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A double headed serine proteinase inhibitor — human plasma kallikrein and elastase inhibitor — from Boophilus microplus larvae
Immunopharmacology 45 Ž1999. 171–177 www.elsevier.comrlocaterimmpharm
A double headed serine proteinase inhibitor — human plasma kallikrein and elastase inhibitor — from Boophilus microplus larvae Aparecida S. Tanaka a,) , Renato Andreotti b, Alberto Gomes b, Ricardo J.S. Torquato a , Misako U. Sampaio a , Claudio A.M. Sampaio a
a
Departamento de Bioquımica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020, Sao Paulo, SP, Brazil ´ b EMBRAPA-CNPGC, C. Grande, MS, Brazil Accepted 4 March 1999
Abstract Preying on cattle, the hard tick Boophilus microplus causes heavy economical losses to Brazil. Tick proteins are a good target to be used as tools for tick control. Serine protease inhibitors from B. microplus larvae ŽBmTI. were preliminarily characterized. One-week-old larvae were the source of a 2% protein solution in 5 mM Tris–HCl, 20 mM NaCl, pH 7.4. The inhibitors were purified by affinity chromatography on trypsin-Sepharose, and ion-exchange chromatography on Resource Q column, and they separated in two major active peaks, corresponding to 10-kDa and 18-kDa proteins ŽBmTI-B and BmTI-A, respectively.. Both purified proteins inhibited trypsin with K i of 0.3 and 3.0 nM, respectively, but only the 18-kDa protein inhibited elastase Ž K i 1.4 nM. and plasma kallikrein Ž K i 120 nM.. BmTI-A did not change prothrombin time ŽPT. and thrombin time ŽTT., but it increased activated partial thromboplastin time ŽAPTT. was dose-dependent. The partial amino acid sequence indicated that BmTI-A belongs to the bovine pancreatic trypsin inhibitor ŽBPTI.-Kunitz type inhibitor family. These inhibitors Žby their properties. play a role in the feeding process of the tick. Development of antibodies against these proteins may be used to impair the normal feeding and consequently, the parasite would be no longer viable. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Serine proteinase inhibitors; Tick; Blood coagulation; APTT
1. Introduction The tick Ž Boophilus microplus . is an important cattle parasite in South and Central America, Australia, Asia and Africa. One cause of death in cattle is the heavy infestation by ticks, and the transmission
)
Corresponding author. Tel: q55-11-576-4444; fax: q55-11572-3006; e-mail: [email protected]
of diseases such as anaplasmosis and babesiosis. Tick control can be achieved by application of chemical products, but the development of resistance to many acaricides has created problems in this approach ŽRoulstan et al., 1981.. Cattle acquire partial immunity to the ectoparasite after extensive natural exposure, due largely to an immediate hypersensitivity reaction to the tick which is, nevertheless, unable to prevent serious losses in cattle production ŽRodrıguez et al., 1994.. Recently tick control has ´
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 0 7 4 - 0
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A.S. Tanaka et al.r Immunopharmacology 45 (1999) 171–177
been done by the use of natural components, which created immunological resistance by cattle to the tick. This resistance is acquired after an extended exposure to the tick and this is mainly due to cutaneous allergic reactions ŽWilladsen, 1980.. More recently, the first ectoparasite vaccine has been developed in Australia where it is commercially released under the name ‘TickGARD’. The publication of the gene sequence enabled the development of a similar vaccine in Cuba ŽWilladsen et al., 1995.. The vaccine active antigen, which was named Bm86 for the species of origin and the year of the first identification, is an 89-kDa glycoprotein with an extracellular location on the digest cells of the tick gut ŽGough and Kemp, 1993.. The identification and characterization of other protective antigens can be important in developing recombinant vaccines having long term protection. It is possible that a vaccine containing more than one antigen can elicit a cooperative effect on protection ŽWilladsen, 1990.. Following this idea, the biochemical function of many molecules have been characterized in order to understand the complex interactions which occur between parasites and their hosts. Several proteinaceous components, including proteinase inhibitors present in tick eggs and larvae have been described ŽWilladsen and Riding, 1980; Willadsen and McKenna, 1983.. A part of this activity was connected with toxicity observed in guinea pigs ŽVermeulen et al., 1988.. The eggs and unfeeding larvae of the ectoparasite, B. Microplus, contain at least two proteolytic enzyme inhibitors which inhibit trypsin andror chymotrypsin ŽVermeulen et al., 1988.. Considering the versatility of these inhibitors present in the various stages of tick, it would be interesting to determine the primary structure of the trypsin inhibitors to understand the mechanism of inhibitor–enzyme interaction and their possible role in the protection of the larvae against host proteases. The presence of many proteinase inhibitors in the larvae of B. microplus was a problem in the early stages of this work but this communication focuses on the plasma kallikrein and elastase inhibitor, which interferes in blood coagulation. The plasma kallikrein is a serine protease which participates in the intrinsic pathway of blood coagulation and also produces bradykinin from kininogen. Bradykinin is an important cell-independent edemapromoting substance. The edema can be the signifi-
cant component in tick rejection reactions ŽRibeiro, 1989.. The present work describes the purification, characterization and determination of the partial primary structure of a serine proteinase inhibitor present in the larvae of B. microplus ŽBmTI-A., which is a member of the bovine pancreatic trypsion inhibitor ŽBPTI.-Kunitz type family. It inhibits trypsin, elastase and human plasma kallikrein. It also interferes in the blood coagulation time by increasing the activated partial thromboplastin time ŽAPTT.. 2. Materials and methods 2.1. Materials Bovine trypsin ŽEC 3.4.21.4., bovine thrombin ŽEC 3.4.21.5. and N-a-benzoyl-D,L-arginyl-p-nitroanilide ŽBz-Arg-pNA. were obtained from Sigma ŽSt. Louis, MO, USA.; a-chymotrypsin was from Worthington Biochemical ŽNJ, USA.; plasmin, Hageman factor Žfactor XIIa., S 2302 ŽH-D-Pro-PheArg-pNA., S 2251 ŽH-D-Val-Leu-Lys-pNA., S2266 ŽH-D-Val-Phe-Arg-pNA. were purchased from Chromogenix ŽMolndal, Sweden.; human plasma ¨ kallikrein ŽEC 3.4.32.34. was purified by affinity chromatography as described previously ŽSampaio et al., 1974; Oliva et al., 1982; Giusti et al., 1988.. Dr. L. Juliano from the Department of Biophysics ŽEPM. kindly donated the substrates Suc-Phe-pNA and AcPhe-Arg-pNA. Factor XIIa deficient plasma was from Baxter-Dade ŽDeerfield, USA.; Silimat Žbuffered suspension of cephalin and micronized silica. was obtained from BioMerieux-Biolab ŽMarcy-l’Etoile, France. and replastin Žthromboplastin. was purchased from Labtest Diagnostica ŽMG, Brazil.. Columns: Resource Q or High trap Q, Superose 12 and PD-10 ŽSephadex G-25. were purchased from Pharmacia. 2.2. B. microplus serine proteinase inhibitor purification Larvae ticks Ž1.13 g. were grinded in a mortar with 5 mM Tris–HCl buffer ŽpH 7.4. contains 20 mM NaCl Ž50 ml.. The resulting homogenate was centrifuged at 10 000 = g for 30 min in a centrifuge ŽHitachi model SCR20B.. The resultant supernatant Ž42 ml. was filtrated using the 0.45-mM filter ŽMilli-
A.S. Tanaka et al.r Immunopharmacology 45 (1999) 171–177
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2.3. Isolation of the BmTI by ion exchange chromatography on high trap Q The active material from trypsin-Sepharose was applied to a High Trap Q column and the bound inhibitors were eluted by a linear NaCl gradient Ž0–0.8 M.. The isolated BmTI-A and BmTI-B was stored at y208C. 2.4. SDS-PAGE SDS polyacrylamide gel Ž15%. electrophoresis in the presence and absence of dithiotreitol was carried out as described by Laemmli Ž1970.. 2.5. Assay of trypsin inhibitory actiÕity
Fig. 1. Isolation and characterization of BmTI forms. ŽA. Ion exchange chromatography on High Trap Q of BmTIs purified in trypsin-Sepharose. The inhibitors Ž ;1.5 mg. were applied to the column in buffer 0.05 M Tris–HCl, pH 8.0 and eluted with NaCl linear gradient Ž0–0.8 M.. Flow rate: 0.5 mlrmin. ŽB. SDS-PAGE Ž15%.. Lanes: 1, inhibitors purified by trypsin-Sepharose; 2, flow through proteins of High Trap Q; 3, Low Mr markers; 4, BmTI-A; 5, BmTI-B.
pore.. The filtrate Ž8 ml. was applied to trypsin-Sepharose Ž6 ml., then after washing with 0.05 M Tris–HCl buffer ŽpH 8.0., the bounded inhibitors were eluted with 0.2 M KCl solution ŽpH 2.0. and immediately neutralized with 1 M Tris–HCl buffer ŽpH 8.0.. The fractions containing the inhibitory activity from several chromatography were pooled and lyophilized. The concentrated material Ž1.8 ml. was desalted in a PD-10 column and eluted with distillate water. The active pool was used to separate the inhibitors by ion exchange chromatography on High Trap Q column ŽFPLC system..
Trypsin inhibitory activity was measured by the remaining hydrolytic activity of trypsin on the substrate N-a-benzoyl-D,L-arginyl-pNA or Ac-Phe-ArgpNA at pH 8.0 after pre-incubation with the inhibitor ŽErlanger et al., 1961.. The actual concentration of active trypsin was determined by active site titration with p-nitrophenyl-pX-guanidino-benzoate as described by Chase and Shaw Ž1970.. The other enzymes were tested using suitable chromogenic substrates and optimal conditions ŽpH, temperature and salt concentration. in each case. 2.6. Automatic sequence determination The amino acid sequences were determined on an Applied Biosystems model 477A protein gas–liquid
Table 1 Dissociation constants K i ŽnM. of BmTI-A and -B for different serine proteinases. n.i., Not inhibited Enzyme
BmTI-A
BmTI-B
Bovine trypsin Bovine chymotrypsin Human neutrophil elastase Human plasma kallikrein Human plasmin Bovine thrombin Human factor Xa Porcine pancreatic kallikrein
3.0 33 1.4 120 590 n.i. n.i. n.i.
0.3 n.i. n.i. n.i. n.i. n.i. n.i. n.i.
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sequencer. Phenylthiohydantoin ŽPTH. amino acids were identified in a model 120A PTH amino acid analyzer ŽApplied Biosystems.. 2.7. Coagulation tests — thrombin time (TT), prothrombin time (PT) and actiÕated partial thromboplastin time (APTT) The clotting tests were performed by standard procedures ŽLourenc¸o et al., 1990.. The reagents used were: replastin Ž200 ml. and silimat Ž100 ml. for PT and APTT, respectively.
3. Results and discussion B. microplus larvae have at least two forms of trypsin inhibitors, they were previously reported by Willadsen and Riding Ž1979. and Willadsen and Riding Ž1980.. They were described as double-headed inhibitors that are able to inhibit two enzyme molecules at the same time for trypsin and chymotrypsin ŽWilladsen and Riding, 1979.. In this work, they were partially purified by affinity chromatography on trypsin-Sepharose but the final separation was achieved only by ion exchange chromatography on High-trap Q column ŽFig. 1A.. The inhibitors can be divided in two groups according to
molecular masses, which were around 10 and 18 kDa, respectively ŽFig. 1B.. These results differ from the 21.9-kDa molecular mass of larval inhibitor from the same ectoparasite studied only by amino acid composition, published by Willadsen and McKenna Ž1983.. These B. microplus trypsin inhibitors ŽBmTIs. are concentrated in the eggs and in the larvae but the isolation of different forms suggests that BmTIs are at least partially stage-specific ŽWilladsen and McKenna, 1983.. The larval inhibitors are rapidly lost after the initial stages of the parasite life cycle, suggesting that they would be secreted into the host ŽWilladsen and Riding, 1980.. These evidences contend that these inhibitors must be important in the initiation of a successful feeding ŽWilladsen and McKenna, 1983.. These results encouraged us to structurally characterize one of the larval inhibitors. The inhibitor was named BmTI-A and was isolated from the other forms by High trap Q column at 0.056 M of NaCl. Its molecular mass was approximately 18 kDa in SDS-PAGE ŽFig. 1B.. The dissociation constants Ž K i . of BmTI-A to different serine proteinases are shown in Table 1. The inhibition of trypsin and neutrophil elastase was in the nM range, but BmTI-A also inhibits plasma kallikrein with K i of 120 nM ŽFig. 2A.. This result suggests that it may play a role of blocking blood coagulation during the larvae fixation on cat-
Fig. 2. Human plasma kallikrein inhibition curves. ŽA. HupK inhibition curve using BmTI-A. HupK Ž0.7 nM. was pre-incubated with different concentration of BmTI-A Ž0–1 mM. and the residual activity was detected using S2302 — HD-Pro-Phe-Arg-pNa — Ž0.2 mM., as a substrate. ŽB. Clotting time — APTT. Different concentration of BmTI-A Ž0–264 nM. was added to the standard APTT assay, and the clotting time was measured and compared to the normal plasma time Ž23.1 s..
A.S. Tanaka et al.r Immunopharmacology 45 (1999) 171–177
tle. Plasma kallikrein is a serine protease that participates in the intrinsic pathway of contact phase of blood coagulation. It is important to generate the coagulation factors to maintain the clot formation. Once the enzyme is inhibited the coagulation time will be prolonged, which can be an advantage to the larvae tick in the initial stages of the feeding process. The APTT results in the presence of BmTI-A was a prolongation in a dose-dependent curve when compared with normal plasma in the absence of inhibitor ŽFig. 2B.. This finding is a good indication that BmTI-A may play an important role in blood coagulation via plasma kallikrein inhibition since it does not inhibit thrombin and factor Xa, enzymes that
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have been targets of some blood-sucking animals. The inhibition of plasma kallikrein can also help the tick, avoiding edema formation by inhibition of local bradykinin releases ŽRibeiro, 1989.. The structural characterization of the BmTI-A purified by ion exchange chromatography was carried out after reduction and alkylation. The reduced BmTI-A was purified by reverse-phase chromatography on C18 column and the N-terminal amino acid sequence was determined. The result showed a high homology with serine proteinase inhibitors from BPTI-Kunitz type family. The reduced and alkylated inhibitor was digested with Lys-C protease. The formed peptides were separated on C18 column, and
Fig. 3. Comparison of BmTI-A partial amino acid sequence with other BPTI-Kunitz type inhibitors. BPTI, amino acid sequence according to Kassel and Laskowski Ž1965., Orn1, Ornithodorin, thrombin inhibitor Žvan de Locht et al., 1996. and Tick anticoagulant peptide ŽTAP; Waxman et al., 1990.. The arrows indicate the reactive sites. The BmTI-A amino acid sequence is underlined.
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A.S. Tanaka et al.r Immunopharmacology 45 (1999) 171–177
some of the isolated peptides were sequenced. The result of the partial amino acid sequence of BmTI is shown in Fig. 3. The BmTI-A sequence showed that it belongs to the BPTI-Kunitz type family, and has two domains explaining hypothesis of the double-headed inhibitor ŽWilladsen and Riding, 1979.. Its partial amino acid sequence also suggested, by comparison with other BPTI-Kunitz type members, the presence of residues Arg — Ala and Leu — Ala at positions P1–P1X in the reactive sites, of both the first and second domains, respectively. The sequence of BmTI is homologous to ornithodorin Žthrombin inhibitor. Žvan de Locht et al., 1996. and tick anticoagulant peptide ŽTAP; factor Xa inhibitor. ŽWaxman et al., 1990., two anticoagulant inhibitors from the same family that were also isolated from the tick, Ornithodoros moubata. Our results may suggest that these small blood-sucking animals have an ancestral gene that was specialized during evolution. Since our results are restricted to the larval stage, it is possible that adult forms have similar inhibitors with different specificity. It is clear that larvae need an efficient system to prevent blood clotting and inflammation response. The hypersensitivity in the larvae fixation site was found, so the inhibition of elastase-like enzyme may lessen this response. We can speculate that the double action of this inhibitor on both clotting and inflammatory enzymes might be an economical solution to the feeding process.
Acknowledgements We thank M.A.E. Noguti ŽDepartamento de Medicina, Disciplina de Hematologia, UNIFESPEPM, Sao ˜ Paulo, Brazil. for performing the coagulation time experiments and R. Mentele ŽAbt. fur ¨ Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt der LMU, Munchen, Germany. for amino ¨ acid sequence determination. This work was supported by FAPESP and CNPq ŽBrazil.; The Sonderforschungsbereich 207 ad 469 of Ludwig-Maximilians — Universitat ¨ Munich and the VolkswagenStiftung project Ir71045 ŽGermany..
References Chase, T., Shaw, E., 1970. Titration of trypsin, plasmin and thrombin with p-nitro-phenyl-p-guanidinobenzoate HCl. Methods Enzymol. 19, 20–27. Erlanger, B.F., Kokowsky, N., Cohen, E., 1961. Preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95, 271–278. Giusti, E.P., Sampaio, C.A.M., Michelacci, Y.M., Stella, R.C.R., Oliveira, L., Prado, E.S., 1988. Horse urinary kallikrein: I. Complete purification and characterization. Biol. Chem. Hoppe-Seyler 369, 387–396. Gough, J.M., Kemp, D.H., 1993. Localization of a low abundance membrane protein ŽBm86. on the gut cells of the cattle tick Boophilus microplus by immunogold labeling. J. Parasitol. 79, 900–907. Kassel, B., Laskowski, M., 1965. The basic inhibitor of bovine pancreas: V. The dissulfide linkages. Biochem. Biophys. Res. Commun. 20, 463–468. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680– 685. Lourenc¸o, D.M., Sampaio, M.U., Sampaio, C.A.M., 1990. Estimation of plasma kallikrein in sickle cell anemia, and its relation to the coagulation and fibrinolytic systems. Adv. Med. Exp. Biol. B 247, 553–557. Ribeiro, J.M., 1989. Role of saliva in tickrhost interactions. Exp. Appl. Acarol. 7, 15–20. Oliva, M.L.V., Grisolia, D., Sampaio, M.U., Sampaio, C.A.M., 1982. Properties of highly purified human plasma kallikrein. Agents Actions 9, 52–57. Rodrıguez, M., Rubiera, R., Penichet, M., Montesinos, R., Cre´ mata, J., Falcon, G., Bringas, R., Cordoves, ´ V., Sanchez, ´ ´ C., Valdes, ´ M., Lleonart, R., Herrera, L., la Fuente, J., 1994. High level expression of the B. Microplus Bm86 antigen in the yeast Pichia pastoris forming highly immunogenic particles for cattle. J. Biotechnol. 33, 135–146. Roulstan, W.J., Wharton, R.H., Nolan, J., Kerr, J.D., Wilson, J.T., Thompson, P.G., Schotz, M., 1981. A survey for resistance in cattle ticks to acaricides. Aust. Vet. J. 57, 362–371. Sampaio, C.A.M., Wong, S.C., Shaw, E., 1974. Human kallikrein. Purification and preliminary characterization. Arch. Biochem. Biophys. 165, 133–139. van de Locht, A., Stubbs, M., Bode, W., Friedrich, T., Bolllschweiler, C., Hoffken, W., Huber, R., 1996. The or¨ nithodorin–thrombin crystal structure, a key tho the TAP enigma?. EMBO J. 15, 6011–6017. Vermeulen, N.M.J., Viljoen, G.J., Bezuidenhout, J.D., Visser, L., Neitz, A.W.H., 1988. Kinetic properties of toxic protease inhibitors isolated from tick eggs. Int. J. Biochem. 20, 621– 631. Waxman, L., Smith, D.E., Arcuri, K.E., Vlasuk, G.P., 1990. Tick anticoagulant peptide ŽTAP. is a novel inhibitor of blood coagulation factor Xa. Science 248, 593–596. Willadsen, P., Riding, G.A., 1979. Characterization of a proteolytic enzyme inhibitor with allergenic activity. Multiple functions of a parasite-derive protein. Biochem. J. 177, 41–47.
A.S. Tanaka et al.r Immunopharmacology 45 (1999) 171–177 Willadsen, P., 1980. Immunity to ticks. Adv. Parasitol. 18, 293– 313. Willadsen, P., 1990. Perspectives for subunit vaccines for the control of ticks. Parassitologia ŽRome. 32, 195–200. Willadsen, P., McKenna, R.V., 1983. Trypsin–chymotrypsin inhibitors from the tick, Boophilus microplus. Aust. J. Exp. Biol. Med. Sci. 61, 231–238.
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Willadsen, P., Riding, G.A., 1980. On the biological role of a proteolytic-enzyme inhibitor from the ectoparasitic tick Boophilus microplus. Biochem. J. 189, 295–303. Willadsen, P., Bird, P., Cobon, G.S., Hungerford, J., 1995. Commercialisation of a recombinant vaccine against Boophilus microplus. Parasitology 110, S43–S50.
A double headed serine proteinase inhibitor — human plasma kallikrein and elastase inhibitor — from Boophilus microplus larvae Aparecida S. Tanaka a,) , Renato Andreotti b, Alberto Gomes b, Ricardo J.S. Torquato a , Misako U. Sampaio a , Claudio A.M. Sampaio a
a
Departamento de Bioquımica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020, Sao Paulo, SP, Brazil ´ b EMBRAPA-CNPGC, C. Grande, MS, Brazil Accepted 4 March 1999
Abstract Preying on cattle, the hard tick Boophilus microplus causes heavy economical losses to Brazil. Tick proteins are a good target to be used as tools for tick control. Serine protease inhibitors from B. microplus larvae ŽBmTI. were preliminarily characterized. One-week-old larvae were the source of a 2% protein solution in 5 mM Tris–HCl, 20 mM NaCl, pH 7.4. The inhibitors were purified by affinity chromatography on trypsin-Sepharose, and ion-exchange chromatography on Resource Q column, and they separated in two major active peaks, corresponding to 10-kDa and 18-kDa proteins ŽBmTI-B and BmTI-A, respectively.. Both purified proteins inhibited trypsin with K i of 0.3 and 3.0 nM, respectively, but only the 18-kDa protein inhibited elastase Ž K i 1.4 nM. and plasma kallikrein Ž K i 120 nM.. BmTI-A did not change prothrombin time ŽPT. and thrombin time ŽTT., but it increased activated partial thromboplastin time ŽAPTT. was dose-dependent. The partial amino acid sequence indicated that BmTI-A belongs to the bovine pancreatic trypsin inhibitor ŽBPTI.-Kunitz type inhibitor family. These inhibitors Žby their properties. play a role in the feeding process of the tick. Development of antibodies against these proteins may be used to impair the normal feeding and consequently, the parasite would be no longer viable. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Serine proteinase inhibitors; Tick; Blood coagulation; APTT
1. Introduction The tick Ž Boophilus microplus . is an important cattle parasite in South and Central America, Australia, Asia and Africa. One cause of death in cattle is the heavy infestation by ticks, and the transmission
)
Corresponding author. Tel: q55-11-576-4444; fax: q55-11572-3006; e-mail: [email protected]
of diseases such as anaplasmosis and babesiosis. Tick control can be achieved by application of chemical products, but the development of resistance to many acaricides has created problems in this approach ŽRoulstan et al., 1981.. Cattle acquire partial immunity to the ectoparasite after extensive natural exposure, due largely to an immediate hypersensitivity reaction to the tick which is, nevertheless, unable to prevent serious losses in cattle production ŽRodrıguez et al., 1994.. Recently tick control has ´
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 0 7 4 - 0
172
A.S. Tanaka et al.r Immunopharmacology 45 (1999) 171–177
been done by the use of natural components, which created immunological resistance by cattle to the tick. This resistance is acquired after an extended exposure to the tick and this is mainly due to cutaneous allergic reactions ŽWilladsen, 1980.. More recently, the first ectoparasite vaccine has been developed in Australia where it is commercially released under the name ‘TickGARD’. The publication of the gene sequence enabled the development of a similar vaccine in Cuba ŽWilladsen et al., 1995.. The vaccine active antigen, which was named Bm86 for the species of origin and the year of the first identification, is an 89-kDa glycoprotein with an extracellular location on the digest cells of the tick gut ŽGough and Kemp, 1993.. The identification and characterization of other protective antigens can be important in developing recombinant vaccines having long term protection. It is possible that a vaccine containing more than one antigen can elicit a cooperative effect on protection ŽWilladsen, 1990.. Following this idea, the biochemical function of many molecules have been characterized in order to understand the complex interactions which occur between parasites and their hosts. Several proteinaceous components, including proteinase inhibitors present in tick eggs and larvae have been described ŽWilladsen and Riding, 1980; Willadsen and McKenna, 1983.. A part of this activity was connected with toxicity observed in guinea pigs ŽVermeulen et al., 1988.. The eggs and unfeeding larvae of the ectoparasite, B. Microplus, contain at least two proteolytic enzyme inhibitors which inhibit trypsin andror chymotrypsin ŽVermeulen et al., 1988.. Considering the versatility of these inhibitors present in the various stages of tick, it would be interesting to determine the primary structure of the trypsin inhibitors to understand the mechanism of inhibitor–enzyme interaction and their possible role in the protection of the larvae against host proteases. The presence of many proteinase inhibitors in the larvae of B. microplus was a problem in the early stages of this work but this communication focuses on the plasma kallikrein and elastase inhibitor, which interferes in blood coagulation. The plasma kallikrein is a serine protease which participates in the intrinsic pathway of blood coagulation and also produces bradykinin from kininogen. Bradykinin is an important cell-independent edemapromoting substance. The edema can be the signifi-
cant component in tick rejection reactions ŽRibeiro, 1989.. The present work describes the purification, characterization and determination of the partial primary structure of a serine proteinase inhibitor present in the larvae of B. microplus ŽBmTI-A., which is a member of the bovine pancreatic trypsion inhibitor ŽBPTI.-Kunitz type family. It inhibits trypsin, elastase and human plasma kallikrein. It also interferes in the blood coagulation time by increasing the activated partial thromboplastin time ŽAPTT.. 2. Materials and methods 2.1. Materials Bovine trypsin ŽEC 3.4.21.4., bovine thrombin ŽEC 3.4.21.5. and N-a-benzoyl-D,L-arginyl-p-nitroanilide ŽBz-Arg-pNA. were obtained from Sigma ŽSt. Louis, MO, USA.; a-chymotrypsin was from Worthington Biochemical ŽNJ, USA.; plasmin, Hageman factor Žfactor XIIa., S 2302 ŽH-D-Pro-PheArg-pNA., S 2251 ŽH-D-Val-Leu-Lys-pNA., S2266 ŽH-D-Val-Phe-Arg-pNA. were purchased from Chromogenix ŽMolndal, Sweden.; human plasma ¨ kallikrein ŽEC 3.4.32.34. was purified by affinity chromatography as described previously ŽSampaio et al., 1974; Oliva et al., 1982; Giusti et al., 1988.. Dr. L. Juliano from the Department of Biophysics ŽEPM. kindly donated the substrates Suc-Phe-pNA and AcPhe-Arg-pNA. Factor XIIa deficient plasma was from Baxter-Dade ŽDeerfield, USA.; Silimat Žbuffered suspension of cephalin and micronized silica. was obtained from BioMerieux-Biolab ŽMarcy-l’Etoile, France. and replastin Žthromboplastin. was purchased from Labtest Diagnostica ŽMG, Brazil.. Columns: Resource Q or High trap Q, Superose 12 and PD-10 ŽSephadex G-25. were purchased from Pharmacia. 2.2. B. microplus serine proteinase inhibitor purification Larvae ticks Ž1.13 g. were grinded in a mortar with 5 mM Tris–HCl buffer ŽpH 7.4. contains 20 mM NaCl Ž50 ml.. The resulting homogenate was centrifuged at 10 000 = g for 30 min in a centrifuge ŽHitachi model SCR20B.. The resultant supernatant Ž42 ml. was filtrated using the 0.45-mM filter ŽMilli-
A.S. Tanaka et al.r Immunopharmacology 45 (1999) 171–177
173
2.3. Isolation of the BmTI by ion exchange chromatography on high trap Q The active material from trypsin-Sepharose was applied to a High Trap Q column and the bound inhibitors were eluted by a linear NaCl gradient Ž0–0.8 M.. The isolated BmTI-A and BmTI-B was stored at y208C. 2.4. SDS-PAGE SDS polyacrylamide gel Ž15%. electrophoresis in the presence and absence of dithiotreitol was carried out as described by Laemmli Ž1970.. 2.5. Assay of trypsin inhibitory actiÕity
Fig. 1. Isolation and characterization of BmTI forms. ŽA. Ion exchange chromatography on High Trap Q of BmTIs purified in trypsin-Sepharose. The inhibitors Ž ;1.5 mg. were applied to the column in buffer 0.05 M Tris–HCl, pH 8.0 and eluted with NaCl linear gradient Ž0–0.8 M.. Flow rate: 0.5 mlrmin. ŽB. SDS-PAGE Ž15%.. Lanes: 1, inhibitors purified by trypsin-Sepharose; 2, flow through proteins of High Trap Q; 3, Low Mr markers; 4, BmTI-A; 5, BmTI-B.
pore.. The filtrate Ž8 ml. was applied to trypsin-Sepharose Ž6 ml., then after washing with 0.05 M Tris–HCl buffer ŽpH 8.0., the bounded inhibitors were eluted with 0.2 M KCl solution ŽpH 2.0. and immediately neutralized with 1 M Tris–HCl buffer ŽpH 8.0.. The fractions containing the inhibitory activity from several chromatography were pooled and lyophilized. The concentrated material Ž1.8 ml. was desalted in a PD-10 column and eluted with distillate water. The active pool was used to separate the inhibitors by ion exchange chromatography on High Trap Q column ŽFPLC system..
Trypsin inhibitory activity was measured by the remaining hydrolytic activity of trypsin on the substrate N-a-benzoyl-D,L-arginyl-pNA or Ac-Phe-ArgpNA at pH 8.0 after pre-incubation with the inhibitor ŽErlanger et al., 1961.. The actual concentration of active trypsin was determined by active site titration with p-nitrophenyl-pX-guanidino-benzoate as described by Chase and Shaw Ž1970.. The other enzymes were tested using suitable chromogenic substrates and optimal conditions ŽpH, temperature and salt concentration. in each case. 2.6. Automatic sequence determination The amino acid sequences were determined on an Applied Biosystems model 477A protein gas–liquid
Table 1 Dissociation constants K i ŽnM. of BmTI-A and -B for different serine proteinases. n.i., Not inhibited Enzyme
BmTI-A
BmTI-B
Bovine trypsin Bovine chymotrypsin Human neutrophil elastase Human plasma kallikrein Human plasmin Bovine thrombin Human factor Xa Porcine pancreatic kallikrein
3.0 33 1.4 120 590 n.i. n.i. n.i.
0.3 n.i. n.i. n.i. n.i. n.i. n.i. n.i.
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sequencer. Phenylthiohydantoin ŽPTH. amino acids were identified in a model 120A PTH amino acid analyzer ŽApplied Biosystems.. 2.7. Coagulation tests — thrombin time (TT), prothrombin time (PT) and actiÕated partial thromboplastin time (APTT) The clotting tests were performed by standard procedures ŽLourenc¸o et al., 1990.. The reagents used were: replastin Ž200 ml. and silimat Ž100 ml. for PT and APTT, respectively.
3. Results and discussion B. microplus larvae have at least two forms of trypsin inhibitors, they were previously reported by Willadsen and Riding Ž1979. and Willadsen and Riding Ž1980.. They were described as double-headed inhibitors that are able to inhibit two enzyme molecules at the same time for trypsin and chymotrypsin ŽWilladsen and Riding, 1979.. In this work, they were partially purified by affinity chromatography on trypsin-Sepharose but the final separation was achieved only by ion exchange chromatography on High-trap Q column ŽFig. 1A.. The inhibitors can be divided in two groups according to
molecular masses, which were around 10 and 18 kDa, respectively ŽFig. 1B.. These results differ from the 21.9-kDa molecular mass of larval inhibitor from the same ectoparasite studied only by amino acid composition, published by Willadsen and McKenna Ž1983.. These B. microplus trypsin inhibitors ŽBmTIs. are concentrated in the eggs and in the larvae but the isolation of different forms suggests that BmTIs are at least partially stage-specific ŽWilladsen and McKenna, 1983.. The larval inhibitors are rapidly lost after the initial stages of the parasite life cycle, suggesting that they would be secreted into the host ŽWilladsen and Riding, 1980.. These evidences contend that these inhibitors must be important in the initiation of a successful feeding ŽWilladsen and McKenna, 1983.. These results encouraged us to structurally characterize one of the larval inhibitors. The inhibitor was named BmTI-A and was isolated from the other forms by High trap Q column at 0.056 M of NaCl. Its molecular mass was approximately 18 kDa in SDS-PAGE ŽFig. 1B.. The dissociation constants Ž K i . of BmTI-A to different serine proteinases are shown in Table 1. The inhibition of trypsin and neutrophil elastase was in the nM range, but BmTI-A also inhibits plasma kallikrein with K i of 120 nM ŽFig. 2A.. This result suggests that it may play a role of blocking blood coagulation during the larvae fixation on cat-
Fig. 2. Human plasma kallikrein inhibition curves. ŽA. HupK inhibition curve using BmTI-A. HupK Ž0.7 nM. was pre-incubated with different concentration of BmTI-A Ž0–1 mM. and the residual activity was detected using S2302 — HD-Pro-Phe-Arg-pNa — Ž0.2 mM., as a substrate. ŽB. Clotting time — APTT. Different concentration of BmTI-A Ž0–264 nM. was added to the standard APTT assay, and the clotting time was measured and compared to the normal plasma time Ž23.1 s..
A.S. Tanaka et al.r Immunopharmacology 45 (1999) 171–177
tle. Plasma kallikrein is a serine protease that participates in the intrinsic pathway of contact phase of blood coagulation. It is important to generate the coagulation factors to maintain the clot formation. Once the enzyme is inhibited the coagulation time will be prolonged, which can be an advantage to the larvae tick in the initial stages of the feeding process. The APTT results in the presence of BmTI-A was a prolongation in a dose-dependent curve when compared with normal plasma in the absence of inhibitor ŽFig. 2B.. This finding is a good indication that BmTI-A may play an important role in blood coagulation via plasma kallikrein inhibition since it does not inhibit thrombin and factor Xa, enzymes that
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have been targets of some blood-sucking animals. The inhibition of plasma kallikrein can also help the tick, avoiding edema formation by inhibition of local bradykinin releases ŽRibeiro, 1989.. The structural characterization of the BmTI-A purified by ion exchange chromatography was carried out after reduction and alkylation. The reduced BmTI-A was purified by reverse-phase chromatography on C18 column and the N-terminal amino acid sequence was determined. The result showed a high homology with serine proteinase inhibitors from BPTI-Kunitz type family. The reduced and alkylated inhibitor was digested with Lys-C protease. The formed peptides were separated on C18 column, and
Fig. 3. Comparison of BmTI-A partial amino acid sequence with other BPTI-Kunitz type inhibitors. BPTI, amino acid sequence according to Kassel and Laskowski Ž1965., Orn1, Ornithodorin, thrombin inhibitor Žvan de Locht et al., 1996. and Tick anticoagulant peptide ŽTAP; Waxman et al., 1990.. The arrows indicate the reactive sites. The BmTI-A amino acid sequence is underlined.
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some of the isolated peptides were sequenced. The result of the partial amino acid sequence of BmTI is shown in Fig. 3. The BmTI-A sequence showed that it belongs to the BPTI-Kunitz type family, and has two domains explaining hypothesis of the double-headed inhibitor ŽWilladsen and Riding, 1979.. Its partial amino acid sequence also suggested, by comparison with other BPTI-Kunitz type members, the presence of residues Arg — Ala and Leu — Ala at positions P1–P1X in the reactive sites, of both the first and second domains, respectively. The sequence of BmTI is homologous to ornithodorin Žthrombin inhibitor. Žvan de Locht et al., 1996. and tick anticoagulant peptide ŽTAP; factor Xa inhibitor. ŽWaxman et al., 1990., two anticoagulant inhibitors from the same family that were also isolated from the tick, Ornithodoros moubata. Our results may suggest that these small blood-sucking animals have an ancestral gene that was specialized during evolution. Since our results are restricted to the larval stage, it is possible that adult forms have similar inhibitors with different specificity. It is clear that larvae need an efficient system to prevent blood clotting and inflammation response. The hypersensitivity in the larvae fixation site was found, so the inhibition of elastase-like enzyme may lessen this response. We can speculate that the double action of this inhibitor on both clotting and inflammatory enzymes might be an economical solution to the feeding process.
Acknowledgements We thank M.A.E. Noguti ŽDepartamento de Medicina, Disciplina de Hematologia, UNIFESPEPM, Sao ˜ Paulo, Brazil. for performing the coagulation time experiments and R. Mentele ŽAbt. fur ¨ Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt der LMU, Munchen, Germany. for amino ¨ acid sequence determination. This work was supported by FAPESP and CNPq ŽBrazil.; The Sonderforschungsbereich 207 ad 469 of Ludwig-Maximilians — Universitat ¨ Munich and the VolkswagenStiftung project Ir71045 ŽGermany..
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