Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph.код для вставкиСкачать
186 Bania et al. Archives of Insect Biochemistry and Physiology 62:186�6 (2006) Specificity of an Extracellular Proteinase From Conidiobolus coronatus and Its Inhibition by an Inhibitor From Insect Hemolymph 1 2 Jacek Bania, * Jaroslaw Samborski, 2 Mieczyslawa Bogus, 3 and Antoni Polanowski The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000�,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala2-Pro-Phe-pNa with a Km-1.36 10 � � M and Kcat-11 s 17,030s � M � � 10 � M and Kcat-24 s � , and N-Suc-Ala2-Pro-Leu-pNa with Km-6.65 � . The specificity index kcat/Km for the tested substrates was calculated to be 176,340s M � � and , respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide 1 2 bonds: Phe -Val , Leu 15 16 -Tyr , and Gly 23 -Phe 24 . The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 � 10 10 � M ), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase. Arch. Insect Biochem. Physiol. 62:186�6, 2006. � 2006 Wiley-Liss, Inc. KEYWORDS : Conidiobolus coronatus; proteinase; substrate specificity; proteinase inhibitor; insect hemolymph INTRODUCTION gal virulence (Bidochka and Khachatourians, 1994; Cole et al., 1993; Gupta et al., 1992; Samuels and Unlike other microbial pathogens, e.g., bacte- Paterson, 1995; St Leger et al., 1987, 1988). ria, rickettsiae, protozoa, and viruses, that invade On the other hand, it has been known for a num- insects after being ingested, fungi can infect insects ber of years that insect hemolymph contains sev- by direct penetration of the host integument. eral different types of polypeptides that are able to Entomopathogenic fungi produce an array of ex- quench the catalytic function of proteolytic enzymes tracellular digestive enzymes, such as proteinases, (Eguchi, 1993; Polanowski et al., 1992; Polanowski lipases, and chitinases, which are involved in de- and Wilusz, 1996). Without a clear elucidation of grading cuticular compounds. Among the enzymes the specificity and mode of action of both fungal taking part in breaching the cuticle, the protein- extracellular proteinases and insect host proteinase ases are believed to play a major role, and have, inhibitors, we can never hope to have a complete therefore, been consider a significant factor of fun- understanding of the pathogenic processes. 1 2 3 Department of Food Hygiene and Consumer Protection, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Wroclaw, Poland W. Stefanski Institute of Parasitology, Polish Academy of Sciences, Warsaw, Poland Institute of Biochemistry and Molecular Biology, University of Wroclaw, Wroclaw, Poland *Correspondence to: Jacek Bania, Department of Food Hygiene and Consumer Protection, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Norwida 31, 50-375 Wroclaw, Poland. E-mail: firstname.lastname@example.org Received 1 October 2005; Accepted 6 February 2006 � 2006 Wiley-Liss, Inc. DOI: 10.1002/arch.20134 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. Specificity of an Extracellular Proteinase From C. coronatus The relatively little-investigated entomopatho- creas was purified in our laboratory. 187 Purified gen Conidiobolus coronatus secretes a range of pro- Beauveria bassiana proteinase was kindly provided teinases to culture broth (Phadatare et al., 1992; by Dr. Maria Kolaczkowska. All other reagents used Sutar et al., 1991; Tanksale et al., 2000). In our were of analytical grade. Conidiobolus coronatus, iso- studies, using a combination of ion-exchange and late number 3491, originally isolated from Dendro- size-exclusion chromatography, we have purified laelaps spp., was obtained from the collection of into homogeneity a serine proteinase of Mr 30,000� Prof. Balazy (Polish Academy of Sciences, Research 32,000, as assessed by SDS-PAGE. The purified en- Center for Agricultural and Forest Environment, zyme exhibits subtilisin-like activity. We show that Poznan), was routinely maintained in 90-mm Petri two serine proteinases derived from the entomo- dishes at 20 C with cyclic changes of light (LD12:12) pathogens Conidiobolus � coronatus and Beauveria on Sabouraud agar medium with the addition of bassiana are strongly inhibited by an inhibitor from homogenized G. mellonella larvae to a final con- Apis mellifera hemolymph. This finding supports centration of 10% wet weight. an as yet little documented hypothesis about the involvement of proteinase inhibitors from hemo- Growth Conditions lymph in insect innate immunity. Conidiobolus coronatus was grown on liquid medium containing 0.1% (NH4)2SO4, 0.45% KH2PO4, MATERIALS AND METHODS 1.05% K2HPO4, 0.05% sodium citrate dihydrate, Chemicals and Entomopathogen Strains 0.2% glucose, and 0.025% MgSO4. The medium was sterilized by autoclaving and its pH adjusted Sephadex G-75 was from Pharmacia Chemicals, Uppsala, Sweden. Bovine chymotrypsin, subtilisin Fine b-trypsin, a- Carlsberg, leupeptin, chymostatin, soybean trypsin inhibitor (STI), Bowman-Birk trypsin inhibitor (BBI), the p-nitroanilide (-pNa) substrates a N-Suc-Ala-Ala-Pro-Phe-pNa, NSuc-Ala-Ala-Pro-Leu-pNa, benzoyl-Arg-pNa (BApNa), � to 7.0. Cultivation was performed at 26 C for 10 days in 750-ml Erlenmeyer flasks containing 250 ml of medium, with mechanical shaking at 150 rpm (rotary shaker). The mycelia were removed by filtration through Whatman no. 1 filter paper and the cell-free filtrate served for proteinase preparation. N-Suc-Ala-Ala-Pro-Val-pNa, N-Suc-Ala-Ala-Ala-pNa, and N a-Tosyl-L-lysine chloromethyl ketone hy- Protein Assay drochloride (TLCK), N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), diisopropyl flouro- Protein content was determined either with phosphate (DFP), trinitrobenzenesulfonic acid bicinchoninic acid (Smith et al., 1985) or by the (TNBS), and phenyl-methylsulfonyl fluoride (PMSF) biuret micro-method (Goa, 1953) using bovine se- were obtained from the Sigma Chemical Company rum albumin as a standard. (St. Louis, MO). N,N,N�,N�-tetramethylethylenediamine (TEMED), N,N�-metylenebisacrylamide, 4- Proteinase Activity nitrophenyl 4-guanidinobenzoate hydrochloride (NPGB) and HPLC grade reagents were from Fluka AG, Buchs, Switzerland. Basic pancreatic trypsin inhibitor (BPTI) Trascolan�, was purchased from Proteolytic activity was measured using the chromogenic substrate: N-suc-Ala-Ala-Pro-Phe-p-Na in 100 mM Tris-HCl buffer, pH 8.3, containing 20 Pharmaceutical Co. Jelfa (Jelenia Gora, Poland). mM CaCl2 and 0.005% Triton X-100. The reactions Turkey ovomucoid (OMTKY) and a chymotrypsin/ were performed in polystyrene cuvettes containing cathepsin G inhibitor from the hemolymph of Apis the appropriate amount of proteinase, 50 mellifera (AMCI-1) were purified according to strate concentration, and reaction buffer to a final mM sub- Bogard et al. (1980) and Bania et al. (1999), re- volume of 2 ml. Progress curves at A412, after start- spectively. Kazal-type inhibitor from bovine pan- ing a reaction by adding substrate, were followed Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. 188 Bania et al. using an HP8452 diode-array spectrophotometer. gel and 4% stacking gel with 0.1% sodium dodecyl A unit of activity was defined as the amount of sulfate (SDS), pH 8.9. Coomassie brilliant blue enzyme that caused an increase in the absorbance staining was used to visualize protein bands. Mo- by 0.1 per min. lecular weight standards bovine serum albumin (66 kDa), egg ovoalbumin (43 kDa), soybean trypsin inhibitor (21 kDa), and basic pancreatic trypsin in- Proteinase Purification hibitor (6.5 kDa) served as reference proteins. The The cell-free filtrate was concentrated about 30- apparent Mr value of purified protein was calcu- fold with an Amicon hollow fiber cartridge concen- lated from semi-log plots of standard protein Mr trator (model H1P10). The retentate was applied vs. migration distances. to a Sephadex G-75 column (45 � 600 mm) equili- brated with 50 mM sodium acetate buffer, pH 5.0. Proteinase pH Stability The column was eluted at of 8 ml/h. Fractions (3 ml) were collected and assayed for protein at 280 nm and for enzyme activity. Fractions exhibiting proteinase activities were pooled, concentrated, and then desalted using an Amicon YM10 membrane. This preparation of active proteins was subjected to HPLC using a Waters SP 5PW 8 � 75 mm ion- exchange column, equilibrated with 50 mM Tris- The enzyme (1 � 10 � M) was incubated for 30 min in 100 mM buffers of pH 2� (citrate-phosphate buffer for pH抯 2� Tris-HCl buffer for pH抯 7� carbonate-bicarbonate buffer for pH抯 9�). After incubation, the remaining activity of the proteinase was measured by the method described above. HCl buffer, pH 8.0. Proteins were eluted with a linear gradient of 0�M NaCl in the equilibrating buffer for 50 min at 0.7 ml/min. Absorbance was monitored at 280 nm. pH Optimum The effect of pH on the proteinase activity toward N-suc-Ala-Ala-Pro-Phe-p-Na was assayed with 100 mM buffers of pH 7�. The reaction was car- Determination of Proteinase Concentration ried out in polystyrene cuvettes in 2 ml of the appropriate buffer, containing proteinase and substrate The C. coronatus proteinase concentration was determined by spectrophotometric titration with the inhibitor AMCI-1. The following procedure was used to estimate the concentration of AMCI-1: the at concentrations of 1 � 10 � M and 50 mM, respec- tively. Activity was determined as the release of pnitroaniline, and was continuously monitored at 412 nm. molar concentration of a stock solution of bovine b-trypsin (dissolved in 1 mM HCl, 20 mM CaCl ), 2 Effects of Inhibitors was determined by titration with p-nitrophenyl pguanidinobenzoate HCl (NPBG) according to The enzyme (1 � 10 � M) was allowed to com- Chase and Shaw (1969). This standardized trypsin plex with an inhibitor (10 mM) in a final volume solution was used to titrate BPTI, which in turn of 2 ml in 100 mM Tris-HCl buffer, pH 8.3, con- served as a secondary standard for determining the taining 20 mM CaCl2 and 0.005% Triton X-100. activity of bovine After preincubation for 30 min, the residual pro- a-chymotrypsin that was used for determining AMCI-1 concentration. teinase activity was measured toward 50 mM N-suc- Ala-Ala-Pro-Phe-p-Na as described above. SDS-Polyacrylamide Gel Electrophoresis and Molecular Mass Determination Gel electrophoresis was Kinetic Constants kcat and Km the Kinetic constants were determined for the hy- method of Laemmli (1970) using 15% separating carried out by drolysis of N-suc-Ala-Ala-Pro-Phe-p-Na and N-suc- Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. Specificity of an Extracellular Proteinase From C. coronatus Ala-Ala-Pro-Leu-p-Na by Conidiobolus proteinase. � � 189 tilisin Carlsberg were added to a 5% ground cu- M ticle suspension in 0.1 M Tris-HCl, pH 8.3. A unit of Conidiobolus proteinase and increasing concen- of subtilisin Carlsberg activity was defined as the The 2-ml reaction mixtures contained 1 � � 10 M in 100 mM amount of enzyme that caused 0.1 A412 per min Tris-HCl buffer, pH 8.3, with 20 mM CaCl2 and increase under the conditions described for deter- 0.005% Triton X-100. After starting the reaction by mining C. coronatus proteinase activity. The mix- adding the appropriate substrate, the release of p- tures were incubated at 37 C for 3, 6, and 24 h, nitroaniline was continuously monitored at A412. then 0.5 ml of 1% SDS was added and the tubes Absorbance rates were converted to the substrate were heated at 75 C for 15 min. The enzyme was concentration changes using p-nitroaniline molar added to the negative control (5% ground cuticle extinction coefficient of 8,800/M/cm, and a graphic suspension in 0.1 M Tris-HCl, pH 8.3), after pre- representation of substrate concentration vs. reac- heating the tube to 75 C. The reaction mixtures tion rate was prepared. The Km was determined were centrifuged at 13,000 rpm, and supernatants using nonlinear regression analysis of the result- were subsequently assayed for free amino groups ing curve fitted to the Michaelis朚enten equation according to Adler-Nissen (1979). An appropriate by GraFit software. kcat was calculated as a quo- volume of the reaction mixture was completed to tient of Vmax and the enzyme concentration (E0). 250 trations of substrate up to 2 10 � � � ml ml of 0.1 M sodium 500 ml of 0.1% trinitro- with water, then 250 The Vmax value was calculated by analysis of the tetraborate, pH 9.6, and curve representing experimental points fitted to the benzenesulfonic acid were added. The samples Michaelis� Menten equation by GraFit software. were incubated at 37 C for 1 h in the dark and The E0 value was determined in an independent the A420 was read. The absorbance of the negative experiment as described previously. controls was subtracted from the samples. A unit � of proteolytic activity was defined as the amount Digestion of the Oxidized Insulin B-Chain of enzyme able to release free amino groups corresponding to 1 mg of alanine. Oxidized insulin B chain 1 mg (Sigma Chemical Company) dissolved in 0.5 ml of Tris-HCl Association Constant of Conidiobolus coronatus buffer, pH 8.0, and was incubated with 1 mol% of Proteinase and Beauveria bassiana Basic Proteinase Conidiobolus proteinase at 37 C for an appropriate With AMCI-1 � reaction time. At intervals of 15, 35, and 60 min, tein was withdrawn and added to 100 mg of digested proml 10% TFA timated using the method described by Empie and to stop the reaction. Products of digestion were Laskowski (1982). The reactions were conducted an aliquot containing about 50 separated via HPLC using a Waters m-Bondpak 3.9 � 150 mm, C-18 column. Each fraction of the pep- The equilibrium association constants were es- in a polystyrene cuvette in a final volume of 2.0 ml of 0.1 M Tris-HCl buffer, pH 8.3, with 20 mM tide peaks was collected and analyzed using an CaCl2 and 0.005% Triton X-100. The enzyme con- amino-acid sequencer (Applied Biosystems) to lo- centrations used [E0] complied with the equation: 碖 calize cleavage sites on the oxidized insulin B- 2 < [E0] chain. tor [I0] ranged from 0 to 2 a < 50 and the concentration of inhibi- �[E ]. A constant amount 0 of an enzyme reacted with an increasing amount � Hydrolysis of Insect Cuticle With of inhibitor at 22 C for a predetermined period of C. coronatus Proteinase time to reach equilibrium, followed by the addition of substrate, whose final concentration in the Exuviae from Dendrolimus pini were washed with reaction did not exceed 0.2 碖 . The hydrolysis of m 0.1 M Tris-HCl, pH 8.3, then air dried for 24 h. C. p-nitroanilide was monitored at 412 nm for 120 coronatus proteinase (20 U) or an equivalent of sub- sec and the free enzyme concentration was calcu- Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. 190 Bania et al. lated. The association constant Ka was calculated by fitting the experimental data to the following equation: E = 1 2 � ([E0] � F[I0] � K a + 2 � ([E0] � F[I0] � K�a ) � 4[E0]F[I0]) where [E] is the residual enzyme concentration, [E0] and [I0] are the total enzyme and inhibitor concentrations, respectively, and F is an enzyme-inhibitor equimolarity factor. RESULTS Purification of Conidiobolus Proteinase The C. coronatus grown as described above produced an extracellular proteinase that reached its maximal activity on the 10th day of cultivation. The level of activity did not increase beyond 10 days of cultivation. Enrichment of the culture medium with casein or yeast extract as a source of carbon resulted in a significant decrease in proteinase concentration (data not shown). The proteinase was purified from the culture broth after removing the fungi by filtration through filter paper. The filtrate volume was reduced with an Amicon Hollow Fiber Cartridge H1P10-20 to about 100 ml, then concentrated using an Amicon YM10 membrane to a final volume of 5 ml. The purification method is summarized in Table 1. The concentrated cell-free medium was first applied on a Sephadex G-75 column at pH 5.0. The concentrated medium was resolved into several peaks, of which only one showed proteinase activity (Fig. 1). SDS-PAGE analysis of the proteolytically active fractions showed several bands (data not shown); Fig. 1. A: Elution profile of C. coronatus proteins from a Sephadex G-75 column at pH 5.0. The protein peak exhibiting proteolytic activity is marked by a horizontal bar. B: Elution profile of proteinase collected from a Sephadex G-75 on SP 5PW ion-exchange column at pH 8.0. Horizontal bar indicates proteinase activity. therefore, they were pooled, concentrated using an Amicon YM10 membrane, and applied to an HPLC ing all the proteinase activity was eluted from the Waters SP 5PW ion-exchange column at pH 8.0. column after about 16 min post-injection. SDS- As shown in Figure 1B, the protein peak contain- PAGE analysis of this peak revealed a single band TABLE 1. Summary of Conidiobolus Proteinase Purification Protein concentration Purification step Culture fluid concentrate Sephadex G-75 HPLC ion-exchange Volume (ml) Specific activity Yield (%) Purification factor 5 3.6 410 22 � � 10 0.4 240 60 58 0.33 185 616 45 0.9 (mg/ml) Activity (U) (U/mg protein) Archives of Insect Biochemistry and Physiology 2.7 28 August 2006 doi: 10.1002/arch. Specificity of an Extracellular Proteinase From C. coronatus 191 for 30 min at pH抯 of 6 to 10 had no effect on its activity. A significant loss of activity could be observed during incubation of proteinase at pH below 5. Proteinase incubations at pH 3 or below irreversibly loses its activity (Fig. 3). The pH dependence of the amidase activity for Fig. 2. SDS-PAGE of Con- purified enzyme is shown in Figure 4. The opti- idiobolus protease after ion- mal pH抯 for the digestion of N-Suc-Ala-Ala-Pro- exchange chromatography Phe-pNa by on to be 8� At pH below 8 and above 9, its activity an 5PW 8 HPLC � Waters SP 75 mm column. Conidiobolus proteinase was determined drastically decreases (Fig. 4). The protein was resolved in 15% polyacrylamide gel, and then stained with Coomasie brilliant blue. of a Mr of 30 kDa (Fig. 2). The enzyme was purified to homogeneity with an overall purification factor of 28-fold and a final yield of over 45%. Effect of Proteinase Inhibitors on Conidiobolus Proteinase Activity A range of synthetic and naturally occurring inhibitors was used to determine the nature of the active site of Conidiobolus proteinase. The protein- ase was inhibited by PMSF and DFP, indicating the Effect of pH on the Activity and Stability of the presence of serine and histidine residues in the ac- Conidiobolus Proteinase tive site. As expected, specific inhibitors of other The Conidiobolus classes of proteinases, i.e., aspartyl, metallo, and proteinase was stable over a wide range of pH values. Incubation of proteinase cysteine proteinases, did not affect its activity. These results confirm that Conidiobolus Fig. 3. proteinase belongs Effect of pH on the stabil- ity of proteinase from C. coronatus. Proteinase in a concentration of 1 � 10 � M was incubated for 30 min at the pH indicated. Then the remaining activity was assayed with N-suc-Ala-Ala-Pro-Phe-p-Na in 100 mM Tris-HCl buffer, pH 8.3, containing 20 mM CaCl2 and 0.005% Triton X-100. Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. 192 Bania et al. Fig. 4. Determination of pH opti- mum for C. coronatus proteinase activity. A constant amount of proteinase was assayed toward N-SucAla-Ala-Pro-Phe-pNa in a range of pH values (100 mM buffers containing 20 mM CaCl2 and 0.005% Triton X-100). to the family of serine proteinases. Some protein teract irreversibly with serine proteinases exhibit- inhibitors of serine proteinases are useful in dem- ing chymotrypsin-like and trypsin-like specificities, onstrating the preferences of proteinase for the in- respectively, were examined. In view of previous hibitor P1 position, and also help in determining results, the lack of inhibition by trypsin-specific the enzyme specificity. From the range of inhibi- TLCK was not surprising, although it was striking tors tested, AMCI-1, OMTKY, and the microbial- that TPCK also appeared to be ineffective. derived inhibitors leupeptin and chymostatin all An additional set of inhibitors known to in- showed strong inhibition. The inhibition profile hibit chymotrypsin-like enzymes, namely BPTI, Conidiobolus proteinase has a subtili- STI, and BBI at concentrations of 10 mM, inhib- sin-like specificity. To prove this, the effect of two ited proteinase activity by 50, 60, and 80%, re- synthetic inhibitors, TLCK and TPCK, known to in- spectively (Table 2). suggests that In contrast, the Kazal-type inhibitor from boTABLE 2. Effect of Selected Inhibitors on the Activity of Conidiobolus Proteinase* trypsin-like proteinases, had no effect on Inhibitors % proteinase activity DFP Conidio- bolus proteinase activity. 0 PMSF 0 TPCK 100 TLCK 100 Leupeptin 0 STI 40 BBI 20 BPTI 50 OMTKY 10 AMCI-1 0 Kazal type inhibitor from bovine pancreas *The enzyme (1 Proteinase Specificity 10 Chymostatin � 10 � From the range of synthetic substrates tested, Conidiobolus proteinase hydrolyzed only chymotrypsin-specific substrates with phenylalanine and leucine residues at the P1 position, and did not generate observable cleavage of the substrates with 100 arginine, valine, or alanine at that position. An M) was allowed to complex with an inhibitor (10 mM). After preincubation for 30 min, the residual proteinase activity was measured toward vine pancreas, known to inhibit exclusively the N-suc-Ala-Ala-Pro-Phe-p-Na. analysis of the enzyme kinetics showed that Conidio- bolus proteinase hydrolyzed N-Suc-Ala-Ala-Pro-Phe- Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. Specificity of an Extracellular Proteinase From C. coronatus pNa with Km = 1.36 s � � � 10 � M and a kcat value of 24 and N-Suc-Ala-Ala-Pro-Leu-pNa with Km = 6.65 � 10 M and a kcat value of 11 s � . The specificity 193 Association Constant of Conidiobolus coronatus Proteinase and Beauveria bassiana Basic Proteinase With AMCI-1 indices kcat/Km for the tested substrates were calculated to be 176,340 s � � Inhibition studies showed (Table 2) that the purified enzyme exhibited the highest sensitivity proteinases, such as BApNa (trypsin), N-Suc-Ala-Ala- to AMCI-1, a chymotrypsin/cathepsin G inhibitor Pro-Val-pNa (human leukocyte elastase), and N-Suc- from the larval hemolymph of the honeybee (Apis Ala-Ala-Ala-pNa (porcine pancreatic elastase) were mellifera) (Bania et al., 1999). Considering the not cleaved by Conidiobolus proteinase. physiological significance of AMCI-1 presence in substrates for M � , re- Specific and 17,030 s � serine spectively. M other Cleavage specificity of Conidiobolus proteinase the hemolymph of the honeybee, we studied its was also determined by using oxidized insulin B- possible role in insect anti-fungal responses. Since chain as a substrate. The hydrolyzed peptide bonds proteinase inhibitors in insect hemolymph are be- are indicated by arrows in Figure 5. After 15-min lieved to protect the insect against fungal infection, digestion, it was found that Conidiobolus protein- we tested the susceptibility of fungal proteinases 1 , to AMCI-1. It was shown that both Conidiobolus . Secondary cleavage coronatus proteinase and the basic proteinase, pu- ase cleaved the substrate at positions Phe 朧al 15 Leu 朤yr 16 , and Gly 23 朠he 24 2 24 � rified from another enthomopathogen Beauveria . Incubation times up to 1 h bassiana, were strongly inhibited by AMCI-1. The did not change the elution profile pattern from the association constants of AMCI-1/Conidiobolus pro- C-18 column, seen at 35 min, indicating a lack of teinase and AMCI-1/Beauveria basic proteinase were additional digestion sites (Fig. 5). determined to be 3 sites were found after 35 min at positions Phe 25 Phe and Phe 25 朤hr 26 To prove that C. coronatus proteinase may be � 10 10 M � and 4 � 10 9 M � , re- spectively. involved in the penetration of the insect body via the cuticle, its ability to hydrolyze the insect cu- DISCUSSION ticle was tested. Hydrolysis was assayed by measuring the free amino groups released during the C. coronatus secretes several proteinases into the reaction. It was found that both C. coronatus pro- culture medium with molecular weight ranging teinase and subtilisin Carlsberg, used as a refer- from 6.8 to 32 kDa (Sutar et al., 1991). The mo- ence enzyme, effectively hydrolyzed the insect lecular masses of the better-described proteinase I cuticle. Within 24 h, the enzymes from C. coron- and proteinase II were found to be 23 and 19 kDa, atus and Carlsberg at the amount specified pro- respectively. Their similar properties, including duced a free amino group from 50 mg of cuticle amino-acid composition, substrate specificity, and corresponding to 28 and 20 mg of alanine, re- antigenic properties suggest that both of them are spectively. presumably the products of the same gene, and Fig. 5. Cleavage pattern of oxidized insulin B-chain di- gested by Conidiobolus proteinase. Digestion was performed Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. in 0.1 M. Tris-HCl buffer, pH 8.0, for 30 min. Vertical arrows show cleavage sites after 15 and 35 min of digestion. 194 Bania et al. proteinase II appears to be the result of auto- teinases used confirms that Conidiobolus protein- proteolysis of proteinase I (Phadatare et al., 1992). ase represents a subtilisin-like specificity. The in- Another subtilisin-like proteinase isolated from the hibitors of both trypsin and chymotrypsin (BPTI, culture broth of C. coronatus of molecular weight STI, BBI, and OMTKY, but also AMCI-1 and chymo- 27�.5 kDa has been described by Tanksale et al. statin, specific inhibitors of chymotrypsin-like en- (2000). zymes) efficiently inhibit Conidiobolus proteinase. A novel proteinase of molecular weight 30-32 The Kazal-type inhibitor from bovine pancreas, kDa was purified from the culture supernatant of known to inhibit trypsin-like enzymes exclusively, C. coronatus. The optimization of broth composi- did not affect the activity of Conidiobolus protein- tion towards the proteinase yield was not a major ase. goal of our study, but, in contradiction to other known to inhibit primarily cysteine proteinases, authors demonstrating that the addition of selected has organic compounds clearly enhances the produc- Conidiobolus proteinase activity. Some examples of tion of proteinases (Phadatare et al., 1993; Sutar serine et al., 1991), we noted a significant reduction in known (Fujishiro et al., 1980) and it has been proteinase concentration in the presence of casein shown that a cysteine residue near the active site and/or other organic sources of carbon in the cul- histidine in some subtilisins renders these enzymes ture medium. The proteinase reduction may be the susceptible to cysteine protease inhibitors, such as result of either decreased proteinase production or leupeptin (Barrett and Rawlings, 1991). disturbed proteinase secretion to the medium. Leupeptin, also been a microbial-derived shown proteinases to exert inhibited some by inhibitor, effect leupeptin on are The substrate preferences of Conidiobolus pro- The proteinase inhibition effects, as well as the teinase were determined by its cleavage sites on substrate specificity studies, showed that the new oxidized insulin B-chain. The proteinase affinity Conidiobolus proteinase may be classified as a sub- seems to be restricted exclusively to the aromatic tilisin-like serine proteinase. The proteinase cleaves Phe and aliphatic Leu and Gly residues. Interest- substrates with phenylalanine (N-Suc-Ala-Ala-Pro- ingly, the proteinase cleaved at all the phenylala- Phe-pNa) and leucine (N-Suc-Ala-Ala-Pro-Leu- nine residues present in the peptide, while only pNa) residues at the P1 positions with kcat and Km one of the four leucines present in the insulin B- values similar to known chymotrypsins and sub- chain were cleaved. This may indicate that the se- tilisins (Nakajima et al., 1979; Yamagata et al., quence of the residues neighboring the P1 site of 1995). The Conidiobolus proteinase was unable to leucine may strongly influence the proteinase speci- hydrolyze the amide substrates containing Arg, Val, ficity. In turn, the cleavage at the Gly residue is a or Ala at P1 position. Other known proteinases characteristic trait of pancreatic elastase (Merops from C. coronatus were shown to be able to cleave database http://merops.sanger.ac.uk). One unusual the ester substrates with both Tyr and Arg at P1, cleavage site after the first Phe residue was found. but failed to hydrolyze its amide counterparts The proteinases from pathogenic fungi are poorly (Phadatare et al., 1992). The authors of the study characterized in their ability to cleave such well- conclude that the investigated proteinases simply defined peptide substrates as insulin B-chain, and lack the amidase activity toward the tested sub- consequently the presence of such an activity in strates. We have shown that the amide substrates the class of serine proteinases cannot be excluded. with Phe and Leu at P1 may be successfully cleaved The enzymes currently known to be able to simul- by the novel Conidiobolus proteinase, confirming taneously act as endo- and exopeptidases and to its amidase activity. This may indicate that the ori- cleave the Phe/Val bond in insulin B-chain belong gin of the observed differences may result from dif- to the class of aspartic or metallo proteinases ferent specificities of proteinases and not from the (Merops database http://merops.sanger.ac.uk). lack of its activity towards the amide substrates. The range of natural inhibitors of serine pro- Entomopathogenic fungi secrete a range of hydrolases that facilitate the penetration of the fun- Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. Specificity of an Extracellular Proteinase From C. coronatus 195 gus into the insect body. The insect cuticle is com- these proteinases for AMCI-1 are relatively high. posed of chitin fibers buried in protein matrix. As To our knowledge, the only well-characterized in- was confirmed by a number of studies, the fungal teraction between a fungal proteinase and an in- serine proteinases are able to degrade the insect sect proteinase inhibitor is that of the proteinase cuticle and are thus especially important in fungal from Aspergillus melleus and the FPI-F inhibitor pathogenesis (Bidochka and Khachatourians, 1994; from Bombyx mori. The association constant of this Cole et al., 1993; Gupta et al., 1992; Samuels and interaction was determined to be 1.1 Paterson, 1995; St Leger et al., 1987, 1988). Sub- (Eguchi et al., 1993). AMCI-1 is able to bind pro- strate specificity and inhibition studies show that teinases from Conidiobolus and Beauveria two or- C. coronatus proteinase displays a broad specificity ders of magnitude more tightly than FPI-F, with characteristic of the fungal proteinases involved in Ka抯 of 3 the degradation of insect cuticle (Samuels and This reinforces the hypothesis, that inhibitors from Patterson, 1995). The demonstration of its ability insect hemolymph may act as protective agents to degrade insect cuticle suggests that the protein- against fungal invasions. � 10 10 M � and 4 � 9 10 M � � 10 7 M � , respectively. ase may be a virulence factor of C. coronatus. In contrast, the hemolymph of insects contains LITERATURE CITED a number of serine proteinase inhibitors (Eguchi, 1993; Polanowski et al., 1992; Polanowski and Adler-Nissen, J. 1979. of food Determination of the degree of Wilusz, 1996). Their presumed role is to protect hydrolyzis the insect against unwanted proteolysis. Some in- benzenesulfonic acid. J Agric Food Chem 27:1256�62. protein hydrolyzates by trinitro- hibitors from insect hemolymph are able to quench the activity of fungal proteinases, and are thought to constitute an important element of insect innate immunity (Hoffmann et al., 1996; Kanost, Babin DR, Peanasky RJ, Goos SM. 1984. The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: the primary structure. Arch Biochem Biophys 232:143�1. 1999). To date, there exist a very small number of Bania J, Stachowiak D, Polanowski A. 1999. Primary struc- reports describing the inhibition of fungal protein- ture and properties of the cathepsin G/chymotrypsin in- ases by inhibitors from insect hemolymph (Eguchi hibitor from the larval hemolymph of Apis mellifera. Eur J et al., 1993; Jiang and Kanost, 1997; Vilcinskas and Biochem 262:680�7. Wedde, 1997). Barrett AJ, Rawlings ND. 1991. Types and families of endopep- Previously, we purified and characterized AMCI- tidases. Biochem Soc Trans 19:707�5. 1, chymotrypsin, and cathepsin G inhibitor from the hemolymph of the honeybee, Apis mellifera Bidochka MJ, Khachatourians GG. 1994. Protein hydrolysis (Bania et al., 1999). AMCI-1 is comprised of 56 in grasshopper cuticles by entomopathogenic fungal ex- amino acids and has five disulfide bridges, as was tracellular proteases J Invert Pathol 63:7�. predicted from amino-acid sequence analysis and confirmed by determination of its molecular structure (Cierpicki et al., 2000). The P1 position in Bogard WC, Kato I, Laskowski M. 1980. A Ser162/Gly162 polymorphism in Japanese quail ovomucoid. J Biol Chem 255:6569�74. the reactive center of AMCI-1 is occupied by a methionine residue. The search for sequence homol- Chase T, Shaw E. 1969. Comparison of the esterase activities ogy showed that AMCI-1 belongs to the Ascaris of trypsin, plasmin, and thrombin on guanidinobenzoate family of serine proteinase inhibitors (Babin et al., 1984). It tightly inhibits chymotrypsin and, to a lesser extent, cathepsin G. esters. Titration of the enzymes. Biochemistry 8:2212� 2224. Cierpicki T, Bania J, Otlewski J. 2000. NMR solution struc- In this report, we have shown that AMCI-1 is ture of Apis mellifera chymotrypsin/cathepsin G inhibitor- very active against proteinase from C. coronatus and 1 (AMCI-1): structural similarity with Ascaris protease Beauveria bassiana. The association constants of inhibitors. Protein Sci 9:976�4. Archives of Insect Biochemistry and Physiology August 2006 doi: 10.1002/arch. 196 Bania et al. Cole SCJ, Charnley AK, Cooper RM. 1993. Purification and partial characterization of a novel trypsin-like cysteine pro- nism for physiological regulation of conidial discharge in Conidiobolus coronatus. Eur J Biochem 205:679�6. tease from Metarhizium anisopliae. FEMS Microb Lett 113:189�6. Phadatare SU, Deshpande VV, Srinivasan MC. 1993. High activity alkaline protease from Conidiobolus coronatus (NCL Eguchi M. 1993. Protein protease inhibitors in insects and comparison with mammalian inhibitors. Comp Biochem 86.8.20): enzyme production and compatibility with commercial detergents. Enzyme Microb Technol 15:72�. Physiol 105B:449�6. Polanowski A, Wilusz T. 1996. Serine proteinase inhibitors Eguchi M, Itoh M, Chou L, Nishino K. 1993. Purification and characterization of a fungal protease specific protein inhibitor (FPI-F) in the silkworm haemolymph. Comp Biochem Physiol 104B:537�3. Empie MW, Laskowski M. 1982. Thermodynamics and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor interactions with serine proteinases. Biochemistry 21:2274�84. from insect hemolyph. Acta Biochim Polon 43:445�4. Polanowski A, Wilusz T, Blum MS, Escoubas P, Schmidt JO, Travis J. 1992. Serine proteinase inhibitor profiles in the hemolymph of a wide range of insect species. 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