A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca 2+ in coleopteran and Tenebrio molitor larvae Kum Young Lee 1 , Rong Zhang 1 , Moon Suk Kim 1 , Ji Won Park 1 , Ho Young Park 2 , Shun-ichiro Kawabata 3 and Bok Luel Lee 1 1 College of Pharmacy, Pusan National University, Jangjeon Dong, Korea; 2 Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejeon, Korea; 3 Department of Biology, Kyushu University, Fukuoka, Japan To elucidate the biochemical activation mechanism of the insect pro-phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm-mas) contains a trypsin-like serine proteinase domain in the C-terminal region, exceptfor thesubstitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm-mas was incubated with CM-Toyopearl eluate solution contain- ing pro-PO and other pro-PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca 2+ . This suggests that the purified 45-kDa Tm-mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm-mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca 2+ , a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm-mas converted to 76-kDa PO and 45-kDa Tm-mas, respectively, with detect- able PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca 2+ and b-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm-mas to the 45-kDa protein, was faster than in the presence of Ca 2+ only. These results suggestthat the cleavage ofthe55-kDa zymogen of Tm-mas by a limited proteolysis is necessary for PO activity, and the Tm-mas is a pro-PO activating cofactor. Keywords: innate immunity; insect; Masquerade; pro-phe- noloxidase; serine proteinase homologue. The pro-phenoloxidase (pro-PO) activation system in arthropods is an important part of the host immune defence, where it functions to detect and kill invading pathogens. It is also a good model system to elucidate the pattern recognition mechanism of nonself pattern recogni- tion proteins, such as peptidoglycan recognition protein or b-1,3-glucan binding protein, which are part of the innate immune reaction [1,2]. However, the molecular mechanism of pro-PO activation remains poorly understood. Previously, we reported the structures and functions of two pro-PO activating factors (PPAF-I and PPAF-II) from the coleopt- eran insect, Holotrichia diomphalia larvae [3–5]. PPAF-I is an easter-type serine proteinase and PPAF-II is a masqu- erade-like serine proteinase homologue. We have demon- strated that they are necessary for activating the phenoloxidase (PO) cascade, by in vitro reconstitution experiments. However, we did not determine the biological functions of PPAF-II during Holotrichia pro-PO activation. Two questions remain to be answered: why is a masquer- ade-like serine proteinase homologue a requirement for PO activity, and how and why is there cross-talk between a masquerade-like serine proteinase homologue and an eas- ter-type serine proteinase during the pro-PO activation reaction? Another key question that remains is how these pro-PO activating factors can be activated in response to microbial infection. One hypothesis is that pattern recogni- tion proteins make a complex with pro-PO activating enzyme(s) and microbial cell wall components, and then activation of pro-PO activating enzyme(s) zymogen con- verts pro-PO to active PO by limited proteolysis [6–8]. Recently we reported that in larvae of the coleopteran insect, Tenebrio molitor, pro-PO was activated by b-1,3- glucan and Ca 2+ , and the activated PO was involved in the cell/clump/cell adhesion reaction as well as in the synthesis of melanin [9]. This insect has one kind of pro-PO; however, two kinds of pro-PO were found in H. diomphalia larvae [9,10]. If it is possible to purify pro-PO activating factors from T. molitor larvae, we can explain the difference between pro-PO activation reactions in the one-pro-PO and two-pro-PO systems in T. molitor and H. diomphalia larvae, respectively. Also, we have reported the presence of early staged encapsulation-relating proteins in T. molitor larvae [11,12]. Given the crucial nature of the melanotic Correspondence to B. L. Lee, College of Pharmacy, Pusan National University, Jangjeon Dong, Kumjeong Ku, Busan, 609-735, Korea. Fax: +82 51 581 1508, E-mail: brlee@pusan.ac.kr Abbreviations: PO, phenoloxidase; pro-PO, pro-phenoloxidase; Tm-mas, Tenebrio masquerade-like serine proteinase homologue; TCA, trichloroacetic acid; PVDF, polyvinylidene difluoride; PPAF, pro-phenoloxidase activating factors; P-NPGB, p-nitrophenyl- p¢-guanidinobenzoate; p-APMSF, p-amidinophenyl-methanesulfonyl fluoride. Note: The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Database with the accession number AB084067. (Received 29 April 2002, revised 9 July 2002, accepted 29 July 2002) Eur. J. Biochem. 269, 4375–4383 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03155.x encapsulation response in insect immunity, it is of great importance to purify pro-PO activating factors and to determine the Tenebrio pro-PO activation mechanism at the molecular level. This paper describes the purification and cDNA cloning of a 45-kDa protein that acts as a pro-PO activating cofactor in T. molitor larvae. The deduced amino acid sequence of the 45-kDa protein derived from the cDNA revealed that this protein exhibits high sequence similarity with Drosophila masquerade serine proteinase homologue [13]. The biological function of the purified 45-kDa protein was examined during pro-PO activation reaction. MATERIALS AND METHODS Animals, collection of hemolymph and hemocytes T. molitor larvae (mealworm) were maintained on a labo- ratory bench in terraria containing wheat bran. Vegetables were placed on top of the bran to provide water. Hemo- lymph and hemocytes were collected as described previously [9]. Briefly, to harvest the hemolymph, larvae were injected with 50 lL of modified anticoagulation buffer (30 m M trisodium citrate, 26 m M citric acid, 20 m M EDTA, 15 m M NaCl, pH 5.5) using a 25-G needle. The tail of each larva was cut off using fine scissors and the extruding hemolymph was placed in a test tube on ice. The collected crude hemolymph was centrifuged at 203 000 g for 4 h at 4 °C. The supernatant then stored at )80 °C until use. Assay of PO activity PO activity was determined according to our previously published method [3]. Briefly, 30 lL crude hemolymph (50 lg protein) or fractionated solution from column chro- matography were preincubated in 85 lL20m M Tris/HCl pH 8.0 containing 1 lg b-1,3-glucan for 10 min at 30 °C, andthen400 lL substrate solution (1 m M 4-methylcatechol, 2m M 4-hydroxyproline ethylester in 20 m M Tris/HCl buffer pH 8.0, containing 5 m M CaCl 2 ) was added to the reaction mixture. After incubation at 30 °C for 10 min, the increase in absorbance at 520 nm was measured using a Shimadzu spectrophotometer. PO activity was expressed as the change in absorbance at 520 nm per 10 min incubation (A 520 per 10 min per 30 lL). To examine the effects of the 45-kDa protein on PO activity, a mixture of CM-Toyopearl eluate solution (15 lg) and the purified 45-kDa protein (1 lg) were added to 400 lL substrate solution in the presence or absence of 5 m M CaCl 2 . After incubation at 30 °Cfor 10 min, the increase in absorbance at 520 nm was measured as described above. Purification of 45-kDa Tenebrio masquerade protein (Tm-mas) To purify 45-kDa Tm-mas from Tenebrio larvae, 50 mL hemolymph supernatant solution were collected following ultracentrifugation, and then concentrated by ultrafiltration through a membrane filter (Amicon, YM-10). About 3 mL of the concentrated solution was applied to a Toyopearl HW-55S size exclusion column (1.5 · 50 cm) equilibrated with buffer A (50 m M Tris/HCl pH 6.5, containing 5 m M EDTA), and eluted with buffer A at a flow rate of 12 mLÆh )1 . Fractions specifically showing PO activity in the presence of b-1,3-glucan and Ca 2+ were pooled. The pooled solution was named HW-55S solution and was used to purify 45-kDa protein. Fifty mL HW-55S solution (300 mg) were loaded onto a Toyopearl CM-650M cation exchange column (1 · 11 cm) equilibrated with buffer A, and washed with buffer A until no absorbance could be detected at 280 nm. The flow-through fraction and washing solution were combined, and concentrated by ultrafiltration. The resulting ultrafiltered concentrated solution (3 mL) was diluted 10 times with buffer A and loaded onto a Blue- Sepharose (1.0 · 5.2 cm) hydrophobic column equilibrated with buffer A. The column was washed with the same buffer at a flow rate of 0.2 mLÆmin )1 , until there was no absorbance at 280 nm, and then it was eluted with buffer A containing 0.2 M NaCl. The eluted solution was concen- trated by repeated ultrafiltration to exclude NaCl. To confirm the presence of the 45-kDa protein, PO activity was examined by incubating together the concentrated 0.2 M NaCl elution solutions from the Blue-Sepharose and Toyopearl CM-650M columns. For further purification of the 45-kDa protein, the concentrated elution solution (7 mg) from Blue-Sepharose was loaded onto a Butyl- Toyopearl FPLC column equilibrated with buffer B [50 m M phosphate pH 7.0, containing 1.7 M (NH 4 ) 2 SO 4 ]. The column was eluted with a linear gradient of ammonium sulfate (from 1.7 to 0 M ) at a flow rate of 0.3 mLÆmin )1 .The fractions containing an approximately 45-kDa protein as determined by SDS/PAGE (reducing conditions) were pooled and concentrated by ultrafiltration kit (YM-10 membrane). Fractions containing 45-kDa protein from the Butyl-Toyopearl FPLC column were pooled and applied to a Mono-Q FPLC anion exchange column equilibrated with buffer C (20 m M Tris/HCl pH 7.4). The absorbed proteins were eluted with a linear gradient of 0–1 M NaCl containing buffer C. Fractions exhibiting PO activity when incubated with 0.2 M NaCl eluate from the Toyopearl CM-650M column, were pooled and concentrated by ultrafiltration. The purified Tm-mas was reduced and S-pyridylated according to a previously published method [14]. The S-pyridylated 45-kDa Tm-mas was digested with trypsin, and the resulting peptides were separated by HPLC on a C 18 reverse-phase column (Gilson). To determine the N-termi- nal sequence of the Tm-mas, it was subjected to SDS/PAGE under reducing conditions. The band of Tm-mas was blotted onto a poly(vinylidene difluoride) (PVDF) mem- brane (Millipore), cut out, and subjected to automated amino acid sequence analysis [15]. The protein concentra- tions were determined by the method of Lowry et al.[16] using BSA as a standard. Molecular cloning of 45-kDa Tm-mas A cDNA library from T. molitor larvae was constructed by using a ZAP-cDNA synthesis kit (Stratagene). To screen the Tm-mas clones, we performed immunoscreening by using the affinity-purified 45-kDa Tm-mas antibody. Following isopropyl-b- D -thiogalactoside induction, 5 · 10 4 plaques were screened with an affinity-purified antibody raised against the purified 45-kDa Tm-mas. A secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG, Bio-Rad) was used at a dilution of 1 : 1000. Phage DNA was isolated from phage lysates by using a lambda DNA preparation kit 4376 K. Y. Lee et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (Biometra) according to the manufacturer’s instructions. We analysed all of the plaques showing positive signals on immunoscreening. We sequenced the clones according to the dideoxy chain-termination method of Sanger et al. [17]. The amino acid sequence of the 45-kDa Tm-mas was compared with the protein sequence database of the National Center for Biotechnology Information using GENETYX (Software Development Co., Ltd, Tokyo). Antibody and immunoblotting Antibody against the Tm-mas was raised by injecting 10 lg of the purified protein into a male albino rabbit with complete Freund’s adjuvant and giving a booster injection of the same amount of protein 14 days later [18]. The resulting antibody was affinity-purified as described previ- ously [11]. For immunoblotting, the proteins separated by SDS/PAGE were transferred electrophoretically to a PVDF membrane which was then blocked by immersion in 5% skimmed milk solution containing 1% horse serum for 12 h. The membrane was then transferred to rinse solution I (20 m M Tris/HCl pH 7.5, containing 150 m M NaCl, 0.1% Tween-20, 2.5% skimmed milk) containing the affinity- purified antibody against the 45-kDa protein (50 ngÆmL )1 ) and incubated at 4 °C for 2 h. The bound antibody was identified using the Western Blot Chemiluminescence Reagent kit (NEN TM Life Sciences). The gel mobility changes of 45-kDa Tm-mas and pro-PO during pro-PO activation To examine the biological functions of the 45-kDa Tm-mas during pro-PO activation, the PO activity was measured by using HW-55S solution in the presence of b-1,3-glucan and Ca 2+ . To examine the cleavage of 55-kDa Tm-mas at different times, reaction mixture showing PO activity was precipitated by trichloroacetic acid (TCA) and 45-kDa Tm-mas was identified by Western blotting. To examine the effects of serine proteinase inhibitors on the proteolysis of 55-kDa Tm-mas and PO activity, HW-55S solution was preincubated for 30 min with 200 l M p-nitrophenyl-p¢- guanidinobenzoate (p-NPGB) and 200 l M p-amidino- phenyl-methanesulfonyl fluoride (p-APMSF), and then reaction mixture was further incubated in the presence of b-1,3-glucan and Ca 2+ . As a control, HW-55S solution was preincubated without inhibitors. After 60 min incubation, PO activity was measured and the cleaved 45-kDa Tm-mas was identified by Western blotting. RESULTS Purification of 45-kDa Tm-mas The purification procedures of Tm-mas are shown in Fig. 1A. To isolate the pro-PO activating factors (PPAFs) from Tenebrio larval hemolymph, we first prepared HW- 55S solution showing PO activity in the presence of b-1,3- glucan and Ca 2+ , by using a Toyopearl HW-55S column. As shown in Fig. 1B, HW-55S solution exhibited the most rapid increase in PO activity in the presence of b-1,3-glucan and Ca 2+ . Under the same conditions, PO activity was not observed with b-1,3-glucan only. However, Ca 2+ in the absence of b-1,3-glucan activated pro-PO, but more slowly than a combination of b-1,3-glucan and Ca 2+ . This result suggests that HW-55S solution contained all the necessary PPAFs, pro-PO and b-1,3-glucan recognition protein(s). To purify PPAF, HW-55S solution was first subjected to a Toyopearl CM-650 column chromatography and the flow-through fraction was purified by hydrophobic chro- matography on Blue-Sepharose CL-6B followed on Butyl- Sepharose. The active fractions showing PO activity were further purified by Mono-Q FPLC column (indicated as a bar in Fig. 2A). The purified protein migrated as a single band of  45-kDa on SDS/PAGE (12% acrylamide) under reducing conditions (Fig. 2B). Two-hundred mL hemo- lymph from 4000 larvae with a protein content of 2000 mg yielded 30 lg of the purified 45-kDa Tm-mas. The purified Tm-mas was used for raising polyclonal antibody. The Tenebrio 79-kDa pro-PO was purified according to our previously published methods [9]. Fig. 1. 11 (A) Procedures used to purify 45-kDa Tm-mas from the hemolymph of T. molitor larvae and (B) the PO activity of HW-55S solution. POactivitywasmeasuredbyincubationwith30lLHW-55S solution with Ca 2+ and b-1,3-glucan (d), Ca 2+ only (m), b-1,3-glucan only (j) and with neither Ca 2+ nor b-1,3-glucan (s). Ó FEBS 2002 Masquerade-like serine proteinase homologue (Eur. J. Biochem. 269) 4377 In vitro reconstitution experiments To confirm the possibility of the purified 45-kDa Tm-mas as Tenebrio PPAF, we performed in vitro reconstitution experiments by using collected fractions or the purified proteins from column chromatography. As shown in Fig. 3A, when Toyopearl CM-flow-through and Toyopearl CM-eluate were incubated in the presence of b-1,3-glucan and Ca 2+ , PO activity was clearly shown to be dependent on b-1,3-glucan and Ca 2+ (column 5). Tenebrio pro-PO was localized in Toyopearl CM-eluate solution by Western blotting analysis (data not shown). Toyopearl CM-flow- through solution was further purified by Blue-Sepahrose and Mono-Q FPLC column. When the purified Tenebrio pro-PO from Toyopearl CM-eluate and the purified Tm-mas from Toyopearl CM-flow-through were incubated, PO activity was not observed (Fig. 3B, column 4). However, when Toyopearl CM-eluate and the purified Tm-mas were incubated, PO activity was shown to be independent of Ca 2+ (column 5 and 6). These results suggested two things: first, that another protein(s) in Toyopearl CM-eluate might be necessary for PO activity; second, that the 45-kDa Tm-mas already seemed to be activated from its zymogen form, during the process of column chromatography. We examined this latter possibility by Western blotting analysis. As shown in Fig. 3C, the affinity-purified antibody raised against the 45-kDa Tm-mas recognized a 45-kDa single band in the Mono-Q fraction (lane 4). However, a 55-kDa protein was recognized in crude hemolymph (lane 1) and Toyopearl CM-flow-through (lane 2). Both the 55-kDa and 45-kDa proteins were recognized in the Blue-Sepharose fraction (lane 3). This result suggests that the purified 45-kDa protein is activated from a precursor 55-kDa zymogen form by limited proteolysis during Blue-Sepharose column and Mono-Q FPLC column chromatography. Isolation of cDNA clone for Tm-mas To determine the whole amino acid sequence of the purified Tm-mas, we first determined three partial amino acid sequences by trypsin digestion as follows: NSQGIDFNLI, GNLYNDIALL and NPNRYLQVGIVA. However, an N-terminal sequence could not be obtained due to blockage. To isolate a cDNA clone for the 45-kDa Tm-mas, we immunoscreened the cDNA library of T. molitor larvae Fig. 2. (A) Elution pattern of Mono-Q FPLC column and (B) SDS/ PAGE pattern of each column step during 45-kDa Tm-mas purification. (A) The Mono-Q column fractions indicated by bars were collected and used for subsequent experiments (see Materials and methods). (B) Lane 1, HW-55S solution; lane 2, the eluate solution of Toyopearl CM-650; lane 3, flow-through fractions of Toyopearl CM-650; lane 4, eluate solution of Blue-Sepharose CL-6B column, lane 5, active frac- tion of Mono-Q FPLC column. The proteins were analysed by SDS/ PAGE (10% acrylamide) under reducing conditions. Fig. 3. (A) PO activities of HW-55S and fractions of Toyopearl CM-650 column, (B) PO activities between the purified pro-PO and the eluate solution of Toyopearl CM-650, and (C) Western blotting of products during 45-kDa Tm-mas purification. (C) Proteins were pre- cipitatedwithTCAandsubjectedtoSDS/PAGEandthenimmu- noblotting with affinity-purified antibodies raised against 45-kDa Tm-mas. Lane 1, 10 lg hemolymph protein; lane 2, 10 lgflow- through solution of Toyopearl CM-650 column; lane 3, 5 lgeluate solution of Blue-Sepharose column; lane 4, 1 lg of the active fractions of Mono-Q FPLC column. The molecular size markers are indicated to the right in kDa. 4378 K. Y. Lee et al. (Eur. J. Biochem. 269) Ó FEBS 2002 with an affinity-purified antibody against the 45-kDa Tm- mas, and obtained 10 positive clones. The nucleotide sequence and the deduced amino acid sequence of one of these clones, named Tm-mas, are shown in Fig. 4. This cDNA contained an open reading frame of 1332 nucleotides corresponding to 444 amino acid residues with a precise mass of 48 814 Da. The apparent mass of 55-kDa for Tm- mas on SDS/PAGE is slightly larger than that of the deduced sequence. This mobility pattern is similar to several other masquerade proteinase homlogue’s [5,19] where the mass of the purified masquerade-like proteins is higher under nonreducing than reducing conditions suggesting that disulfide bonding is responsible for the higher mass. The three chemically determined partial amino acid sequences of the 45-kDa Tm-mas coincided with the deduced amino acid sequences in this open reading frame (dotted lines). There- fore, we concluded that this is a cDNA for the Tm-mas. The Tm-mas has two domains, an N-terminal domain and a serine proteinase-like domain. The hydrophobic first 22 amino acids of the N-terminal end of the protein probably constitute a signal peptide sequence with a putative signal peptidase cleavage site between Ala22 and Ile23 (arrow- head) [20]. One putative disulfide-knotted motif is present in the N-terminal domain of the protein. The putative catalytic domain, from Asn176 (arrow) to Glu444, is characteristic of that found in serine proteinase homologues, as is the presence of a Gly residue instead of a Ser residue in the catalytic site. The residues of a serine proteinase substrate binding pocket (open diamond in Fig. 5), which determine the substrate specificity of active serine proteinases, were present in the 45-kDa Tm-mas. The six cysteine residues (closed circles in Fig. 5), which form three disulfide bridges in most serine proteinases, were conserved in this 45-kDa Tm-mas. There were two potential N-glycosylation sites (Asn-Xaa-Set/Thr, indicated by closed-diamonds in Fig. 4). A homology between the Tm-mas and those in the NCBI database showed that the deduced amino acid sequence of the Tm-mas was similar to Holotrichia 45-kDa protein (Hd- PPAF-II, 59.1%) [5], Tachyplus factor D (Td-D, 38.9%) [21], masquerade of Drosophila melanogaster (Dm-mas, 35.2%) [13], masquerade-like protein of crayfish Pacifasta- cus leniusculus (Pl-mas, 41.2%) [22], mosquito infection- response serine proteinase-like protein (Ag-ispl5, 34.1%) [23], Tenebrio 45-kDa (Tm-45, 46.6%) [5], as well as Holotrichia PPAF-I (Hd-PPAF-I, 33.3%) [4] and Tachyplus proclotting enzyme (Td-PCE, 31.8%) [24] as shown in Fig. 5. The proteins Ag-ispl5, Td-D, Pl-mas, and Dm-mas showed amino acid substitutions of Ser with Gly (or Ala) in the active site triad (arrow). In conclusion, a schematic comparison of the main structural features of Tm-mas with Hd-PPAF-II, AP-ispl5, Tm-45, Td-D and Dm-mas showed a modified serine proteinase domain at the C terminus, and a disulfide-knotted motif present in the N terminus. Previously we found that a disulfide-knotted motif of Hd-PPAF-I was present in big defensin [25], an antimicro- bial peptide purified from Tachyleus hemocytes. Six cysteine residues of the disulfide-knotted motif of Tm-PPAF-I are also conserved with those of other disulfide-knotted motifs. Fig. 4. Nucleotide and deduced amino acid sequences of cloned cDNA encoding the 45-kDa Tm-mas. Numbers of nucleotides starting from the first base at the 5¢ end are shown on the left of each line; the deduced amino acid sequence is numbered from the initiating Met residue on the right of each line. The chemi- cally determined three partial amino acid sequences of the 45-kDa Tm-mas are under- lined by dots. The potential attachment sites for N-linked carbohydrate chains are indicat- ed by r sites of the catalytic triad of serine proteinases are shown by s; the arrowhead indicates the putative cleavage site for the signal peptide; the arrow indicates the putative start residue of catalytic domain. Ó FEBS 2002 Masquerade-like serine proteinase homologue (Eur. J. Biochem. 269) 4379 However, the biological function of this motif has not yet been determined. Finally, the purified Tm-mas exhibited no amidase activity against a variety of commercially available peptidyl-NH-Mec substrates tested (data not shown). Determination of Tm-mas localization To examine the localization of Tm-mas, we firstly prepared fat body, plasma, hemocyte lysate and hemolymph from T. molitor larvae.AsshowninFig.6,noappreciable amount of 55-kDa zymogen Tm-mas was detected in the hemocyte lysate or fat body (lanes 2 and 3). A significant amount of protein, however, was detected in the plasma and hemolymph (lanes 4 and 5), indicating that Tm-mas was localized in the plasma. Biochemical characteristics of Tm-mas during pro-PO activation To determine the biological function of the purified 45-kDa Tm-mas in the Tenebrio pro-PO system, we used SDS/ PAGE and Western blotting to assess whether changes in PO activity were reflected in changes in the gel mobility of Tenebrio pro-PO and the 55-kDa zymogen Tm-mas. As showninFig.7A, 25% of Tenebrio pro-PO from HW- 55S was activated to PO in the presence of Ca 2+ after 45 min incubation (lane 6); however, when HW-55S solution was incubated with b-1,3-glucan and Ca 2+ ,  50% of pro-PO was activated after 45 min (lane 11). Under the same conditions, all of the 55-kDa zymogen form of Tm-mas had converted to 45-kDa Tm-mas by 45 min, in the presence of b-1,3-glucan and Ca 2+ (lane 11 in Fig. 7B). The activation ratio of the 55-kDa zymogen form in the presence of b-1,3-glucan and Ca 2+ wasfasterthaninthe presence of Ca 2+ only (compare lane 6 with lane 11 in Fig. 7B). This result suggests that the activation of the 55-kDa zymogen form to the 45-kDa Tm-mas is a requirement for PO activity in the Tenebrio Pro-PO activation system. Furthermore, we examined the effects of serine proteinase inhibitors against proteolysis of the 55-kDa Tm-mas and PO activity by using two kinds of serine proteinase inhibitors, such as p-NPGB and p-APMSF. As shown in Fig. 7C and D, when HW-55S Fig. 6. Localization of the 55-kDa Tm-mas determined by immuno- blotting. Hemolymph was obtained from 150 larvae and centrifuged at 700 g 2 at 4 °C for 10 min. Precipitated hemocytes were washed with 500 lL anticoagulation buffer (pH 5.5) and suspended again with 500 lL anticoagulation buffer. One hundred lL of this suspension provided the total haemocyte component. The remaining solution (400 lL) was sonicated for 15 s at 4 °Candcentrifugedat 15 000 r.p.m. (22 000 g)at4°C for 10 min The supernatant was used as haemocyte lysate. The soluble proteins were precipitated with TCA and subjected to SDS/PAGE and then immunoblotting with affinity- purified antibody raised against the 45-kDa Tm-mas. Lane 1, 10 lg protein of flow-through solution from Toyopearl CM-650 column; lane 2, 10 lg of soluble fat body protein; lane 3, 10 lg of soluble haemocyte lysate protein; lane 4, 10 lgofplasmaprotein;lane5,10 lg of haemolymph protein. Fig. 5. Alignment of the catalytic domains of Tm-mas and Holotrichia 45-kDa protein (Hd-PPAF-II) with catalytic domains of known serine proteinase homologues. Tachyplus factor-D (Tt-D), Drosophila masquerade (Dm-mas), crayfish masquerade (Pl-mas), Anopheles immune response serine proteinase-like protein (Ap-ispl5) and known serine proteinase, such as Holotrichia PPAF-I (Hd-PPAF-I) and Tachyplus proclotting enzyme (Tt-PCE). Numbers refer to the predicted protein sequence. Stars indicate the residues of the catalytic triad of serine proteinase. The conserved cysteine residues are indicated by d; residues conserved in all sequences are shown within boxes; e indicate the positions of residues known to occupy the substrate binding pockets of trypsin; the arrow indicates the substitution residue of the catalytic triad of serine proteinase. Gaps were introduced to obtain maximal sequence similarity. 4380 K. Y. Lee et al. (Eur. J. Biochem. 269) Ó FEBS 2002 solution was preincubated with p-NPGB and p-APMSF, andthencalciumandb-1,3-glucanwereaddedtoHW-55S solution, PO activity and proteolysis of the 55-kDa Tm-mas were not observed (Fig. 7C, column 3 and Fig. 7D, lane 3). However, PO activity and the cleavage of the 55-kDa Tm-mas were clearly shown when calcium and b-1,3-glucan were added to HW-55S solution in the absence of inhibitors (Fig. 7C, column 2 and Fig. 7D, lane 2). These results suggest that the cleavage of the 55-kDa Tm-mas might be induced by unidentified serine proteinase. Also, it was confirmed that PO activity is shown when the 55-kDa Tm-mas is cleaved to the 45 kDa Tm-mas. DISCUSSION In this study, we isolated a novel 45-kDa protein from the hemolymph of T. molitor larvae, which showed high homology (35% sequence identity) with Drosophila mas- querade serine proteinase homologue. We have called this protein Tenebrio masquerade-like proteinase homologue (Tm-mas). This is also the first report to demonstrate that a novel masquerade-like serine proteinase homologue zymo- gen (55-kDa Tm-mas) is cleaved to 45-kDa Tm-mas as a prerequisite for PO activity. Several kinds of serine proteinase homologue have been purified already from vertebrates and invertebrates, and they have been suggested to perform different biological functions, including antim- icrobial activities (e.g. horseshoecrab factor D [21] and human azurocidin [26]), or by acting as adhesion molecules (e.g. Drosophila masquerade [13], Pacifastacus masquerade- like protein [22], glutacin [27] and neurotactin [28]), immune molecules (e.g. mosquito ispl5 [23], Holotrichia PPAF-II [5]), growth factors (e.g. human hepatocyte growth factor [29]) or as pattern recognition proteins in crayfish [19]. As their name indicates, all known serine proteinase homo- logues are very similar to serine proteinases, differing only in the substitution of their catalytic residues. It is suggested that the pro-PO activating system, which functions in nonself recognition and defence responses in invertebrates, is composed of an enzyme cascade consisting of pattern recognition proteins, several serine proteinases and pro-PO [1,2]. Recently our group and three other groups have reported that Drosophila easter-type serine proteinases with disulfide-knotted domain(s) are involved in the pro-PO activation system [4,30–32]. Recently we have reported that Holotrichia masquerade-like PPAF-II (Hd- PPAF-II) is also engaged in Holotrichia pro-PO activation and is cleaved at Arg99–Glu100 by an easter-type serine Fig. 8. Amino acid sequence comparison of the cleavage sites for Tm-mas and Hd-PPAF-II. The arrow indicates the cleavage site of Hd-PPAF-II by Hd-PPAF-I. Fig. 7. Western blotting of (A) Tenebrio 79-kDa pro-PO, and (B) 55-kDa Tm-mas in HW-55S solution by incubation with Ca 2+ and b-1,3-glucan and the effects of serine proteinase inhibitors on (C) PO activity and (D) proteolysis of 55-kDa Tm-mas. PO activity of HW-55S solution was measured as described in Fig. 1. The reaction mixtures at different times were precipitated with TCA and subjected to SDS/ PAGE and then immunoblotting with the affinity-purified antibody raised against the Tenebrio 79-kDa pro-PO and 45-kDa Tm-mas. (A) The 79-kDa and 76-kDa bars indicate Tenebrio pro-PO and PO, respectively. (B) The 55-kDa and 45-kDa bars indicate the zymogen form and cleaved protein of 45-kDa Tm-mas, respectively. Lane 1, 30 lL HW-55S solution was incubated with neither Ca 2+ nor b-1,3- glucan for 60 min; lane 2, incubation with b-1,3-glucan only for 60 min; lane 3, 4, 5, 6 and 7, incubation with Ca 2+ only for 10, 20, 30, 45, 60 min, respectively; lane 8, 9, 10, 11 and 12, incubation with Ca 2+ and b-1,3-glucan for 10, 20, 30, 45, 60 min, respectively. (C) Incuba- tion conditions with serine proteinase inhibitors were described in Materials and methods. PO activity of HW-55S solution with or without inhibitors was measured as described in Fig. 1. Column 1, after 0 min incubation without inhibitors; column 2, after 60 min incubation without inhibitors; column 3, after 60 min incubation with inhibitors. D, The reaction mixtures of Fig. 7C at different times were precipitated with TCA and subjected to SDS/PAGE and then immunoblotting with the affinity-purified antibody raised against the 45-kDa Tm-mas. Ó FEBS 2002 Masquerade-like serine proteinase homologue (Eur. J. Biochem. 269) 4381 proteinase [5]. To address the possibility that Tm-mas also has a similar cleavage site to Holotrichia masquerade-like PPAF-II, we have compared the Tm-mas amino acid sequence with the sequence in the vicinity of the Hd-PPAF- II cleavage site. As shown in Fig. 8, the tentative cleavage site of Lys98-Glu99 in Tm-mas is perfectly conserved in Holotrichia masquerade-like PPAF-II, suggesting that a serine proteinase cleaves the Lys98–Glu99 site of Tm-mas. However, in this study, the identity of the serine proteinase that cleaves the 55-kDa zymogen form, has not been elucidated. Further studies focusing on this unidentified serine proteinase will provide clues to understanding the biological function of serine proteinase homologues in the pro-PO activation system. Previously, we reported the cDNA sequence of another masquerade-like serine proteinase homologue (Tm-45 protein), obtained from a Tenebrio cDNA library that had been cloned following screening with Holotrichia PPAF-II antibody [5]. The deduced amino acid sequence of Tm-45 protein has 46.6% similarity with the purified 45-kDa Tm-mas described in the present report (data not shown). Although the Tm-45 protein has not yet been purified, any differences we may be able to detect between the biological functions of Tm-45 and Tm-mas will be important for understanding the details of the Tenebrio pro-PO system. One interesting point regarding masquerade-like serine proteinase homologues, is that horseshoecrab factor D copurified with a serpin during purification procedures [21], which suggests that masquerade-like serine proteinases might make a complex with serpin. 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A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca 2+ in coleopteran and Tenebrio molitor larvae Kum. et al.[16] using BSA as a standard. Molecular cloning of 45-kDa Tm-mas A cDNA library from T. molitor larvae was constructed by using a ZAP-cDNA synthesis