin, all of which lack the initiation codon methionine. To obtain the complete sequences for the existing isoforms, a further screening was performed with an A

B

Specific activity W/w) 1.3 2.3 31.5

Yield WI 100 54 71

Purification factor 1 1.8 24

oligonucleotidic probe derived from the 5’-region (5’-CCC ACC TTC CGT/C CGC GGT CTC AA-3’). Thus, 30 positive clones out of a total of 6 x lo4 phages were obtained. Five variants of the P. vannamei trypsin were identified. The longest cDNA insert (HPV 30) contained a complete coding sequence of 801 bp including the start and stop codons, and a 51 bp non-coding down-stream sequence (Fig. 2). With the exception of this cDNA, all analysed inserts obtained by the screening of the digestive gland cDNA-library lacked the ATG-start codon. The fifth variant lacked not only the leader sequence, but also 66 bases coding for the N-terminal sequence of the mature protein. Comparison of the nucleotide sequences coding for the mature protein shows five to seven

C kDa

D M

E T2

Tl

Fig. 1. Characterization of crude extract and purified trypsin of digestive gland from Penaeus vannamei. A: Davis-electrophoretic analysis of crude extract: 2.5 pg protein revealed by silver staining. B: Revelation of trypsin activity in crude extract by BANA. C: Purified trypsin revealed by silver staining (1 lg). D: SDS-gel electrophoresis of molecular weight standards. E: Purified trypsin: T, corresponds to the most anionic major isoform and T, to the less anionic major isoform, which could be separated by ion exchange chromatography as described previously for Penaeus juponicus (Galgani et ul., 1985).

556

B. Klein et al. TRYA m

ATG

PstI

Ara I

TAA Poly A tail

Kb

0

0.5

D TRY B

m

0.8

TRY C

b

TRY A

4 4 4

UP

TRY C TRY B

Fig. 2. Map of the shrimp trypsin cDNA and sequencing strategy. The bright shadowed box represents the leader sequence followed by the sequence for the precursor peptide and the subsequent mature protein, The open box shows the 3’-non-coding region. The sequencing strategy which has been respected for the analysed isoenzymes is shown by horizontal arrows. Localization of the hybridizing site for the different oligonucleotide probes (Try A, Try B and Try C), sequencing primers (R: Reverse; U: Universal) and cleavage sites for the most common restriction enzymes @%I, Ava I and Cla I) are indicated. The PstI-restriction site is not present in variants 21 and 42.

different bases between variant 30 and variants 40 and 39. Base changes between variant 30 and variants 21 and 42 are more frequent: variant 30 differs from 21 in 41 nucleotides and from 42 in 35 nucleotides. The 3’ untranslated regions of the first three variants are identical, but very different from those of the variants 21 and 42. All variants contained the AAT(T/A)AA polyadenylation signal. From 28 inserts that have been analysed by sequencing, the various trypsins encoding cDNA were represented in the library with the following percentages: variant 40: 32%, variant 2 1: 28%, variant 39: 11%, variant 30: 21% and variant 42: 7% (Fig. 3). The absence of 5’ untranslated regions in the inserts has also been noticed in cathepsin cDNA (Le Boulay et al., 1995) and amylase cDNA (Van Wormhoudt and Sellos, in press), but not in chymotrypsin cDNA (Sellos and Van Wormhoudt, 1992), thus minimizing the possibility of a defect in the shrimp digestive gland cDNA library. However, the size of the mRNA

does not preclude the existence of such a 5’ region. Structural features and alignments of the jive variants of shrimp trypsin The deduced protein sequence of 256 residues, including the initiation codon methionine, contained a hydrophobic signal peptide of 14 amino acids (numbered - 28 to - 15), followed by a precursor peptide of 14 residues (numbered - 14 to - 1) and the active enzyme (numbered l-227) (Fig. 3). The corresponding molecular weight of this last enzyme was around 22,500 Da, differing from that obtained by SDS gel electrophoresis and suggesting therefore that P. vannamei trypsin is possibly glycosylated (Kimoto et al., 1983; Dendiger and O’Connor, 1990). The N-terminal amino acid sequence determined from the purified shrimp trypsin was observed again (residues 1-16) in the deduced amino acid sequence from the cDNA. The

Fig. 3. Nucleotide sequences of the cDNAs of the five isoforms of trypsin and their deduced amino acid sequences. The amino acid sequences are numbered beginning with the first N-terminal residue of the mature protein. The sense strand sequences are numbered from the first detected nucleotide. The presumptive signal sequences are underlined and the supposed precursor peptide is in bold. The amino acid residues of the catalytic triad are marked with an asterisk. The polyadenylation signal sequence in the 3’ non-coding region is double underlined. The sequences hybridizing to the oligonucleotides Try A, Try B and Try C are printed in bold. The numbers under the sequences follow the established chymotrypsin numbering system.

Sequencing trypsin cDNAs from Penaeus

___-_-_-P---T TTC &CC NE AGC MC GAC AX TCC CTG CTC AAG m _ ---------F 100

vannamei

--Y _ _ . _ K?T CAD ccc cm AGC ml2 ?Ac GAC TP.CD%T CGCGee Al-c GAT ATT ccc GCT CRG 294 -----Y _ _ . _ _ _ . 110 120

557

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B. Klein et

cleavage site Lys/Ile, typically found in trypsinogens (De HaEn et al., 1975), was also present in shrimp protrypsin deduced from the cDNA. The amino terminal sequence of the active enzyme Ile-Val-Gly-Gly is highly conserved in all trypsins. Except for the predominant presence of hydrophobic amino acid residues, there was a high variability of the amino acid composition between the leader peptides of the trypsin isoenzymes, yet the precursor sequences were highly conserved. Homology between the activation peptides of trypsins has been observed for various vertebrate species (Neurath, 1984). However, there is no discernible sequence identity between the known precursor sequences in invertebrate and vertebrate species. This might be due to differences in the mechanism of proteolytic activation. In this respect, the presence of two cleavage sites for trypsin (Arg-Arg and Arg-Lys) within the precursor sequence could be of interest. All variants included AspIs at the base of the substrate-binding crevice, which is an important residue for the trypsin substrate specificity, and the active site residues His”, Asplo and Se?” (chymotrypsin numbering system), which are implicated in the catalytic mechanism of all serine proteases (Neurath, 1984). The sequences flanking these residues substitution were conserved with minor (Ala*-+Gly). At this position alanine is found also in trypsin from crayfish (Titani et al., 1983) and the insect Aedes aegypti (Kalhok et al., 1993). The alignment of the nucleotide sequences coding for the precursor protein of the five variants revealed a sequence identity of about 85%. Differences between the nucleotide sequences encoding the five isoforms occurred in 36 of 243 codons (without the signal peptide), resulting in the modification of 25 amino acids. The comparison of the sequences suggested a division of the trypsin isoenzymes into two families, formed on the one hand by variants 30, 40 and 39 and on the other hand by variants 21 and 42. The amino acid composition of these two families varied in 23 positions, but within each family the amino acid sequences differed in only six positions. (Variants 30139-40: Leu2’+Phe and Leu20+Met; variants 40/39+30: Lys’-+Thr; variants 30/40+39: Asn”+Lys; and variant 42-+21: Asns6+Ser.) The existence of a PstIcleavage site for variants 40, 30 and 39 and

al.

its absence in variants 21 and 42 support this classification of the isoenzymes. The amino acid sequence in mature P. vannamei trypsin was highly similar to that in other invertebrate and vertebrate serine proteases (Fig. 4). The short evolutionary distance between the trypsin of P. vannamei and that of crayfish is reflected by a sequence identity of 73%. The alignment of the entire amino acid sequences revealed that the crustacean (Titani et al., 1983) sequence was more related to the sequence of bovine trypsin (42%) than to that of bacterial trypsin. This finding is in contrast with previous results based only on the N-terminal sequence by Olafson and co-workers (1975) who concluded that the highest degree of identity exists between crayfish and bacterial trypsin (Table 2). The active form of trypsin encompasses eight Cys residues in conserved positions, which might form four disulphide bridges, whereas six Cys residues were found in crayfish and twelve Cys residues are predominant in vertebrate species (Emi et al., 1986; Fletcher et al., 1987). Insects and vertebrates have approximately the same level of sequence similarity confirming an ancient divergence of crustacea and insects within the arthropods. Northern blot analysis mRNA expression and trypsin activity at d@erent moult stages

The size and the expression of trypsin mRNA was determined by Northern blot analysis of total RNA of the digestive gland (Fig. 5). Hybridization with trypsin cDNA probe showed that its mRNA is highly expressed in all moult stages with only one band of an approximate size of about 970 bp. The longest clone with an open reading frame obtained after the screening of the digestive gland cDNA-library was composed of only 854 pb. Therefore the nucleotide sequence is lacking about 110 bp in the 5’ region. The results of the dot blot hybridization showed the propensity of trypsin encoding mRNA to increase during the premoult stage (D,). A peak is observed at Dl”-Dl”’ stages, followed by a steep decline of expressed mRNA in late premoult (D,-D,) (Fig. 6(a)). An increase was then measured in premoult (A and B stages). The elevated mRNA expression in D, corresponded to earlier results pointing out the increase of protein and RNA during the premoult stage, a phenomenon that has been previously

559

Sequencing trypsin cDNAs from Penaeus vannamei

TRYP-PENVA TRYP-ASTFL

TRY5-AKDAK

SLNVGY----SLSGVGSS---

TRYl-ANOGA

SLQYNKR----

TRYP-SQUAC

TRYA-DROHE

---t.fVRLSMG----

TRYP-STRGR

SGNNTS---I

SLNSGY-----H

TRYP-BOVIN

LNQDVDEGNKQTVILSKIIQHEDYNGFTIS LDNSVNEGSEQTITVSKIILHENFDYDLLD HDISANF,GDETYIDSSMVIRHPNYSGYDLD -SSQRASGGQL-I ENKKVNRHPKYDEVTTD -TSRHASGGTV-VRVARWQHPKYDSSSID -STTWSSGGW-AKVSSFKNHEGY GWDLQSSSAVKVRSTKVLQAPGYNGT-DNINVVEGNEQFISASKSIVHPSYNSNTL I

I

I*

80

90

100

TNEGGST-PS

135 135 123 127 127 125 117 123

PLSFNDNVRAIDIPAQGHAA--SGDC SLTFNNNVAPIALPAQGHTA--TGNV PAALNRNVDLISLPTGCAYA--G?Z’MC! TVTFSDSCAPVKLPQKDTPVNKGTCL DELTFSDSVQPVGLPKQDETVKDG SSLSFSSSIKAISLATYNPAN-PI ----NQPTLKIATTTAYN--QGTF SAASLNSRVASISLPTSCASA--GTQCLI *I 110

I

I

120

130

P** 140

201 201 185 191 191 189 184 186

KVTVPIVSDDi&D---&SD&-EDS&E

TKSSGTSYPD 150

160

170

180

YHVDWIK--ANAV YHVDWIK--ANAV HYVSWIHETIASV VRDWVKE-VSGL E-NSGV STANSI IASAARTL YVSWIKQTIASN ,f

****

*;*

****

* I

210

220

237 237 222 227 227 226 223 223

230

I 240

Fig. 4. Sequence homology of vertebrate and invertebrate species to the trypsin of Penaeus vannamei. The 42 completely conserved sites are marked by black boxes. The eight Cys residues that might be imphcated in forming four disulphide bridges are denoted by an arrow. Amino acid sequences are shown for Penaeus vannamei trypsin (TRYP-PENVA), bovine trypsin (TRYP-BOVIN), dogfish trypsin (TRYP-SQWAC), Drosophila melanogaster trypsin (TRYA-DROME), anophih trypsin (TRY I-ANOGA), crayfish win (TRYP-ASTFL), Aedes aegypti trypsin (TRYSAEDAE), Streptumyces griseus trypsin (TRYPJEltGR) [SWISSPROT data-library accession codes]. The numbers under the sequences follow the established chymotrypsin numbering system.

B. Klein et al.

560

demonstrated to be under hormonal control by ecdysteroids (Van Wormhoudt et al., 1985). During the moult cycle, the specific activity of trypsin reached its maximum in premoult (Dl”-Dl”’ stages) and its minimum during B and D3 stages (Fig. 2). The same correlation exists for chymotrypsin mRNA and chymotrypsin specific activity in premoult (Van Wormhoudt et al., 1995). However no increase of trypsin mRNA was observed in postmoult (stage B). Whether this difference is physiological or due to different experimental conditions is not known. Specific activity ranged between 0.25 and 0.92 Units/mg protein. The variation of mRNA expression followed the pattern of trypsin enzyme activity. The amplitude of the variations of specific activities ( x 4) corresponded to the amplitude of the variations of mRNA expression suggesting the regulation of protein synthesis at the level of transcription (Fig. 6(b)).

23,13096% -43612322 2027

564-

Fig. 5. Northern blot analysis of total RNA from the digestive gland of P. vannamei. (A): Total RNA (20 @g/5.6 ~1) was separated on a 1.5% agarose-gel. The blot was hybridized with an EcoRI-ClaI-fragment of a trypsin encoding cDNA. Radioactivity was detected by autoradiography for 1 night. Line M indicates the standard molecular weight markers (Lambda Hind111 DNA fragments) in bp.

Sequencing trypsin cDNAs from Penaeus

vannamei

Southern blot analysis

The results of the Southern blot analysis support the theory that trypsin is encoded by a multigene family (Fig. 7). The enzyme EcoRI did not cleave the trypsin encoding cDNA. A Cla I cleavage site was found in all the five cDNAs and one cleavage site for PstI was determined in the three variants corresponding to the first family (Nos 30,40 and 39). Southern hybridization transfer revealed several bands in each lane. The hypothesis of the existence of several trypsin genes not

b

A

B

A

B

C

DOD,’

Malt

D,,,D,,,,

D,

b

alages

Fig. 6. Variation of the expression of trypsin during the moult cycle A: Variation of mRNA during the moult cycle in P. vannamei digestive gland. - 3.6 and - 1.2 pg RNA were dotted on a nylon membrane and hybridized with a trypsin cDNA probe. The dots were cut and counted. mRNA levels (c.p.m./pg total RNA) were expressed as the mean of n values (see brackets) + SD. The differences between the moult stages were calculated using the Student’s t-test. Different letters indicate significance of the response: P c 0.01. B: Variation of trypsin specific activity. Units of activity correspond to one pmole ofp-nitroanilide liberated in 1 min by the hydrolysis of BAPNA. The statistic analyses were made according to A.

Fig. 7. Southern blot analysis of genomic DNA from the digestive gland of P. vunnamei. The genomic DNA (12 pg) was hydrolysed by single and double digestions. Lane 1 = Eco RI/Cla I, lane 2 = Eco RI, lane 3 = Eco RI/Pst I, lane 4 = Pst I, lane 5 = Pst I/Cla I and lane 6 = Cla I. Blots were hybridized with a cDNA probe derived from the entire trypsin encoding cDNA from clone no. 30. The arrows indicated several bands in each lane. Line M shows the standard molecular weight markers (Lambda Pst I DNA fragments) in bp.

tandemly arranged in the genome based on the presence of several fragments with different sizes for each enzyme. The trypsin gene was mainly contained inside a 1.2 Kb Eco RI fragment (sites which are located in the introns) that was cleaved with Cla I (site seen in the coding sequence). This type of organization is only applicable to part of the genes. For example, for Pst I digestion, several large fragments (4 Kb to 5-6 Kb) were observed and with the double digestion (Pst I/Eco RI) the Eco RI fragment appeared and the large 6 Kb fragment was cleaved, but the other fragments (4Kb) remained unchanged. The hypothesis of the existence of two families of cDNA was confirmed by the existence of two types of trypsin gene. Overall conclusion

The cloning and sequencing of the trypsin cDNA provide the first description of a putative zymogen sequence for a crustacean species. The sequence analysis of the trypsin of P. vannamei and its comparison to the nucleotide sequences

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of other species assist the study of the evolutionary relationship between serine proteases. In P. uannanzei, biochemical and molecular data confirm the existence of a high degree of polymorphism for trypsin. The complexity of the genome organization reported also permits the characterization of shrimp trypsin into two families. The quantification of trypsin mRNA and the measurement of its specific activity during the moult cycle are the first steps in understanding the regulation of trypsin synthesis. Further studies will be nccessary to obtain more information about the regulation of trypsin gene expression, especially in relation to ecdysteroid control in the premoult stage. REFERENCES

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