Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraeamylitta

Insect Biochemistry and Molecular Biology 33 (2003) 1025–1033 www.elsevier.com/locate/ibmb

Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraea mylitta Binita Shrivastava, Ananta Kumar Ghosh ∗ Department of Biotechnology, Indian Institute of Technology, Kharagpur-721302, West Bengal, India Received 17 March 2003; accepted 20 June 2003

Abstract An inhibitor of Aspergillus oryzae fungal protease was purified to homogeneity from the hemolymph of fifth instar larvae of Antheraea mylitta by ammonium sulfate precipitation, anion exchange and gel filtration (FPLC) chromatography, and termed as AmFPI-1. The extent of purification was checked by two-dimensional gel electrophoresis, and the molecular weight of purified inhibitor was determined by SDS-PAGE as 10.4 kDa. Fifteen N-terminal amino acid sequences of this protein were determined, and degenerate oligonucleotides were synthesized on the basis of these sequences. A cDNA library of A. mylitta integument was constructed, and protease inhibitor cDNA was partially amplified by PCR using degenerate oligonucleotides and CDS primers. A full-length inhibitor cDNA clone obtained by screening the library with PCR amplified DNA as probe was sequenced. The cDNA consists of 543 nucleotides with an ORF of 315 bp and encodes a protein of 105 amino acids. The sequence exhibits similarity to several Bombyx mori ESTs, and in particular to N-terminal amino acid sequence of an inducible serine protease inhibitor (ISPI-1) from Galleria mellonella indicating its relatedness to ISPI-1 of G. mellonella. The presence of this protease inhibitor in the hemolymph may play an important role as a natural defense system against invading microorganisms.  2003 Elsevier Ltd. All rights reserved. Keywords: Protease inhibitor; Antheraea mylitta; cDNA library; Cloning; Purification; Fungal protease

1. Introduction Protease inhibitors play a significant role in the regulation of proteolysis, whether the target enzymes are of exogenous or endogenous origin. They are present throughout the range of living organisms and have diverse functions. In plants, they can act by reducing the damage caused by insect pests (Ryan, 1990; Murdock and Shade, 2002). Transgenic plants expressing protease inhibitors have now been produced to provide protection from herbivorous insects (Gatehouse et al., 1991; McManus et al., 1994; Christeller et al., 2002). In animals, the protease inhibitors might prevent unwanted proteolysis by regulating proteases in vivo. The use of protease inhibitors as therapeutic agents, in particular, their use in inhibition of cellular transformation, blood

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0965-1748/$ - see front matter  2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0965-1748(03)00117-6

clotting disorders, osteoporosis, retroviral disease and cancer is under thorough investigation (Hocman, 1992). Due to these applications of protease inhibitors in medicinal and agricultural fields, the search for novel protease inhibitors has received widespread interest. Insect hemolymph, like vertebrate system, contains several protease inhibitors belonging to Kunitz and serpin families, although their properties and functions are not well understood (Eguchi, 1993; Polanowski and Wilusz, 1996). The presence of serine protease inhibitors has been demonstrated in the hemolymph of insects such as Bombyx mori (Eguchi et al., 1982; Sasaki and Kobayashi, 1984; Shinohara et al., 1993; Kurata et al., 2001), Antheraea pernyii (Eguchi et al., 1982), Drosophila melanogaster (Kang and Fuschs, 1980), Manduca sexta (Kanost et al., 1989; Kanost, 1990; Ramesh et al., 1988; Sugumaran et al., 1985; Gan et al., 2001), Locusta migratoria (Boigegrain et al., 1992; Simonet et al., 2002), Galleria mellonella (Frobious et al., 2000), Schistocerca gregaria (Hamdaoui et al., 1998; Gaspari et al.,

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2002), Theromyzon tessulatum (Chopin et al., 2000) and Mythimna unipuncta (Cherqui et al., 2001). It has been suggested that these inhibitors have a role in the regulation of proteolytic-activated processes, such as the phenoloxidase cascade, as well as in metamorphosis, development, and defense against invading pathogens (Ramesh et al., 1988; Sugumaran et al., 1985). Thus, they act as powerful tools for understanding insect immunity at a molecular level (Kanost, 1999). In comparison to the mulberry silkworm, Bombyx mori, protease inhibitors from non-mulberry silkworm have not been widely studied. Antheraea mylitta, an indigenous wild type non-mulberry saturniid silkworm present in India, produces an exotic variety of silk called tasar silk. These silkworms being wild in nature, mostly suffer from viral, bacterial and protozoal infection and not so much by fungi such as Aspergillus oryzae, Penicillium citrinum, Beauveria bassiana, the common entomopathogens of Bombyx mori, and other insects (Jolly et al., 1974). Entomopathogenic fungi utilize a complex of hydrolases (protease, chitinase), which enable them to infect susceptible hosts directly via the exoskeleton as these enzymes digest cuticle proteins (Clarkson and Charnley, 1996). Inhibition of these hydrolases by specific inhibitors of A. mylitta may prevent the onset and spread of fungal infection. But no protease inhibitors with activity against fungal proteases have been studied or characterized from A. mylitta. Here, we report the purification of a low molecular weight fungal protease inhibitor called AmFPI-1 from the hemolymph of A. mylitta larvae, and cloning and characterization of its cDNA.

2. Materials and methods 2.1. Insect and hemolymph Third, fourth, fifth instar and spinning stage larvae, and pupae of Indian non-mulberry silkmoth, A. mylitta, were obtained from local tasar silk farms of Purulia and Jhargram districts of West Bengal state, India, and their hemolymph was collected in a cold tube by cutting the abdominal legs. After removing the hemocytes by centrifugation at 10,000 rpm for 10 min, the supernatant was either used immediately for inhibitor assay or stored frozen at ⫺20 °C for subsequent analysis. 2.2. Protease inhibition assay Protease inhibition assay was done in triplicate in the hemolymph from different stages of larval development as well as after each step of inhibitor protein purification. Initially, protease activity was assayed by caseinolytic method using Aspergillus oryzae protease (Sigma) as enzyme and casein as substrate (Yamashita and Eguchi,

1987). In brief, 0.2 ml of casein solution (1%) was digested by incubation with 0.01 ml (1 mg/ml) of enzyme solution (equivalent 0.36 U) for 30 min at 37 °C in 1 ml reaction buffer (100 mM Tris–HCl buffer, pH 8.0). After digestion, the unhydrolyzed casein was precipitated using 1 ml of 20% trichloroacetic acid and removed by centrifugation. The concentration of digested protein in the filtrate was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. For measuring protease inhibitor activity in the hemolymph, the same amount of protease solution was pre-incubated with 0.075 ml of hemolymph (containing protease inhibitor) for 15 min at 37 °C and then protease activity was measured as described above. One unit of inhibition was defined as the amount of inhibitor that decreases the protease activity to 50% of the original value (Eguchi, 1982). 2.3. Purification of the inhibitor protein from the hemolymph The protease inhibitor was purified from the total protein present in the hemolymph of fifth instar larvae through several steps, as no single step was sufficient to obtain homogenous protein. Initially, the crude hemolymph (50 ml) was precipitated with 60% ammonium sulfate at 4 °C for 16 h. The precipitated proteins were recovered by centrifugation at 10,000 rpm for 10 min, dissolved in 10 ml of 3 mM sodium citrate buffer, pH 6.0 and dialyzed overnight against the same buffer to remove ammonium sulfate. The dialyzed protein solution was heat treated at 80 °C for 2 min, and the precipitated heat denatured proteins were removed by centrifugation at 10,000 rpm for 10 min. The heat stable protein solution present in the supernatant was applied to a DEAE Sephacel (anion exchange) column (45 × 2.5 cm) equilibrated with the above sodium citrate buffer. The unbound proteins were collected as flow through and the bound proteins were eluted from the column with a gradient of NaCl from 0 to 0.5 M at a flow rate of 0.3 ml/min. The optical density of all the fractions are measured at 280 nm and the peak fractions of both the flow through and column bound eluted proteins were pooled together, dialyzed against 3 mM sodium citrate buffer pH 6.0, lyophilized and assayed for inhibitor activity by the caseinolytic method. The active fractions (showing protease inhibitor activity) from anion exchange column were then applied on to a gel filtration superose-12 column (10 × 300 mm), connected to an FPLC system (Pharmacia) and run with equilibration buffer (0.15 M NaCl in 0.05 M phosphate buffer, pH 7.0). One-milliliter fractions were collected at a flow rate of 0.4 ml/min after passing through an online UV detector. The extent of protease inhibitor purification in each step was checked by sodium dodecyl sulfate-15% poly-

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acrylamide gel electrophoresis (SDS-PAGE) (Lamelli, 1970) and the homogeneity of purified protein after FPLC was verified by two-dimensional (2D) gel electrophoresis (O’Farrell, 1975). The first dimension was an isoeletric focusing gel with a pH 3 to pH 10 gradient, and the second dimension was an SDS-PAGE with 12% separating gel. After electrophoresis, the gel was stained with Coomassie brilliant blue, destained and photographed. Besides Aspergillus oryzae protease, the inhibitory activity of this purified inhibitor, AmFPI-1, was checked against five commercially (Sigma) available proteases such as Bacillus licheniformis protease, bovine trypsin, bovine pancreatic chymotrypsin, Rhizopus oryzae protease and papain. The purified inhibitor (0.075 ml) was pre-incubated with a constant amount (0.01 ml) of different protease (0.36 U) and the inhibition assay was done in triplicate by the caseinolytic method as described above. Phenylmethylsulfonyl fluoride (PMSF) was used as a positive control during the assay. 2.4. Amino acid sequencing FPLC purified AmFPI-1 was separated in SDS-15% polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Towbin et al., 1979). The membrane was stained with Coomassie brilliant blue R-250, destained and washed thoroughly to remove any bound glycine to the membrane. The protein sample in the blot was sent to University of Southern California School of Medicine core protein sequencing facility and N-terminal amino acid sequencing was performed using Edman degradation in an Applied Biosystems 1470 sequencer. 2.5. Construction of A. mylitta integument cDNA library Total RNA was isolated from the integument of fifth instar larvae by the guanidium thiocyanate method (Chomczynski and Sacchi, 1987) using an RNA isolation kit (Stratagene) and poly (A) + RNA prepared by oligodT cellulose chromatography (Aviv and Leder, 1972). The first strand cDNA was synthesized from 5 µg of poly (A) + RNA using MuLV reverse transcriptase and a 50 nucleotide long linker primer containing a XhoI site and 18 base poly (dT) sequence according to the procedure established by Gubbler and Hoffman (1983) using a cDNA synthesis kit (Stratagene). Second strand synthesis was carried out by the RNaseH procedure using E. coli DNA polymerase. Subsequently, the cDNAs were blunt ended, ligated with EcoRI linker, digested with EcoRI and XhoI, and ligated to XhoI/EcoRI digested lambda Uni-ZAP XR vector (Stratagene). The ligated DNA was then packaged into bacteriophage particles using Gigapack packaging sys-

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tem (Stratagene) as per the manufacturer’s protocol. The resulting library was amplified in E. coli (XL1-Blue MRF strain), titer determined and stored in the presence of chloroform at 4 °C. 2.6. Preparation of probe and screening of cDNA library To obtain a full-length cDNA clone specific for the AmFPI-1, protease inhibitor cDNA was partially amplified by PCR from the constructed cDNA library and used as a probe for screening the library. Degenerate oligonucleotide primers 5⬘-ATHGGHACHAAYTAYTAYAA KGAYCAYCC-3⬘ were synthesized on the basis of 10 N-terminal amino acid sequence of purified protein starting from isoleucine to proline. These degenerate oligonucleotides were used as forward primer and commercially available CDS (cDNA synthesis) primer (Clontech) (5⬘-AAGCAGTGGTAACAACGCAGAGTACT (30) N⫺1 N-3⬘, where N = A C G or T and N⫺1 = A G or C) was used as the reverse primer for the PCR reaction. Lambda DNA was isolated from the phage cDNA library using the commercially available lambda DNA isolation kit (Qiagen) as per the manufacturer’s protocol. A fragment of the protease inhibitor cDNA was amplified by PCR using 200 ng of lambda phage DNA as a template. PCR was conducted in a 100-µl reaction for 35 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 45 s, and extension at 72 °C for 1 min in a Perkin Elmer model 2400 thermocycler. The PCR product was analyzed on an agarose gel, 450 bp amplified DNA was electroeluted and cloned into PCR 2.1-TOPO vector (Invitrogen). Miniplasmids were prepared from selected transformed E. coli using a miniprep kit (Qiagen), the size of insert was determined by EcoRI digestion, and the DNA was sequenced by an automated DNA sequencer (ABI model 377). Plasmid DNA (20 µg) was then digested with EcoRI, separated through 1% agarose gel and the 450 bp band was eluted from the gel using a gel extraction kit (Qiagen). The eluted DNA was labeled with 32P (Feinberg and Vogelstein, 1983) using a random primer labeling kit (Stratagene) and was used as a probe to screen the A. mylitta integument cDNA library by the standard method (Sambrook et al., 1989). Individual hybridizing plaque was purified after two rounds of screening, and phagemid containing protease inhibitor cDNA was rescued from the lambda ZAP II vector by infection with helper phage and grown as plasmids. The size of the full-length insert was determined by the EcoRI digestion of plasmid and confirmed by sequencing. 2.7. Characterization of inhibitor cDNA The nucleotide sequence of the full-length inhibitor cDNA in pBluescript phagemid was determined in an

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Applied Biosystems automated DNA sequencer using dye terminator mixes according to the procedures specified by the manufacturer. Both strands of cDNA were sequenced using oligonucleotide primers corresponding to the T3 and T7 promoter sequences present in the plasmid vector, and aligned using Sequencher program. Homology searches were done in Genbank, protein sequence and B. mori expressed-sequence-tag (EST) databases using the BLAST program. A hydrophobicity plot was generated using the method of Kyte and Doolittle (1982). Identification of secretion signal peptide and prediction of cleavage site were undertaken using the method of Nielsen et al. (1997).

3. Results 3.1. Protease inhibition assay To determine protease inhibitor activity in different developmental stages, the protease inhibitor activity was measured in the hemolymph collected from third, fourth, fifth, spinning stage larvae and pupae. As seen in Table 1, the protease inhibitor activity in the hemolymph of third and fourth instar larvae was relatively low (5.2 and 8.6 U/ml) in comparison to fifth instar larvae (12 U/ml). The amount of inhibitor activity increased in the hemolymph with the advancement of larval development. At the beginning of the spinning stage, maximum inhibitor activity (15 U/ml) was observed and then declined (10.4 U/ml) at the onset of pupation. 3.2. Purification of protease inhibitor from the hemolymph The purification of protease inhibitor is summarized in Table 2. Assay of protease inhibitor against A. oryzae protease after ammonium sulfate fractionation showed only 1.56-fold purification. After heat treatment, the protease inhibitor was enriched 2.2-fold. The heat-stable protein sample was then loaded onto a DEAE-Sephacel (anion exchange) column. As illustrated in Fig. 1, two protein peaks (one minor and one major) were detected in the flow through fractions and three peaks were observed in the fractions eluted with 0–0.5 M NaCl Table 1 Fungal protease inhibitor in different stages of larval development Stage

Inhibitory activity (U / ml) ± standard error

Third instar Fourth instar Fifth instar Early spinning Pupa

5.2 ± 0.21 8.6 ± 0.26 12 ± 0.52 15 ± 0.67 10 ± 0.49

gradient. Only the major peak in the flow through contained protease inhibitor activity (400 U) and no inhibitor activity was detected in minor peak of the flow through or any other fractions obtained after the elution of bound protein from the column. This suggests that inhibitor protein at pH 6.0 became positively charged and did not bind to the column. A 49.9-fold purification was obtained after this stage (Table 2). The flow through peak fraction having protease inhibitor activity was dialyzed, concentrated and then the sample was applied to a Sepharose-12 column for further purification. As illustrated in Fig. 2, gel filtration resolved the proteins into seven peaks, and protease inhibitor activity (81.9 U) was found only in the last peak (peak seven). After this step, 200-fold purification was achieved with 14.18% recovery (Table 2). The FPLC purified protein showed only one band in SDS-15% PAGE under reducing condition (Fig. 3, lane 2) and the molecular weight was determined as 10.4 kDa. Analysis of this purified inhibitor by 2D gel also showed a single spot (Fig. 4) and confirmed the homogenous purification of a fungal protease inhibitor which we termed as AmFPI-1. 3.3. Inhibition spectrum The inhibitory activity of the purified inhibitor from A. mylitta hemolymph against different proteases is shown in Table 3. The inhibitor strongly suppressed fungal proteases from Aspergillus oryzae and Rhizopus oryzae, as well as bovine trypsin and chymotrypsin, but is less active against bacterial protease from B. licheniformis and papain. 3.4. N-terminal amino acid sequence To determine the primary structure of the 2D gel purified protein, it was transferred to a PVDF membrane, and the sequence of 15 N-terminal amino acids was determined by the Edman degradation. The obtained sequence, Asp–Leu–Ile–x–Gly–The–Asn–Tyr–Tyr– Lys–Asp–His–Pro–Cys–The–Ser (amino acid residue in the fourth position marked “x” could not be determined), when compared by homology search in protein sequence database (SWISS-PROT) using BLAST program showed 46% identity with the published N-terminal amino acid sequences of an inducible serine protease inhibitor (ISPI-1) from G. mellonella (Fig. 7). 3.5. Isolation of protease inhibitor cDNA To isolate a cDNA clone encoding the fungal protease inhibitor, AmFPI-1, a 450 bp fragment of the cDNA was first amplified by PCR using a degenerate oligonucleotide primer synthesized on the basis of the N-terminal amino acid sequence of the purified protein and a CDS primer (which can bind at the poly A tail in the second

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Table 2 Summary of purification of fungal protease inhibitor Purification steps

Total protein (mg) Total inhibitory activity (U)

Specific inhibitory activity (U/mg of protein)

Recovery (%) Purification (fold)

Crude hemolymph 60% (NH4)2SO4 precipitate Heat treatment Anion exchange chromatography (flow through) Gel filtration chromatography (FPLC)

2510 1314.9 941 34.95

577.3 473.38 470.5 400.92

0.23 0.36 0.5 11.47

100 82 81.5 69.4

1.771

81.9

46.2

14.18

1 1.56 2.2 49.9 200.8

Fig. 1. Elution profile from anion exchange column chromatography. (%%) indicates the NaCl gradient (0–0.5 M) and the fractions containing the inhibitor proteins is shown by (↔).

Fig. 2. Elution profile from Superose-12 gel filtration column chromatography (FPLC). Fractions containing the inhibitor is shown as (↔).

strand cDNA). A lambda ZAPII cDNA library constructed from A. mylitta integument mRNA was used as template for this PCR reaction. A single band of 450 bp DNA was amplified by PCR, cloned into a TA cloning vector (pCR 2.1 TOPO), and a 450 bp insert was obtained after digestion of plasmids with EcoRI. The nucleotide sequence of this insert matched the 15 N-terminal amino acid sequence of the purified inhibitor protein (Fig. 5) and confirmed the partial amplification of inhibitor cDNA lacking the complete 5⬘ end. The 450 bp insert from the digested plasmid DNA was then used as a probe for screening the same library to obtain fulllength inhibitor cDNA. After two rounds of screening, one plaque was selected as A. mylitta protease inhibitor cDNA clone.

Fig. 3. Analysis of FPLC purified inhibitor by SDS-15%PAGE. Lane 1, Crude hemolymph; lane 2, Purified inhibitor; lane M, Molecular weight marker.

Fig. 4. Analysis of FPLC purified protease inhibitor by 2-dimensional gel electrophoresis. Arrow indicates the position of purified inhibitor protein.

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Table 3 Activity of the purified AmFPI-1 against a range of proteases Proteases

Inhibition (%)

Bacillus licheniformis Aspergillus oryzae Bovine trypsin Bovine alpha-chymotrypsin Rhizopus oryzae Papain

6 63 80 67 78 12

likely a secretion signal peptide as predicted by neural network program (Fig. 6). The deduced molecular mass of the encoded protein including the first 19 amino acid residues was 11.5 kDa and excluding these 19 amino acid residues was approximately 10 kDa. Besides similarity to 50 N-terminal amino acid sequence of ISPI-1 of G. mellonella, AmFPI-1 shared sequence similarity (60–65% identity) with 34 EST database sequences of B. mori.

4. Discussion

Fig. 5. Nucleotide and deduced amino acid sequence of the inhibitor. The start and stop codon are underlined. Poly A addition sequence is boxed, and the corresponding N-terminal amino acid sequence is double underlined.

3.6. In vivo excision of phagemid and its analysis After in vivo excision of pBluescript phagemid from the lambda phage, restriction endonuclease analysis indicated that the plasmid contained a cDNA insert of approximately 0.5 kb. 3.7. Characterization of inhibitor cDNA The entire nucleotide sequence of both strands of the inhibitor cDNA was determined and is presented along with deduced amino acid sequence in Fig. 5. The cDNA consisted of 543 nucleotides including a 27-adenine tract at the 3⬘ end. An open reading frame of 315 bp encoding 105 amino acid residues began with an ATG codon at nucleotide position 79 and ended with a stop codon TAA at nucleotide 394. Seventy-eight nucleotides upstream of the initiation codon and 147 nucleotides downstream of the termination codon were present as 5⬘ and 3⬘ untranslated sequences. The 3⬘-untranslated region was AT rich, and a putative polyadenylation signal (AATAAA) was observed at nucleotide 491. The deduced amino acid sequence (amino acid 20–34) from this cDNA matched with the N-terminal amino acid sequence of the purified inhibitor (double underlined in Fig. 5) and showed that the first 19 amino acid residues were not present in the mature protein. This sequence is very hydrophobic as revealed by hydropathy analysis (data not shown) and is

In the present study, we purified a low molecular weight fungal protease inhibitor called AmFPI-1 from the hemolymph of tasar silkworm, A. mylitta, and cloned and characterized its cDNA. Although inhibitor activity was present in all stages of larval development starting from third instar to pupa, maximum inhibitory activity was detected at the early spinning stages and then the activity declined. The presence of protease inhibitor in all the stages of larval development indicates that the inhibitor may be important in both metamorphosis and in the defense mechanisms of this insect. Similar developmental changes in inhibitory activity were also reported in B. mori hemolymph against bovine chymotrypsin and fungal protease from Aspergillus melleus (Yamashita and Eguchi, 1987). In holo-metabolus insects, degradation of larval tissues and development of adult tissues proceed simultaneously during metamorphosis (Suzuki and Natori, 1985). Protease leaking into the hemolymph from decomposed larval tissues or autolysed hemocytes during metamorphosis could be harmful to developing adult tissues. Thus, one physiological function of protease inhibitors in the hemolymph is probably to protect adult tissues from proteases during metamorphosis (Suzuki and Natori, 1985). The same may be true when foreign substrates are introduced into the abdominal cavity of larva, since these substances when surrounded by hemocytes are known to die, releasing cellular components (Eguchi and Kanbe, 1982). The protease released from such cells would rapidly be inhibited by the inhibitors in the hemolymph. The other physiological significance of the presence of highly specific inhibitors of fungal and bacterial proteases in insect hemolymph is to play a role in defense against invading microorganisms (Eguchi, 1982), because insect humoral response is characterized by a rapid and transient synthesis of proteins with potent antibacterial and/or antifungal activity (Gillespie et al., 1997; Gan et al., 2001). Two heat stable protease inhibitors were identified from the molting fluid of pharate adult tobacco hornworm, Manduca sexta, and one of them (11 kDa) was found to be highly specific for the fungal enzyme from the entomopathogen, Metarhizium anisopliae, and its role in insect defense was suggested (Samuels and Reynolds, 2000).

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Fig. 6. Prediction of secretion signal peptide and its cleavage site within the first 60 amino acid residues of AmFPI-1 according to Nielsen et al. (1997).

Fig. 7. Comparison of N-terminal amino acid sequence of mature A. mylitta protease inhibitor (AmFPI-1) and G. mellonella protease inhibitor (ISPI-1) (Frobius et al., 2000). Identical amino acids are indicated with vertical lines, conservative amino acid changes are indicated with dot.

The hemolymph of mulberry silkworm, Bombyx mori, has also been reported to contain various protease inhibitors, which not only inhibit endogenous proteases but also trypsin, chymotrypsin (Sasaki, 1978), and proteases from fungus, Aspergillus melleus and the entomopathogen Beauveria bassiana (Eguchi et al., 1993). A specific fungal protease inhibitor named FPI-F was extensively purified, cloned, sequenced and expressed to produce functional inhibitor protein. Based on the position of reactive site and the disulfide bridges, FPI-F was considered to be a member of a new family of a serine protease inhibitor (Pham et al., 1996). Because of easy collection and availability of a large amount of hemolymph from fifth instar larvae, the fungal protease inhibitor has been purified from this hemolymph. Using ammonium sulfate precipitation, heat treatment, anion exchange and FPLC, inhibitor protein has been purified to homogeneity as confirmed by one and two dimensional gel electrophoresis. The inhibitor protein is heat stable and its isoelectric point (pI) is in alkaline range (around pH 9.0–10.0). The purified inhibitor was also found to strongly suppress trypsin, chymotrypsin and Rhizopus oryzae proteases. Since all these were serine proteases (as inhibited by PMSF) and the inhibitory activity against bacterial protease and papain was very low, it indicates that the purified protein might act as a wide spectrum serine protease inhibitor. N-terminal amino acid sequencing of the purified inhibitor protein and homology search in the protein sequence databases suggest that the inhibitor is probably related (46% identity) to an inducible serine protease inhibitor, ISPI-1, of Galleria mellonella. ISPI-1 was syn-

thesized in G. mellonella larvae after the administration of a yeast polysaccharide, zymosan preparation, and may be an anti-fungal humoral immune response to inhibit proteases from entomopathogenic fungus, Metarrhizium anisopliae (Frobious et al., 2000). Although in the present study, purification of protease inhibitor has been done from the hemolymph of uninduced larvae, it may be hypothesized that synthesis of this A. mylitta protease inhibitor may be induced by fungi, but experimental proof is definitely needed. Amplification of a single band PCR product of 450 bp using CDS and degenerate oligonucleotide primers synthesized on the basis of the N-terminal amino acid sequence of the purified protein, and detection of 100% sequence identity in N-terminal amino acid sequence beyond the primer region (10 amino acids) confirms the amplified DNA as the cDNA of purified inhibitor. But since 5⬘ primer was synthesized on the basis of N-terminal amino acid sequences of mature protein, the amplified cDNA was not full-length and lacked its 5⬘ end. By screening the cDNA library with this partial cDNA as probe, a full-length clone of the protease inhibitor cDNA was obtained, as revealed by the insert size of 543 bp (93 base pair more than that of partially amplified cDNA). The cDNA contains an ORF of 105 amino acids and could synthesize a protein of 11.5 kDa with a signal peptide of amino terminal 19 amino acids associated with it. The assumption that the first 19 amino acids would be the signal peptide because (a) these amino acids were not present in the N-terminal amino acid sequence of the mature protein, (b) hydrophobic amino acid residues are present in this region as supported by the hydropathy profile of the amino acid residues deduced from the sequences, (c) molecular weight of the mature protein (10.4 kDa) is less than calculated molecular weight from the total deduced amino acid sequences, and (d) the probable signal peptide cleavage site was found between 19 and 20 amino acid residues. From the deduced amino acid sequence, it is also found that AmFPI-1 contained 12 cysteine residues like ISPI-

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1 of G. mellonella (Frobious et al., 2000) and is likely to form six disulfide bridges. This may account for the high thermal stability of this inhibitor. With 60–65% sequence identity to a number of EST database sequences of B. mori and 46% identity to 50 N-terminal amino acid residues of ISPI-1 of G. mellonella, the protease inhibitor of A. mylitta, AmFPI-1, may be related to ISPI-1 of G. mellonella. However, the complete cDNA sequence of ISPI-1 has not been reported. The cloned cDNA may be expressed in bacteria or insect cells to produce large quantity of functional protease inhibitor to study its anti-fungal activity against a wide range of fungi. References Aviv, H., Leder, P., 1972. Purification of biologically active global messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69, 1408–1412. Boigegrain, R.A., Mattras, H., Brehelin, M., Paroutaud, P., Colettipreviero, M.A., 1992. Insect immunity: two proteinase inhibitors from haemolymph of locusta migratoria. Biochem. Biophys. Res. Commun. 189, 790–793. Cherqui, A., Cruz, N., Simoes, N., 2001. Purification and characterization of two serine protease inhibitors from the hemolymph of Mythimna unipuncta. Insect. Biochem. Mol. Biol. 31, 761–769. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. Chopin, V., Salzet, M., Baert, J., Vandenbulcke, F., Sautie`re, P., Kerckaert, J., Malecha, J., 2000. Therostasin, a novel clotting factor Xa inhibitor from the Rhynchobdellid Leech, Theromyzon tessulatum. J. Biol. Chem. 275, 32701–32707. Christeller, J.T., Burgess, E.P., Mett, V., Gatehouse, H.S., Markwick, N.P., Murray, C., Malone, L.A., Wright, M.A., Philip, B.A., Watt, D., Gatehouse, L.N., Lovei, G.L., Shanon, A.L., Phung, M.M., Wtason, L.M., Laing, W.A., 2002. The expression of a mammalian proteinase inhibitor, bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae. Transgen. Res. 11, 161–173. Clarkson, J.M., Charnley, A.K., 1996. New insights into the mechanism of fungal pathogenesis in insects. Trends. Microbiol. 4, 197–203. Eguchi, M., 1982. Inhibition of the fungal protease by haemolymph protease inhibitors of the silkworm, Bombyx mori L (lepidoptera: bombycidae). Appl. Ent. Zool. 17 (4), 589–590. Eguchi, M., 1993. Protein protease inhibitors in insects and comparison with mammalian inhibitors. Comp. Biochem. Physiol. 105B, 449–456. Eguchi, M., Kanbe, M., 1982. Changes in haemolymph protease inhibitors during metamorphosis of the silkworms, Bombyx mori L. (Lepidoptera: Bombycidae). Appl. Ent. Zool. 17, 179–187. Eguchi, M., Haneda, I., Iwamoto, A., 1982. Properties of protease inhibitors from the haemolymph of silkworms, Bombyx mori, Antheraea pernyii and Philosamia Cynthia ricini. Comp. Biochem. Physiol. 71B, 569–576. Eguchi, M., Itoh, M., Chou, L., Nishino, K., 1993. Purification and characterization of a fungal proteinase specific protein inhibitor (FPI-F) in the silkworm haemolymph. Comp. Biochem. Physiol. 104B, 537–543. Feinberg, A.P., Vogelstein, B., 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.

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