Cloning of the cDNA encoding Scg-SPRP, an unusual Ser-protease-related protein from vitellogenic female desert locusts (Schistocerca gregaria)

Insect Biochemistry and Molecular Biology 28 (1998) 801–808

Cloning of the cDNA encoding Scg-SPRP, an unusual Serprotease-related protein from vitellogenic female desert locusts (Schistocerca gregaria) Shean-Jaw Chiou a, Jozef Vanden Broeck a,*, Ine Janssen a, Dov Borovsky b, Frank Vandenbussche a, Gert Simonet a, Arnold De Loof a a

Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute, K. U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium b University of Florida-IFAS, Florida Medical Entomology Laboratory, 200 9th Street S.E., Vero Beach, FL 52962 USA Received 19 November 1997; accepted 27 May 1998

Abstract The cDNA coding for a Ser-protease-related protein (Scg-SPRP)1 was cloned from desert locust (Schistocerca gregaria) midgut. The derived amino acid sequence consists of 260 residues and shows strong sequence similarity to insect trypsin-like molecules. It is, however, likely that Scg-SPRP is not a proteolytically active enzyme and that it plays another physiologically relevant role, since two out of three residues which are indispensable for catalytic activity of Ser-proteases are replaced. Northern analysis revealed that the Scg-SPRP gene is expressed in midgut tissue and that this expression is strongly induced in adult female locusts. Moreover, the occurrence of the transcript (1.2 kb) fluctuates during the molting cycle and during the female reproductive cycle. Juvenile hormone (JH III) dependence of transcription was investigated by chemical allatectomy (precocene I) of adult females. This resulted in inhibition of vitellogenesis and in disappearance of the Scg-SPRP transcript. Expression of Scg-SPRP in precocene-treated locusts could be reinduced by additional treatment with JH III or with 20-OH-ecdysone.  1998 Elsevier Science Ltd. All rights reserved. Keywords: Chymotrypsin; Ecdysone; Insect; Juvenile hormone (JH); Molting; Protease; Trypsin; Vitellogenesis

1. Introduction In all metazoan species, Ser-proteases play a prominent role in a variety of important physiological processes, such as food digestion, blood clotting, embryogenesis, tissue reorganization (e.g. wound healing, regeneration, molting, metamorphosis), defense mechanisms and immune responses. Activation and inactivation of protease cascades are controlled at different regulatory levels: protease gene transcription, mRNA translation, zymogen activation, substrate selectivity, binding to inhibitors, etc. In insects, the enzymes for food protein digestion, most of which belong to the group of Ser-proteases, are mainly produced in the midgut. Moreover, in some species, the ingestion of a * Corresponding author. Tel.: + 1-32-16-323912; Fax: 32-16323902; E-mail: [email protected] 1 The cDNA sequence encoding Scg-SPRP was submitted to the EMBL-database and received the accession number Y09607. 0965-1748/98/$19.00  1998 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 9 8 ) 0 0 0 5 4 - X

protein-rich meal triggers the onset of female vitellogenesis and folliculogenesis: e.g. female mosquitoes take a blood meal and this is digested following an intense increase in midgut trypsin production. The resulting free amino acids are required for vitellogenin synthesis in the fat body. It has been demonstrated that mosquitoes have multiple trypsin genes (Barillas-Mury et al., 1991; Kalhok et al., 1993; Muller et al., 1993, 1995; Noriega et al., 1996a). An ‘early trypsin gene’ transcript is already present before the blood meal is taken (Noriega et al., 1996b). Therefore, ‘early trypsin’ synthesis appears to be regulated at a translational level, whereas the production of ‘late trypsin’ is the result of a transcriptional activation of the corresponding ‘late trypsin’ genes (Barillas-Mury and Wells, 1993; Noriega et al., 1994; Muller et al., 1995). At the end of vitellogenesis, the ovary of the yellow fever mosquito, Aedes aegypti, releases a feedback factor in order to block trypsin biosynthesis and oocyte growth (Borovsky et al., 1990). This factor is a decapeptide and was called trypsin mo-

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dulating oostatic factor (Aea-TMOF). In the grey fleshfly, Neobellieria (Sarcophaga) bullata, a similar situation was observed (Bylemans et al., 1994). A trypsin transcript is present before the protein-rich (flesh or liver) meal is taken. Neobellieria trypsin production is regulated at the level of mRNA translation, which is triggered by the ingestion of a protein meal and blocked by Neb-TMOF at the end of vitellogenesis (Borovsky et al., 1996). More recently, Jiang et al. (1997) have demonstrated the existence of an adult, female-specific chymotrypsin from mosquito (Aedes aegypti) midgut and the production of this protease is also induced by ingestion of a blood meal at a posttranscriptional level. It is not excluded that in many other insect species, including non-carnivorous ones, the female reproductive cycle is coupled to an increase in protease production by the midgut and that there are feedback factors released by the mature ovaries (De Loof et al., 1995). In this respect, we have to mention that very recently, an ovarian inhibitor of midgut Ser-protease production was isolated and partially sequenced from the phytophagous desert locust, Schistocerca gregaria (Janssen, 1997). This article presents the cloning and identification of the cDNA encoding a locust Ser-protease-related protein, Scg-SPRP, induced in vitellogenic female midguts. Tissue- and stage-dependent gene expression, as well as a possible influence of JH and ecdysteroids, have been investigated via northern blot analysis.

2. Materials and methods 2.1. Rearing of desert locusts Schistocerca gregaria (Forsk.) was reared under stable temperature conditions (32 ± 1°C). The animals were kept in special cages and fed daily with fresh grasses, rolled oats and cabbage leaves. Mature females deposit their eggs in pots filled with slightly humidified sand. After oviposition these pots were collected at given time intervals resulting in synchronized pools of hatched first instar hoppers.

stranded cDNA was obtained in two steps by following the protocol included in the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). 2.3. Polymerase chain reaction (PCR) PCR primer sequences were based on conserved amino acid sequences in Ser-proteases. The primers (Eurogentec, Seraing, Belgium) had the following sequences: (1) →5⬘-AT(A,T,C)GTIGGIGGITT(T,C)GAG-3⬘, was derived from the N-terminal amino acid sequence of mosquito trypsins (IVGGFE); (2) ←5⬘-ATCICC(T,C)TG(A,G)CAIGA(A,G)TC(T,C)TT-3⬘, was derived from an amino acid sequence present in many trypsins (KDSCEGD). Fifty microliter PCR reactions were performed containing 5 ␮l of the 10 × PCR buffer delivered with the enzyme, 0.2 mM of each dNTP, 0.2 ␮M of each primer, 5 ␮l of cDNA template ( ⬍ 0.1 ␮g of cDNA), 0.4 ␮l of Amplitaq (Perkin Elmer) heat-stable DNA polymerase. Hot-start PCR was run for 35 cycles. One cycle consisted of denaturation (for 60 s at 94°C), primer annealing (for 60 s at 55°C) and extension (for 60 s at 68°C) steps. After the last cycle an additional extension time of 7 min at 68°C was applied. PCR reactions were then analyzed by horizontal agarose gel electrophoresis and bands were visualized by ethidium bromide fluorescence. PCR products were subcloned and sequenced as outlined below. 2.4. RACE protocol In order to obtain a complete cDNA sequence, rapid amplification of cDNA ends (RACE) was employed. This RACE protocol was performed according to the instructions of the Marathon cDNA Amplification Kit (Clontech). The adapter primers were provided with the kit, whereas the specific 5⬘- and 3⬘-RACE primers (Eurogentec, Belgium) were derived from the sequence of the original PCR fragment:

2.2. RNA isolation and cDNA synthesis

(1) →5⬘-GGCGACCTACGTGATCAGGAACGTG-3⬘

Locusts were dissected under a binocular microscope and tissues were collected and directly frozen in liquid nitrogen. Total RNA was extracted by using TRI-zol reagent (BRL, Bethesda). Poly-A RNA was extracted from pooled tissue samples in RNase-free conditions by employing the QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech, Uppsala, Sweden). Midgut mRNA from vitellogenic females was used as a template (0.5 ␮g) for cDNA synthesis. Double

(2) ←5⬘-TTCGTGGATGTACCTGCACGCAGCG-3⬘ 5⬘- and 3⬘-RACE fragments were analyzed by horizontal agarose electrophoresis and ethidium bromide fluorescence. 2.5. Control PCR After sequencing the first PCR and both RACE products, the obtained cDNA sequence was verified by per-

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forming a PCR of the open reading frame of the protein. This was done by using an enzyme mix (50 × Klentaq polymerase mix, Clontech) containing a proofreading enzyme. (1) →5⬘-GCCTCGAGATAAATATGTTTCGTCTGGCAGTACTG-3⬘ (2) ←5⬘-GCTCTAGATTACTTTCAGACTCCTGAGACCTCGCT-3⬘ Again, the obtained band was visualized via horizontal agarose electrophoresis, cut out from the gel, subcloned via TA-cloning and sequenced using the protocols outlined below. 2.6. Cloning, sequencing and sequence analysis PCR and RACE products were cloned into pCR2.1 vector by using the Original TA Cloning Kit (Invitrogen, Carlsbad, CA). Recombinant plasmids were isolated and the inserts were sequenced by following the protocols outlined by the Sequenase Version 2 sequencing kit (USB-Amersham). The universal M13 (− 40) and reverse M13 primers were used to obtain the terminal cDNA sequences. Nucleotide and amino acid sequence analyses and comparisons were performed by using pc-gene software (intelligenetics). Sequence alignments were obtained by employing the clustal and align programs. Database searches were run with the blast or fasta programs. 2.7. Hormonal treatment Female locusts were treated at the time of adult emergence by topical administration of precocene I (500 ␮g in 15 ␮l of acetone per animal). Control animals were treated with acetone only. At day 8, the animals were treated with JH III (per animal: 10 ␮g by topical treatment, 5 ␮g by injection into the abdomen; JH III was dissolved at 1 ␮g/␮l in ethanol), with 20-OH ecdysone (two times per day during 3 consecutive days, injection of 2 ␮g dissolved in ethanol at 1 ␮g/␮l), or with solvent only (control). Fat body and ovaries were collected at day 12. Hemolymph samples were analyzed by measuring total protein concentration (Bradford, 1976) and by SDS–PAGE in order to monitor vitellogenin appearance and to evaluate the effects of precocene and hormone treatments. Tissues were pooled ( ⭓ 6 animals per pool) and mRNA was extracted for northern analysis. 2.8. Northern blot analysis Poly-A RNA was prepared from locust tissues or whole animals corresponding to different developmental or physiological conditions. Two micrograms of each

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sample (derived from pooled insects) were loaded for RNA electrophoresis. The agarose gel electrophoresis was run under denaturing conditions by including formaldehyde in the gel (Sambrook et al., 1989). The RNA in the gel was then transferred onto Hybond N (Amersham) nylon membranes by capillary blotting with DEPC-treated 20 × SSPE buffer (Sambrook et al., 1989) and irreversibly blocked by drying and baking in an oven at 80°C for 2 h. The blot was prehybridized for 4 h at 42°C in a solution (20 ml) containing 0.5 mg of denatured heterologous herring sperm DNA, 50% deionized formamide, 5 × SSPE, 10 × Denhardt’s solution and 0.5% SDS. A specific cDNA probe was prepared by labelling the cloned RACE fragments via the Rediprime system (Amersham) with 32P (Redivue dCTP, Amersham). The denatured probe was added to the prehybridization solution and the hybridization reaction was incubated for at least 16 h. The hybridized nylon membrane was rinsed in 2 × SSPE, 0.1% SDS at room temperature and washed at room temperature and at 65°C in 1 × SSPE, 0.1% SDS during 15 min for each condition. High stringency washing was performed at 65°C in 0.1 × SSPE, 0.1% SDS for 10 min. Then the filter was removed, wrapped in SaranWrap and positioned in an X-ray cassette (KODAK) with intensifying screens. Autoradiography was performed by exposure of a hyperfilm-MP (Amersham) to the hybridized blot during a period of 24 h. Blots were rehybridized with an actin probe as a control to check if the mRNA content in each lane was comparable.

3. Results 3.1. Cloning and sequencing results The cDNA sequence contains an open reading frame encoding a Ser-protease-like protein. The derived amino acid sequence consists of 260 residues (Fig. 1). Comparison with other Ser-proteases revealed strongest sequence similarity to other insect trypsin-like molecules. Surprisingly, two out of three catalytic amino acid residues which are highly conserved in most Ser-protease enzymes are absent in Scg-SPRP. His and Ser are replaced into Leu and Phe respectively, whereas the Asp-residue remains unchanged (Fig. 2). This sequencing result was found to be very consistent in all subcloned sequences that were analyzed and also in additional fragments which were amplified via proofreading enzymes in control PCR-reactions. Therefore, Scg-SPRP has most probably lost its ability to act as a protease, although its structure must be very similar to that of many Ser-proteases. Although its amino acid sequence is closest to that of insect trypsins, Scg-SPRP misses the residue that is

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Fig. 1. Nucleotide sequence (cDNA) and translated amino acid sequence of Scg-SPRP. The nucleotide sequences for the startcodon, stopcodon and a possible polyadenylation consensus sequence (AATAAA) are underlined. An arrow (⇑) indicates the expected cleavage site of the signal peptide (according to Von Heijne, 1991). The predicted pro-enzyme cleavage site is shown by ↑.

important for determining trypsin-specific activity (Asp) since this is also changed (into a Gly). 3.2. Northern analysis 3.2.1. Tissue distribution (Fig. 3) In a first experiment, RNAs derived from different desert locust tissues were analyzed via northern blot hybridization with the above mentioned cDNA probes. The transcript of Scg-SPRP has an approximate length of 1.2 kb and was only found to be present in midgut mRNA. 3.2.2. Staging (Fig. 4) In a further test, mRNAs prepared from whole animals of different stages were analyzed. The Scg-SPRP is encountered in all larval stages (I–V), but it is much more abundant in adult female locusts.

3.2.3. Reproductive cycle (Fig. 5) To follow the time course of expression of the gene during the reproductive cycle, locust midgut mRNA was prepared at given times after adult emergence and the mRNAs of pooled tissues corresponding to the same stage were prepared and analyzed via northern hybridization. In the midgut, the expression level of the Scg-SPRP gene appeared to be high before and during vitellogenesis, reaching peak values during early and mid-vitellogenesis, whereas in the late vitellogenic stages and at the time of oviposition (egg laying), at days 19–21, the transcript almost disappeared. Scg-SPRP mRNA again increased starting from day 23 (at the start of the next reproductive cycle). The same blot was rehybridized with a ␤-actin probe to show that mRNA was present in all lanes.

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Fig. 2. Sequence comparison of Ser-protease-like proteins, some of which are lacking one or more of the catalytic triad residues (䊏). The specificity pocket residue (D in trypsins) is shown by 쐌. Conserved positions are shown by asterisks. SPRP—SG, desert locust SPRP, Schistocerca gregaria; CAP7—H, human azurocidin; CFAD—H, human complement factor D; EL2A—H, human elastase 2A; ELNE—H, human leukocyte elastase; CTR1—PV, shrimp chymotrypsin B1, Penaeus vannameii; HGFL—H, human hepatocyte growth factor-like protein; HGF—H, human hepatocyte growth factor; KAL—H, human plasma kallikrein; PLMN—H, human plasminogen; TRY1—AG, African malaria mosquito trypsin 1, Anopheles gambiae; TRYA—D, fruitfly trypsin alpha, Drosophila melanogaster; TRYA—H, human alpha-tryptase.

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Fig. 3. Tissue-dependent expression analyzed via northern blot hybridization. Mg, midgut; Fb , male fat body; Fb , female fat body; T, testis; Ov, ovary; kb, kilobases.

Fig. 6. Northern analysis of Scg-SPRP transcripts during the last molting cycle (starting at the fourth molt, i.e. in the fifth larval stage). Samples were prepared from pooled whole body extracts at 1, 2, 4 and 6 days after the fourth molt (lanes V + 1, + 2, + 4, + 6) and at a few hours after the final molt (in the adult stage, Ad). a: Scg-SPRP; b, actin control.

3.2.4. Molting cycle (Fig. 6) Desert locusts were set apart at the start of the fifth instar. Messenger RNA was prepared from whole animals ( > 6 animals pooled per condition) which were taken at certain time intervals (1 day, 2 days, 4 days, 6 days) after fifth instar emergence and at a few hours (approx. 3 h) after adult emergence. Northern analysis was performed with 3 ␮g mRNA per lane by hybridizing with radio-labelled Scg-SPRP cDNA as a probe. The detectable amount of mRNA coding for ScgSPRP was much higher at 2 and 4 days after fifth instar emergence than at 1 or 6 days. At adult emergence the transcript even virtually disappeared. Fig. 4. Stage-dependent expression analyzed via northern blot hybridization. The mRNA samples were derived from whole bodies. Larval stages: I, II, III, IV, V; , adult males and , adult females; kb, kilobases.

Fig. 5. Northern analysis of Scg-SPRP transcripts during the adult female reproductive cycle. Samples were prepared from pooled female midgut extracts at 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 days (d) after the final molt. a: Scg-SPRP; b: actin control.

3.2.5. Precocene I and hormonal treatment (Fig. 7) Precocene I treatment resulted in a strong inhibition of Scg-SPRP gene expression in the locust midgut (compared to the acetone treated control). Additional treatment with JH III reinduced the transcript and treatment with 20-OH-ecdysone almost completely restored the effect of precocene I.

Fig. 7. Northern analysis of female midgut mRNA samples taken after treatment of the animals with precocene I and/or hormonal factors. Samples were prepared from pooled tissues at 12 days after the final molt. Lanes: p + JH, precocene I and juvenile hormone treatment; c + JH, juvenile hormone control treatment; p + E, precocene I and 20-OH-ecdysone treatment; c + E, 20-OH-ecdysone control treatment; p, precocene I treatment; c, control condition.

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4. Discussion This paper describes the cloning of a cDNA for Schistocerca gregaria trypsin-like protein, Scg-SPRP, a Serprotease-related protein. The corresponding mRNA is highly abundant in the midgut of vitellogenic female desert locusts, whereas in larval stages and in males the level of transcription is lower. This is in agreement with our recent results (Janssen, 1997) which demonstrate the existence of a significant increase of Ser-proteases in the gut of vitellogenic female S. gregaria by using the irreversible inhibitor di-isopropyl-fluorophosphate (DFP). Males of the same age as the vitellogenic females have a significantly lower amount of DFP-binding proteases in the gut. The situation in locusts appears to be similar to the one in mosquitoes and fleshflies. There is a close relationship between protease production in the midgut and vitellogenesis, suggesting that this is a process which is linked to the development of adequately matured eggs. It is however doubtful that the Scg-SPRP cDNA encodes a truly active protease. Its derived amino acid sequence lacks two out of three residues which are indispensable for catalytic activity of Ser-proteases. When searching sequence databases, a few other protease-like proteins were found to carry similar changes at their ‘active’ site (Fig. 2). All proteins carrying such changes indeed have extremely low or completely undetectable proteolytic activities, but, nevertheless, have acquired other, physiologically relevant functions (Nakamura et al., 1989; Han et al., 1991; Morgan et al., 1991; Almeida et al., 1991). In a related locust species, Locusta migratoria, trypsin- and chymotrypsin-like enzymes have already been purified and partially characterized, but these enzymes do not seem to correspond to ScgSPRP when comparing the available amino acid analysis data (Sakal et al., 1988, 1989). These observations indicate that Scg-SPRP is probably not a true protease, and might play a different role. Interestingly, the Scg-SPRP transcript appears to be regulated during the molting cycle (at least during the last one) and during the female reproductive cycle. These observations suggest that the transcript might be controlled by hormones such as JH III and 20-OH-ecdysone. To analyze the possible involvement of JH III in the control of Scg-SPRP, locusts were treated with the allatectomizing agent precocene I. This study showed that the Scg-SPRP gene is repressed after chemical allatectomy. Moreover, JH III treatment reinduces the expression and 20-OH-ecdysone can even fully restore the inhibiting effect of precocene I. Interestingly, ‘early trypsin’ gene transcription in Aedes aegypti also appears to be controlled by JH (Noriega et al., 1997). Although it is not yet clear whether the mechanisms by which JH or ecdysone are regulating transcription of Scg-SPRP are direct or indirect (via additionally induced

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factors), the results suggest that the observed developmental fluctuations (males/females, molting cycle) in expression of the Scg-SPRP gene might be caused by hormones. In this context, it is important to refer to the existence of a feedback factor which is produced in the ovaries of female locusts at the end of vitellogenesis. This (Scg-C/T-M-O-F) factor inhibits the production of chymotrypsin and/or trypsin in the midgut (Janssen, 1997). Once this peptidic factor is fully characterized, it will be possible to investigate whether it can control the expression of Scg-SPRP. The Scg-SPRP transcript is indeed present in lower amounts at the end of vitellogenesis (before and during oviposition). This period probably coincides with high levels of circulating ScgC/TMOF.

Acknowledgements The authors thank the FRS (Fund for Scientific Research-Flanders, Belgium), the NATO (North Atlantic Treaty Organization, CRG 940057, granted to D.B.) and the European Union (TS3*-CT93-0208) for funding this research. J.V.B. is a Senior Research Associate of the FRS-Flanders. I.J. has received a Postdoctoral Fellowship of the FRS-Flanders. R. Jonckers and J. Gijbels are gratefully acknowledged for technical assistance. The authors also thank H. Van den bergh, M. Van Der Eeken, J. Puttemans and M. Christiaens for their help with text and figure editing.

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