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A Novel Protease in the Pupal Yellow Body of Sarcophaga peregrina (Flesh Fly)

1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês

10.1074/jbc.272.38.23805

1083-351X

Yuki Nakajima, Yumiko Tsuji, Ko‐ichi Homma, Shunji Natori,

Insect symbiosis and bacterial influences

We purified a novel serine protease with a molecular mass of 26 kDa from Sarcophaga pupae. This protease appeared almost exclusively in the yellow body, an organ that develops temporarily in the pupae of dipteran insects and expands to form the adult midgut by engulfing the larval midgut. cDNA analysis revealed that this protease consists of 239 amino acid residues and has significant structural similarity with bovine trypsin (about 40% sequence identity). The 26-kDa protease gene was transiently activated in 1-day-old pupae. The protease was found to cross-react immunologically with antibody against sarcotoxin IA, an antibacterial protein produced by this insect. It is suggested that this protease participates in the decomposition of the larval midgut in the yellow body during metamorphosis. We purified a novel serine protease with a molecular mass of 26 kDa from Sarcophaga pupae. This protease appeared almost exclusively in the yellow body, an organ that develops temporarily in the pupae of dipteran insects and expands to form the adult midgut by engulfing the larval midgut. cDNA analysis revealed that this protease consists of 239 amino acid residues and has significant structural similarity with bovine trypsin (about 40% sequence identity). The 26-kDa protease gene was transiently activated in 1-day-old pupae. The protease was found to cross-react immunologically with antibody against sarcotoxin IA, an antibacterial protein produced by this insect. It is suggested that this protease participates in the decomposition of the larval midgut in the yellow body during metamorphosis. In holometabolous insects, most larval tissues disintegrate during the pupal stage, and new adult structures develop from imaginal discs (1Bodenstin D. Demerce M. Biology of Drosophila. Hafner Publishing Co, Inc., New York1950: 275-367Google Scholar). However, little is known about the molecular mechanism underlying the selective decomposition of larval tissues (2Lockshin R.A. Kerkut G.A. Gilbert L.I. Comprehensive Insect Physiology, Biochemistry, and Pharmacology. 2. Pergamon Press Ltd., Oxford1985: 301-317Google Scholar). Recently, we suggested that hemocyte cathepsin B is responsible for decomposition of the fat body in Sarcophaga peregrina (flesh fly) (3Kurata S. Saito H. Natori S. Insect Biochem. 1990; 20: 461-465Crossref Scopus (21) Google Scholar, 4Kurata S. Saito H. Natori S. Dev. Biol. 1992; 153: 115-121Crossref PubMed Scopus (63) Google Scholar). We purified this enzyme and isolated its cDNA (5Kurata S. Saito H. Natori S. Eur. J. Biochem. 1992; 204: 911-914Crossref PubMed Scopus (38) Google Scholar, 6Takahashi N. Kurata S. Natori S. FEBS Lett. 1993; 334: 153-157Crossref PubMed Scopus (60) Google Scholar).Sarcophaga cathepsin B differs from its mammalian counterparts in several respects. It does not seem to be a typical lysosomal enzyme, as its optimum pH is higher (pH 6.0) (5Kurata S. Saito H. Natori S. Eur. J. Biochem. 1992; 204: 911-914Crossref PubMed Scopus (38) Google Scholar). Moreover, production of this enzyme was shown to be regulated at the translational level, since larval hemocytes contained a significant amount of cathepsin B mRNA but not the enzyme itself (7Yano T. Takahashi N. Kurata S. Natori S. Eur. J. Biochem. 1995; 234: 39-43Crossref PubMed Scopus (20) Google Scholar). The mRNA started to be translated on pupation, and cathepsin B then accumulated in the hemocytes. When pupal hemocytes containing cathepsin B interacted with the fat body, the enzyme was released and digested the basement membrane of the fat body, resulting in its decomposition (4Kurata S. Saito H. Natori S. Dev. Biol. 1992; 153: 115-121Crossref PubMed Scopus (63) Google Scholar). This paper reports the purification, cDNA cloning, and some characteristics of a novel serine protease that is likely to participate in the decomposition of the larval midgut during metamorphosis. This enzyme was discovered incidentally during a study of sarcotoxin IA, a cecropin-type antibacterial protein produced bySarcophaga (8Okada M. Natori S. Biochem. J. 1983; 211: 727-734Crossref PubMed Scopus (116) Google Scholar). We purified a protein that reacted immunologically with anti-sarcotoxin IA antibody from an extract ofSarcophaga pupae. This protein was revealed to be a novel serine protease, which was preferentially induced in the yellow body, in which disintegration of the larval midgut takes place during metamorphosis. During remodeling of the midgut, the yellow body is known to be formed as a first step (1Bodenstin D. Demerce M. Biology of Drosophila. Hafner Publishing Co, Inc., New York1950: 275-367Google Scholar). It surrounds the larval midgut at the pupal stage. A morphological change of the yellow body is induced to form the adult midgut simultaneously with the disintegration of the larval midgut. Therefore, we assume that this novel protease is responsible for disintegration of the larval midgut within the yellow body. Taking into account our previous results (3Kurata S. Saito H. Natori S. Insect Biochem. 1990; 20: 461-465Crossref Scopus (21) Google Scholar, 4Kurata S. Saito H. Natori S. Dev. Biol. 1992; 153: 115-121Crossref PubMed Scopus (63) Google Scholar), it is suggested that several proteases participate in tissue remodeling during insect metamorphosis. Flesh flies (S. peregrina) were reared according to the method of Ohtaki (9Ohtaki T. Jpn. J. Med. Sci. Biol. 1966; 19: 97-104Crossref Scopus (103) Google Scholar). The physiological age of third instar larvae can be synchronized by dry-wet treatment; when larvae were transferred from wet conditions to dry conditions at 27 °, they started to pupate 16 h later. Thereafter, we defined them as 1-day-old pupae. Adults emerged 10 days after pupation under our rearing conditions. Therefore, 10-day-old pupae are synonymous with newly emerged adults. Pupae were used after removing their pupal shells. About 200 pupae were homogenized in 40 ml of 10 mm Tris-HCl buffer (pH 7.9) containing 130 mm NaCl, 5 mm KCl, 1 mmCaCl2, 1 mm phenylmethylsulfonyl fluoride, 100 μg/ml leupeptin, and 0.1 μg/ml pepstatin. The homogenate was centrifuged at 10,000 × g for 20 min, and the supernatant was collected and used as the pupal extracts. Antiserum was raised against synthetic sarcotoxin IA as described previously (10Yamada K. Nakajima Y. Natori S. Biochem. J. 1990; 272: 633-636Crossref PubMed Scopus (51) Google Scholar). As sarcotoxin IA is a small peptide consisting of 39 amino acid residues, we first coated reverse-phase column packing material (C18) with sarcotoxin IA according to the method of Flyg et al. (11Flyg C. Dalhammat G. Rasmuson B. Boman H.G. Insect Biochem. 1987; 17: 153-160Crossref Scopus (38) Google Scholar), then immunized albino rabbits with the sarcotoxin IA-coated particles. The resulting antiserum was affinity-purified with sarcotoxin IA. For this, sarcotoxin IA (300 μg) was first covalently linked to Affi-Gel 10 (Bio-Rad). A 2-fold dilution of anti-sarcotoxin IA antiserum (4 ml) was then mixed with this gel and kept overnight at 4 °C. After washing with phosphate-buffered saline (130 mm NaCl, 3 mm KCl, 8 mmNa2HPO4·12H2O, 1.5 mmKH2PO4, pH 6.8), the gel was packed in a column and washed stepwise with 0.2 m glycine-HCl buffer (pH 2.8) without and with 4 m urea. Anti-sarcotoxin IA antibody was eluted from the column at the second step. The eluate was neutralized with 1 m Tris solution, and fetal calf serum was added at a final concentration of 10% (w/v). On immunoblotting of the immunized larval extract, this antibody specifically recognized sarcotoxin IA. Electrophoresis on SDS-polyacrylamide slab gel was carried out by the method of Laemmli (12Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Samples were denatured by heating them for 20 min at 75 °C in 1% (w/v) SDS and 2% (v/v) 2-mercaptoethanol. After electrophoresis, the gel was stained according to the method of Fairbanks et al. (13Fairbanks G. Steck T.L. Wallach D.L.H. Biochemistry. 1971; 10: 2606-2617Crossref PubMed Scopus (6177) Google Scholar). For immunoblotting, proteins separated by electrophoresis were transferred electrophoretically from the gel onto a filter (Immobilon P; Millipore). The filters were immersed in 20 mm Tris-HCl buffer (pH 7.9) containing 5% (w/v) skim milk for 1 h. After washing with rinsing solution (10 mmTris-HCl buffer (pH 7.9) containing 150 mm NaCl, 1 mm EDTA, 0.1%(v/v) Triton X-100, 0.01% (w/v) sodium azide, and 0.25% skim milk), they were dipped in rinsing solution containing affinity-purified antibody against sarcotoxin IA and kept overnight at 4 °C. They were then washed well with rinsing solution, transferred to 5 ml rinsing solution containing radioiodinated anti-rabbit IgG (3.7 kBq), and kept at room temperature for 3 h. Finally, they were washed well with rinsing solution, dried, and subjected to autoradiography with Kodak XAR film. Extracts of 3-day-old pupae were found to contain a 26-kDa protein that reacts immunologically with anti-sarcotoxin IA antibody. To purify this protein, we defined 1 unit of the protein as the amount that gave the same immunoreactivity as 0.25 ng of sarcotoxin IA on immunoblotting followed by densitometric scanning of the 26-kDa protein band. The 26-kDa protein in the pupal extract was precipitated by ammonium sulfate between 40–65% (w/v) saturation. The resulting precipitate was collected, dissolved in 10 mm sodium phosphate buffer (pH 6.0) containing 25 mm NaCl, and dialyzed extensively against the same buffer. The dialyzate was applied to a CM-cellulose column (1.5 × 6 cm) that had been equilibrated with the same buffer. The column was washed well, then the adsorbed materials were eluted stepwise with 10 mm phosphate buffers containing 120 and 350 mmNaCl, respectively. The 26-kDa protein was eluted by the latter buffer. This fraction was subjected to reverse-phase high performance liquid chromatography (HPLC) 1The abbreviations used are: HPLC, high performance liquid chromatography; MCA, methylcoumaryl-7-amide; Boc, butyloxycarbonyl; E-64,trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane. with a Synchropak RP-P C18 column. The 26-kDa protein was eluted with 37% (v/v) acetonitrile when the column was developed with a linear gradient of 0–100% acetonitrile in 0.05%(v/v) trifluoroacetic acid solution. The 26-kDa protein was almost pure at this stage and gave a single band when analyzed by SDS-polyacrylamide gel electrophoresis. Purified 26-kDa protein was digested with lysyl endopeptidase, and the resulting peptides were separated by HPLC on a C18 column (Gilson). The fractions containing each peptide were lyophilized and the sequences of 5 peptides, including the 10 amino-terminal residues, were determined. Using these peptide sequences, oligodeoxyribonucleotides corresponding to MHPQYDPV and DAIVAGWG were synthesized. Their sequences were 5′-ATGCA(T/C)CCICA(A/G)TA(T/C)GA(T/C)CCIGTICA-3′ and 5′-CCCCAICC(T/C/A/G)GCIAC(A/G/T)ATIGC(A/G)TC-3′, respectively. These oligodeoxyribonucleotides were mixed and labeled with [γ-32P]ATP by the method of Sgaramella and Khorana (14Sgaramella V. Khorana H.G. J. Mol. Biol. 1972; 72: 427-444Crossref PubMed Scopus (85) Google Scholar). Total RNA was extracted from homogenates of 2-day-old pupae by the guanidine thiocyanate method of Chirgwin et al. (15Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar); poly(A)-rich RNA was prepared with (dT)30 latex beads. A cDNA library of this poly(A)-rich RNA was constructed with the expression vector Zap (Stratagene). This cDNA library in bacteriophage Uni-Zap XR vector converted into cDNA library in plasmid pBluescript by the method of Short et al. (16Short J.M. Fernandez J.M. Sorge J.A. Huse W.D. Nucleic Acids Res. 1988; 16: 7583-7600Crossref PubMed Scopus (1080) Google Scholar). We screened 5 × 104 colonies and obtained nine hybridization-positive clones. We analyzed the two plasmids with the longest insert. These clones yielded identical restriction maps, and their inserts were found to contain one determined amino acid sequence. Therefore, one of these clones was selected for further analysis. For nucleotide sequencing of the cDNA, various deletion derivatives of the DNA fragment were prepared using exonuclease III and mung bean nuclease. Each deletion derivative was sequenced, and the nucleotide sequences of both strands were determined. Northern blot hybridization was performed in 50% formamide, 5 × SSPE (1 × SSPE equals 0.15m NaCl, 10 mm NaH2PO4, 1 mm EDTA), 1 × Denhardt's solution (0.02% (w/v) each of Ficoll 400, bovine serum albumin, and polyvinylpyrrolidone), 0.1% SDS, and sonicated salmon sperm DNA solution (200 μg/ml) for 18 h at 42 °C. The filters were then washed for 15 min each time with 0.1 × SSC (1 × SSC equals 0.15 mNaCl, 0.05 m sodium citrate) containing 0.1% SDS at room temperature and 42 °C and then autoradiographed at −80 °C. The DNA used as a probe was a 26-kDa protein cDNA-labeled with [α-32P]dCTP using the BcaBEST random primer labeling kit (Takara, Tokyo). Protease activity was assayed with various peptidylmethylcoumaryl-7-amide (peptidyl-MCA) substrates. The enzyme reaction was performed in 0.25 ml of 0.1 mTris-HCl buffer (pH 7.9) containing 0.1 mm substrate, 1 mm EDTA, and the enzyme sample. After incubation for 10 min at 27 °C, the reaction was terminated by adding 0.375 ml of 17% aqueous acetic acid. The fluorescence was measured at excitation and emission wavelengths of 380 and 460 nm, respectively. An antibody against the 26-kDa protein was raised by injecting the purified protein (15 μg) in complete Freund's adjuvant into two male albino rabbits with three booster injections of 10 μg of protein and incomplete Freund's adjuvant at intervals of 14 days. Affinity purification of the antibody was achieved in essentially the same way as the preparation of a specific antibody against regenectin (17Kubo T. Kawasaki K. Nonomura Y. Natori S. Int. J. Dev. Biol. 1991; 35: 83-90PubMed Google Scholar). Previously, we demonstrated that the sarcotoxin IA gene was transiently activated during the early pupal stage in Sarcophaga (18Nanbu R. Nakajima Y. Ando K. Natori S. Biochem. Biophys. Res. Commun. 1988; 150: 540-544Crossref PubMed Scopus (26) Google Scholar). To prove that sarcotoxin IA is in fact present in the pupae, we performed immunoblotting using pupal extracts prepared from pupae harvested at various developmental stages. As shown in the lower panel of Fig. 1 A, sarcotoxin IA was detected in 3–6-day-old pupae, and the content was estimated to be approximately 10 ng/mg of protein. Synthesis of sarcotoxin IA corresponded with expression of the sarcotoxin IA gene that we reported previously (18Nanbu R. Nakajima Y. Ando K. Natori S. Biochem. Biophys. Res. Commun. 1988; 150: 540-544Crossref PubMed Scopus (26) Google Scholar). In addition to sarcotoxin IA, the antibody was found to cross-react with a protein with a molecular mass of 26 kDa (26-kDa protein) (top panel). When the antibody was treated with sarcotoxin IA-coated silica beads to absorb specific antibody, the remaining antibody reacted with neither sarcotoxin IA nor with the 26-kDa protein (Fig. 1 B), indicating that the 26-kDa protein cross-reacted immunologically with anti-sarcotoxin IA antibody. Other signals in the high molecular mass region are nonspecific background ones, since they were independent of the first antibody. The 26-kDa protein was detected exclusively in 3–7-day-old pupae; thereafter it disappeared rapidly. The 26-kDa protein signal was very strong, and its content did not seem to change throughout this period. This protein is not a precursor of sarcotoxin IA, because cDNA analysis has shown that there is no precursor protein for sarcotoxin IA (19Matsumoto N. Okada M. Takahashi H. Ming Q.X. Nakajima Y. Nakanishi Y. Komano H. Natori S. Biochem. J. 1986; 239: 717-722Crossref PubMed Scopus (69) Google Scholar). To study the structural relationship between sarcotoxin IA and the 26-kDa protein, we purified the 26-kDa protein from pupal extracts. Purification was monitored by immunoblotting followed by densitometric scanning of the 26-kDa protein band using anti-sarcotoxin IA antibody. 1 unit of 26-kDa protein was defined as the amount that gave the same immunoreactivity as 0.25 ng of sarcotoxin IA on immunoblotting. The typical purification is summarized in TableI, and HPLC, SDS-polyacrylamide gel electrophoresis, and immunoblotting profiles of the sample during the final purification step are shown in Fig.2. About 250 μg of pure protein was obtained from the extracts of about 200 pupae.Table ISummary of purification of a 26-kDa proteinPurification step26-kDa proteinTotal proteinSpecific contentYieldunitsmgunits/mg%Crude homogenate supernatant87,50094193100Ammonium sulfate, 40–65% saturation43,2004709249.4CM-cellulose14,4000.68421,00016.5Reverse-phase HPLC10,0000.24541,00011.4 Open table in a new tab To determine the amino acid sequence of the 26-kDa protein, we isolated its cDNA. For this, we first determined the amino acid sequences of five peptides derived from the 26-kDa protein, including its amino-terminal sequence. These were VIMHPQYDPVHITNDVALLR, DAIVAGWGLFK, FLDWIHNNSR, YPWTAQLVK, and IVGGTQVRQN (amino terminus). Having obtained this sequence information, we synthesized DNA probes, used them to screen the cDNA library of Sarcophaga pupae, and obtained nine hybridization-positive clones. The nucleotide sequence of the insert and the deduced amino acid sequence of one of these clones are shown in Fig.3. The amino acid sequence contained all the sequences determined for the five peptides derived from the 26-kDa protein, indicating that it was a cDNA clone for the 26-kDa protein. We designated the Ile at position 87 as its amino-terminal residue starting from the first Met residue. Therefore, the 26-kDa protein consists of 239 amino acid residues. The 86 residues from the first Met are thought to be a prosegment that includes a signal sequence, possibly consisting of 18 residues. No significant sequence similarity was found between the 26-kDa protein and sarcotoxin IA. Therefore, it is uncertain why the antibody against sarcotoxin IA cross-reacted with the 26-kDa protein. However, as shown in Fig. 4, the 26-kDa protein was found to have significant sequence similarity with bovine trypsin, and the His, Asp, and Ser residues conserved in many serine proteases are also present in the 26-kDa protein, suggesting that the 26-kDa protein is a serine protease. The maximal sequence identity between the 26-kDa protein and bovine trypsin was about 40%. As it became evident that the 26-kDa protein was likely to be a novel serine protease, we assayed its protease activity using various peptidyl-MCA substrates. As shown in Table II, purified 26-kDa protein clearly hydrolyzed substrates with Arg or Lys at their carboxyl termini. Therefore, the substrate specificity of this protease is vary similar to that of trypsin. Other substrates, including those of chymotrypsin and prolyl endopeptidase, were not hydrolyzed by this enzyme.Table IISubstrate specificity of the 26-kDa proteaseSubstrateActivityμmol of 7-amino-4-methyl coumarin released/mgBoc-Gln-Ala-Arg-MCA258Boc-Leu-Thr-Arg-MCA203Boc-Phe-Ser-Arg-MCA131Pro-Phe-Arg-MCA77Carbobenzoxy-Phe-Arg-MCA69Boc-Val-Leu-Lys-MCA60Pyr-Gly-Arg-MCA17Carbobenzoxy-Arg-Arg-MCA7Benzoyl-Arg-MCA3Succinyl-Leu-Leu-Val-Tyr-MCA<1Succinyl-Ala-Ala-Pro-Phe-MCA<1Succinyl-Ala-Pro-Ala-MCA<1Succinyl-Gly-Pro-MCA<1Protease activity was assayed with the indicated substrates under the standard conditions. Open table in a new tab Protease activity was assayed with the indicated substrates under the standard conditions. We examined the characteristics of this protease using Boc-Gln-Ala-Arg-MCA as a substrate. The optimum pH range for this protease was rather broad, between pH 6 and 8. However, no enzyme activity was detected above pH 10 or below pH 4, suggesting that it is not a lysosomal enzyme. This protease was sensitive to diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, leupeptin, and antipain but not to E-64, indicating that it is a typical serine protease. To estimate the biological role of the 26-kDa protease, we examined its localization within pupae. For this, we prepared affinity-purified antibody against the 26-kDa protease using purified protein. In contrast to sarcotoxin IA antibody, this antibody reacted only with the 26-kDa protease and not with sarcotoxin IA. Significant fluorescence was detected in the pupal midgut (the so-called "yellow body") when pupal sections were subjected to an immunofluorescence study. Localization in other regions was not clear because of high background fluorescence (data not shown). To confirm this observation, we prepared homogenates of the whole body, head, thorax and abdomen, and yellow body of 3-day-old pupae and subjected them to immunoblotting. As shown in Fig.5 A, the 26-kDa protease signal was detectable in homogenates of the whole body, thorax and abdomen, and yellow body but not in that of head. Moreover, several other signals besides that of the 26-kDa protease were detected in the homogenate of the yellow body, suggesting that various proteases that cross-react immunologically with the anti-26-kDa protease antibody are concentrated in the yellow body. The yellow body appears at the pupal stage and engulfs the larval midgut, finally developing into the adult midgut after the larval midgut has been disintegrated in situ. Therefore, we examined the expression of these proteases in the yellow body in parallel with adult development within the pupal case. As is evident from Fig. 5 B, no appreciable signal was detectable when the midguts of third instar larvae or the yellow bodies of 1-day-old pupae were examined. However, at least four signals, including that of the 26-kDa protease, became detectable in the yellow bodies of 2-day-old pupae. Many of these persisted for several days but disappeared rapidly during the late pupal stage. It is likely that various proteases that cross-react immunologically with anti-26-kDa protease antibody are synthesized simultaneously in the pupal yellow body. Therefore, we examined changes in the expression of the 26-kDa protease gene with time after pupation by Northern blotting. As shown in Fig. 6, mRNA for the 26-kDa protease was detected exclusively in RNA extracted from 1-day-old pupae, and no appreciable signal was detected in RNA from pupae at other stages. Thus, it is clear that the 26-kDa protease gene is transiently activated for a restricted period during development, but the protease translated from the resulting mRNA persists in the yellow body for several days. We found and purified a 26-kDa protein that cross-reacts immunologically with anti-sarcotoxin IA antibody in the pupal extracts of Sarcophaga. This protein was found to be a novel serine protease with significant sequence similarity to bovine trypsin. Northern blotting and immunoblotting experiments revealed that the 26-kDa protease gene was transiently activated during the early pupal stage, and the 26-kDa protease was found to accumulate in the yellow body. In dipteran insects, the larval midgut is known to disintegrate, and the adult midgut develops during the pupal stage (1Bodenstin D. Demerce M. Biology of Drosophila. Hafner Publishing Co, Inc., New York1950: 275-367Google Scholar). This process proceeds in a temporary organ named the yellow body. Adult midgut cells derived from the imaginal island on the larval midgut surround the larval midgut to form a primordial adult midgut. This swells to form the yellow body. The larval midgut is engulfed by the yellow body and disintegrates in situ. The yellow body itself then extends to form the adult midgut during the development of adult structures. Therefore, the 26-kDa protease and other related proteases that transiently appear in the yellow body at a specific developmental stage are likely to play a role in the disintegration of the larval midgut and construction of the adult one. These proteases are not simply digestive enzymes, because the larvae stop feeding as soon as they reach the third instar, and no food remains in the digestive tract during the pupal stage. We found that the 26-kDa protease gene is transiently activated before induction of the enzyme, and that the timing of gene activation seems to depend strictly upon the developmental stage of the adult in the pupal case. The expression of the genes for other proteases that cross-react immunologically with the 26-kDa protease are likely to be regulated in the same way. Although we were able to identify the timing of the activation of the 26-kDa protease gene, the cells synthesizing the mRNA for this enzyme remained to be identified. As it is localized in the yellow body, adult midgut cells are most probable candidate cells. The 26-kDa protease was first detected immunologically using an antibody against sarcotoxin IA. Sarcotoxin IA is an antibacterial protein produced by Sarcophaga, which consists of 39 amino acid residues (19Matsumoto N. Okada M. Takahashi H. Ming Q.X. Nakajima Y. Nakanishi Y. Komano H. Natori S. Biochem. J. 1986; 239: 717-722Crossref PubMed Scopus (69) Google Scholar). We raised the antibody using chemically synthesized sarcotoxin IA. No significant sequence similarity was found between sarcotoxin IA and the 26-kDa protease. During remodeling of the midgut from the larval to the adult type during metamorphosis, normalSarcophaga flora living in the larval midgut are killed and replaced with adult flora. It is possible that the 26-kDa protease contains an epitope with a similar secondary or tertiary structure to sarcotoxin IA, and that this epitope has sarcotoxin IA-like antibacterial activity that might kill the normal larval flora that are dispersed in the yellow body when the larval midgut disintegrates in it. Another possibility to be noted is that sarcotoxin IA is a covalent inhibitor of the 26 kDa protease, and the covalently bound sarcotoxin IA reacted with the antibody. This possibility needs further examination.