Veterinary Parasitology, 45 (1992) 133-140 Elsevier Science Publishers B.V., Amsterdam
133
Proteolytic enzymes from Trichinella spiralis larvae A. Criado-Fornelio, C. de Armas-Serra, C. Gim6nez-Pardo, N. CasadoEscribano, A. Jim6nez-G6nzalez and F. Rodriguez-Caabeiro Parasitology Laboratory, Faculty of Pharmacy, Universityof Alcald de Henares, 28871 Alcala de Henares, Madrid, Spain (Accepted 7 May 1992)
ABSTRACT Criado-Fornelio, A., de Armas-Serra, C., Gim6nez-Pardo, C., Casado-Escribano, N., Jimdnez-G6nzalez, A. and Rodriguez-Caabeiro, F., 1992. Proteolytic enzymes from Trichinella spiralis larvae. Vet. Parasitol.,45: 133-140.
Trichinella spiralis larvae infect their hosts by the penetration of small intestine enterocytes. The exact mechanism of penetration is unknown, but the presence of proteolytie enzymes is suspected. In this study, whole worm extracts and excretory-secretory (ES) components were obtained and their proteolytic enzymes examined. Enzymes from worm extracts were capable of hydrolysing azocoll, a general protease substrate in a wide range of pH (2-8), with maximal activity at pH 5. Trichinella spiralis larval enzymes were sensitive to metalloprotease and serine protease inhibitors. Three proteases were identified in worm extracts at molecular weight (MW) 48, 54 and 62 kDa by incorporating a gelatine substrate into a standard or a modified sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) set-up, in which we used low SDS concentration in gel and electrophoresis buffer (0.01%). Intact larvae incubated in a medium containing azocoll showed azocollytic activity. Subsequent analysis of ES products by modified SDS-PAGE in gels containinggelatine demonstrated the presence of three protease of apparent MW 33, 62 and 230 kDa.
INTRODUCTION
Although the histopathology of trichinellosis has been described in detail (see review by Weatherly, 1983 ), little is known about the biochemical mechanisms of invasion of the host tissue. The detection of a proteinase in worm extracts has been reported in Trichinella spiralis (Niimura et al., 1988 ), but few details have as yet become available. According to microscopical observations by Bruce (1970), T. spiralis larvae do not possess a buccal stylet, so Correspondence to: A. Criado-Fornelio, Parasitology Laboratory, Faculty of Pharmacy, University of Alcal~ide Henares, 28871 Alcala de Henares, Madrid, Spain.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
134
A. CRIADO-FORNELIO ET AL.
that it seems reasonable to assume that tissue-degrading proteases may be elaborated by the parasite to facilitate larval penetration of host enterocytes. This assumption is more likely if we consider that other nematodes, such as Anisakis simplex, have proteases that are important factors in facilitating the penetration of the stomach and intestinal walls of their hosts (Sakanari and McKerrow, 1990). For these reasons, we have undertaken some work to detect the presence of proteinases in extracts and excretory-secretory products (ES) of T. spiralis larvae. MATERIALS AND METHODS
Parasite In this investigation muscle-stage larvae of T. spiralis (GM-1 isolate) were used. Larvae were isolated by the standard pepsin digestion method (Brand et al., 1952).
Azocoll degradation assay by intact larvae Decapsulated T. spiralis larvae (0.1 ml) were incubated at 37°C in 0.8 ml of a modified Hanks' solution using citrate buffer (0.1 M, pH 5 ) instead of phosphate buffer. The incubation medium also contained azocoll (2.5 mg m l - 1), penicillin ( 1 mg m l - ~), streptomycin sulphate (2 mg m l - ~) and 5fluorocytosine (0.1 mg m l - ~). Incubation was conducted in Eppendorf tubes placed in an orbital mixer at a speed of 15 rev m i n - 1 for 20 h. After incubation the tubes were centrifuged at 1000×g for 5 min and the supernatant removed for spectrophotometric measurements. Control medium without larvae was used as the blank. Further details on colorimetric assays are described under the section Proteinase assays and inhibition studies.
Collection of ES products from larvae Secreted proteins from larvae were obtained from the medium used in the azocoll degradation assay after 20 h of incubation. The medium was concentrated using a Centricon-10 protein concentrator (Amicon, Beverly, MA) with a size exclusion of MW 10 000 and used immediately for electrophoretic analysis.
Proteinase assays and inhibition studies Muscle-stage larvae were homogenised in 0.2 M phosphate buffer (pH 7.2 ) and centrifuged at 100 0 0 0 × g for 30 rain. The supernatant was recovered
PROTEOLYTIC ENZYMES FROM TRICHINELLA SPIRALIS LARVAE
135
and recentrifuged. The supernatant protein content was determined according to Lowry et al. ( 1951 ) and the protein concentration in the crude extract was adjusted to 6 mg ml-~ by appropriate dilution with homogenisation buffer. Crude extracts were used subsequently or frozen at -70°C. Proteinase assays were colorimetric measurements of hydrolysis of azo-dye-coupledhide powder (azocoll) as described by Monroy et al. ( 1989 ). Incubation time was 24 h in assays. The following buffers were used: 0.1 M citric acid/sodium citrate, pH 3, 4 and 5, 0.1 M phosphate-buffered saline (PBS), pH 6, 7 and 8. Proteolytic activity was also measured in the presence of the following inhibitors: phenylmethylsulphonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), chymostatin, sodium dodecyl sulphate (SDS), soybean trypsin inhibitor (STI), MgC12, leupeptin and pepstatin A. All of these inhibitors were dissolved in water except PMSF and pepstatin A, which were solubilised in dimethyl sulphoxide (DMSO).
Substrate gel electrophores& In a first series of experiments, we tried an electrophoretic analysis of T.
spiralis larvae extracts or ES products in gelatin-containing polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-gelatin-PAGE) as described by Sakanari and McKerrow (1990). Detection of proteolytic activity in these conditions was difficult, as weak protease bands were observed in some gels and others showed no activity at all. Suspecting possible SDS inhibition ofproteolytic activity, we changed electrophoresis conditions, using no SDS or reducing the SDS concentration (in gel and electrophoresis buffer) to 0.01% in order to facilitate the detection of proteolytic bands in gels. Electrophoretic analysis was conducted in polyacrylamide gels copolymerisedwith a gelatin substrate (the final gelatin concentration in gels was 0.15%). Samples were diluted 3:1 in sample buffer and electrophoresed in a Bio-Rad Mini Protean apparatus at 50 V per gel. Afterwards, gels were washed in 2.5% Triton X-100 for 1 h to remove SDS, incubated in 0.1 M citrate buffer, pH 5, 2 mM CaCI2 for 24 h and stained with Coomasie blue. After destaining, proteases were located as clear bands in a blue gel.
Chemicals Azocoll, gelatin, proteinase inhibitors and electrophoresis calibration kits were purchased from Sigma (St. Louis, MO). All other chemicals were of analytical grade.
136
A. CRIADO-FORNELIOET AL.
RESULTS
The colorimetric assay using azocoll as substrate has been used for detection of proteinases in T. spiralis crude extracts. Azocoll hydrolysis was found over a relatively broad pH range (Fig. 1 ). Maximal activity was obtained at pH 5. Intact larvae incubated in an azocoll-containing m e d i u m at pH 5 showed a strong azocollytic activity, degrading 35.1% of the azocoll present in the m e d i u m after 20 h of incubation. Electrophoresis of T. spiralis crude extracts and ES products under non-
100
3am
~-.
50
,q
I,-,.,.i
0
I
I
2
4
I
i
6
I
I
8
pH
Fig. l. pH activity curves of T. spiralis larvae proteases on azocoll (measured in crude extracts). Each point represents the mean of three experiments. The point of maximal azocoll hydrolysis was taken as 100%. TABLE1 The effect of inhibitors on T. spiralis proteases measured in crude extracts. Assays were conducted at pH 5, except in SDS experiments, performed at pH 3. All of the experiments were repeated at least three times. Results are expressed as a percentage of reduction in absorbanee compared with an inhibitor-free control Inhibitor
Concentration
Inhibition (%)
SDS Leupeptin Pepstatin A PMSF EDTA MgC12 STI Chymostatin DMSO
0.1% 100/zM 1 mM 5 mM 5 mM 10 mM 50/lg m l - l 500/gg m l - ~ 0.5%
100 5.6 12.9 60.1 39.5 34.0 3.1 68.5 53.1
Abbreviations: SDS, sodium dodecyl sulphate; PMSF, phenylmethylsulphonyl fluoride; EDTA, ethylenediaminetetraacetic acid; STI, soybean trypsin inhibitor; DMSO, dimethyl sulphoxide.
137
PROTEOLYTIC ENZYMES FROM TRICHINELLA SPIRALIS LARVAE
MW kDa
2A
MW kDa
2B
27266-
66-
45-
45-
29-
Fig. 2. Electrophoretic analysis of T. spiralis proteases in polyacrylamide gels. Gels were incubated for 24 h at 37 °C. Molecular weight markers are indicated. (A) Worm extract sample (50 #g of protein) run in a 7.5% gel containing gelatin. No SDS was present in gel or electrophoresis buffer. (B) ES products sample (60 #g of protein) run in an 11% gel containing gelatin. SDS concentration in gel and electrophoresisbuffer was 0.01%.
reducing conditions and low SDS concentration in buffer and gel (0.01%) revealed several proteinases. Crude extract proteinases showed apparent molecular weights ( M W ) o f 48, 54 and 62 kDa (Fig. 2A). In ES products we found three proteases at M W 33, 62 and 230 kDa (Fig. 2B). No additional bands could be observed by ovedoading the gels with samples or by changes in the acrylamide percentage o f the gels. Trials for detection o f proteolytic activity in electrophoresis performed with 0.1% SDS in buffer and gels led to poor results, as proteases generally showed low or no activity at all, even when gels were overloaded with samples. These data suggested that proteases were not totally resistant to SDS. Studies on the inhibition o f azocoll degradation by SDS and other inhibitors were carried out using w o r m crude extracts. The results obtained are shown in Table 1. In standard assays at pH 5, protease
138
A. CRIADO-FORNELIOET AL.
activity of T. spiralis extracts was decreased by DMSO, EDTA, MgClz, PMSF, chymostatin and pepstatin A. In inhibition assays at pH 3 with SDS (0.1% w / v ) a strong inhibition ofproteolytic activity was observed. DISCUSSION
Significant proteolytic activity was observed in T. spiralis extracts at pH 3, 4 and 5. As azocoll assay measures net proteolytic activity of the extract, this result may reflect the presence of multiple proteases, each with its own optimum pH. Trichinella spiralis proteases have an optimum pH of 5, which is similar to that of the proximal portion of the small intestine, where most of the adults have been found in experimental infections in mice (De ArmasSerra et al., 1987). This fact, together with the finding that several proteases are excreted by the larvae, and the absence of an oral styler (Bruce, 1970) may be indicative of their function as lytic enzymes used to facilitate larval penetration into host enterocytes. Niimura et al. ( 1988 ) pointed out that a 160 kDa molecule obtained from T. spiralis extracts was active as a proteinase at pH 4, but further details have not been published. The present data did not show any proteolytic enzyme at that molecular weight, but this fact may be explained by the incubation of gels at pH 5 in our experiments instead of pH 4. Another possibility is the existence of biochemical differences between Trichinella isolates (RodriguezCaabeiro et al., 1985; La Rosa et al., 1991 ). The azocoll degradation assay demonstrated that T. spiralis larvae secreted proteases and subsequent electrophoretic analysis demonstrated their presence in ES products and worm extracts. It is interesting to note that two proteases of MW 230 and 33 kDa could only be detected in ES products. The reasons for this are unknown, but possible speculative explanations are that these enzymes are associated with the moulting process and start to be synthesised after decapsulation. Therefore, they are only detectable in ES products when they are accumulated in the medium, or the proteases are found within the worm cells as inactive forms and are "activated" when secreted out of the cells. The 48 and 54 kDa proteases, which seem to be present only in worm extracts, could merely be enzymes used for digestive functions inside the worm. Furher investigation is needed to confirm this assumption. Finally the 62 kDa enzyme, which is common to ES products and worm extracts is likely to be an enzyme used for penetration into host enterocytes, as it does not need to be activated or synthesised after decapsulation and might be quickly used for lysis of host cells membranes. Further studies will be conducted to ascertain these speculative hypotheses. In studies on the inhibition of azocollytic activity, partial inhibition was noted with DMSO, EDTA, PMSF, chymostatin, pepstatin A and MgCla, in assays conducted at pH 5. A strong inhibition was found for SDS at pH 3.
PROTEOLYTIC ENZYMES FROM TRICHINELLA SP1RALIS LARVAE
139
DMSO is a solvent widely used to test the effect of insoluble inhibitors on proteolytic activity (Dresden et al., 1984; Monroy et al., 1989). Unfortunately, DMSO showed some inhibitory action on T. spiralis proteases, so that the effects reported here for PMSF and pepstatin A (tested as a solution in DMSO) could have been obscured by interference of the solvent. Likewise, the effect of 0.1% SDS on T. spiralis proteases was difficult to assess at pH 4 and 5, because significant degradation of azocoll was observed in SDS controls without homogenate at these pHs. However, SDS did not degrade azocoll at pH 3 and, at least at this pH, our results seem to confirm that this detergent is a strong inhibitor of proteolytic activity in T. spiralis larvae. The sensitivity to the inhibitors mentioned above indicated that certain enzymes were metaUoproteases and serine proteases. The presence of serine proteases seems to be a very common feature in parasitic nematodes. They have been found in Dirofilaria immitis (Tamashiro et al., 1987), Nematospiroides dubius (Monroy et al., 1989), Onchocerca spp. (Lackey et al., 1989) and Anisakis simplex (Sakanari and McKerrow, 1990). The present studies on T. spiralis proteases have provided us with new data about the biochemical basis for penetration in their hosts. Besides their activity in penetration, however, another possible role of proteolytic enzymes is as a means of escaping the host's immune response. Dirofilaria immitis proteases have been found to cleave IgG (Tamashiro et al., 1987). Additional studies on T. spiralis proteases are underway to ascertain if proteolytic enzymes are used in worm digestion and evasion of the immune response. Finally, further investigations on inhibition of protease activity by chemicals or antibodies would be, at least theoretically, an effective trend in anthelmintic therapy. ACKNOWLEDGEMENTS This investigation received financial support from the Scientific and Technical Assessor Committee (Ministry of Education and Science, Spain), No. PM 89-0211 and from the Vicerrectorado de Investigaci6n (University of Alcal~t de Henares, Spain), No. 91 A/22. REFERENCES Brand, T., Weistein, P., Mehlan, B. and Weinbach, E., 1952. Observation on the metabolism of bacteria-free larvae of Trichinella spiralis. Exp. Parasitol., 1: 245-255. Bruce, R.G., 1970. Structure of the esophagus of the infective juvenile and adult Trichinella spiralis. J. Parasitol., 56: 540-549. De Armas-Serra, C., Martinez-Fernandez, A.R., Bolas-Fernandez, F. and Gomez-Barrio, A., 1987. A comparative study of the endogenous biological behaviour of six Trichinella isolates (in Spanish, abstract in English). Rev. Ib6r. Parasitol., Vol. extra: 153-157.
140
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Dresden, M.H., Rege, A.A. and Murrell, K.D., 1985. Strongyloides ransomi: Proteolytic enzymes from larvae. Exp. Parasitol., 59: 257-263. Lackey, A., James, E.R., Sakanari, J.A., Resnick, S.D., Brown, M., Bianco, A.E. and McKerrow, J.H., 1989. Extracellular proteases of Onchocerca. Exp. Parasitol., 68:176-185. La Rosa, G., Pozio, E. and Rossi, P., 1991. Biochemical resolution of European and African isolates of Trichinella nelsoni Britov and Boev, 1972. Parasitol. Res., 77:173-176. Lowry, O., Rosebrough, N., Farr, A. and Randall, R., 1951. Protein measurement with the Folin fenol reagent. J. Biol. Chem., 193: 265-275. Monroy, F.G., Cayzer, C.J.R., Adams, J.H. and Dobson, C., 1989. Proteolytic enzymes in excretory-secretory products from adult Nematospiroides dubius. Int. J. Parasitol., 19:129131. Niimura, M., Kobayashi, M. and Kojima, S., 1988. A mouse monoclonal antibody that binds to an alpha-stichocyte of Trichinella spiralis. Parasitol. Res., 74: 271-276. Rodriguez-Caabeiro, F., Criado-Fornelio, A. and Jimenez-Gonzalez, A., 1985. A comparative study of the succinate dehydrogenase-fumarate reductase complex in the genus Trichinella. Parasitology, 91: 577-583. Sakanari, J.A. and McKerrow, J., 1990. Identification of the secreted neutral proteases from Anisakis simplex. J. Parasitol., 76: 625-630. Tamashiro, W.L., Rao, M. and Scott, A.L., 1987. Proteolytic cleavage of IgG and other protein substrates by Dirofilaria immitis microfilarial enzymes. J. Parasitol., 73:149-154. Weatherly, N.F., 1983. Anatomical Pathology. In: W.C. Campbell (Editor), Trichinella and trichinellosis. Plenum Press, New York, pp. 173-208.
133
Proteolytic enzymes from Trichinella spiralis larvae A. Criado-Fornelio, C. de Armas-Serra, C. Gim6nez-Pardo, N. CasadoEscribano, A. Jim6nez-G6nzalez and F. Rodriguez-Caabeiro Parasitology Laboratory, Faculty of Pharmacy, Universityof Alcald de Henares, 28871 Alcala de Henares, Madrid, Spain (Accepted 7 May 1992)
ABSTRACT Criado-Fornelio, A., de Armas-Serra, C., Gim6nez-Pardo, C., Casado-Escribano, N., Jimdnez-G6nzalez, A. and Rodriguez-Caabeiro, F., 1992. Proteolytic enzymes from Trichinella spiralis larvae. Vet. Parasitol.,45: 133-140.
Trichinella spiralis larvae infect their hosts by the penetration of small intestine enterocytes. The exact mechanism of penetration is unknown, but the presence of proteolytie enzymes is suspected. In this study, whole worm extracts and excretory-secretory (ES) components were obtained and their proteolytic enzymes examined. Enzymes from worm extracts were capable of hydrolysing azocoll, a general protease substrate in a wide range of pH (2-8), with maximal activity at pH 5. Trichinella spiralis larval enzymes were sensitive to metalloprotease and serine protease inhibitors. Three proteases were identified in worm extracts at molecular weight (MW) 48, 54 and 62 kDa by incorporating a gelatine substrate into a standard or a modified sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) set-up, in which we used low SDS concentration in gel and electrophoresis buffer (0.01%). Intact larvae incubated in a medium containing azocoll showed azocollytic activity. Subsequent analysis of ES products by modified SDS-PAGE in gels containinggelatine demonstrated the presence of three protease of apparent MW 33, 62 and 230 kDa.
INTRODUCTION
Although the histopathology of trichinellosis has been described in detail (see review by Weatherly, 1983 ), little is known about the biochemical mechanisms of invasion of the host tissue. The detection of a proteinase in worm extracts has been reported in Trichinella spiralis (Niimura et al., 1988 ), but few details have as yet become available. According to microscopical observations by Bruce (1970), T. spiralis larvae do not possess a buccal stylet, so Correspondence to: A. Criado-Fornelio, Parasitology Laboratory, Faculty of Pharmacy, University of Alcal~ide Henares, 28871 Alcala de Henares, Madrid, Spain.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
134
A. CRIADO-FORNELIO ET AL.
that it seems reasonable to assume that tissue-degrading proteases may be elaborated by the parasite to facilitate larval penetration of host enterocytes. This assumption is more likely if we consider that other nematodes, such as Anisakis simplex, have proteases that are important factors in facilitating the penetration of the stomach and intestinal walls of their hosts (Sakanari and McKerrow, 1990). For these reasons, we have undertaken some work to detect the presence of proteinases in extracts and excretory-secretory products (ES) of T. spiralis larvae. MATERIALS AND METHODS
Parasite In this investigation muscle-stage larvae of T. spiralis (GM-1 isolate) were used. Larvae were isolated by the standard pepsin digestion method (Brand et al., 1952).
Azocoll degradation assay by intact larvae Decapsulated T. spiralis larvae (0.1 ml) were incubated at 37°C in 0.8 ml of a modified Hanks' solution using citrate buffer (0.1 M, pH 5 ) instead of phosphate buffer. The incubation medium also contained azocoll (2.5 mg m l - 1), penicillin ( 1 mg m l - ~), streptomycin sulphate (2 mg m l - ~) and 5fluorocytosine (0.1 mg m l - ~). Incubation was conducted in Eppendorf tubes placed in an orbital mixer at a speed of 15 rev m i n - 1 for 20 h. After incubation the tubes were centrifuged at 1000×g for 5 min and the supernatant removed for spectrophotometric measurements. Control medium without larvae was used as the blank. Further details on colorimetric assays are described under the section Proteinase assays and inhibition studies.
Collection of ES products from larvae Secreted proteins from larvae were obtained from the medium used in the azocoll degradation assay after 20 h of incubation. The medium was concentrated using a Centricon-10 protein concentrator (Amicon, Beverly, MA) with a size exclusion of MW 10 000 and used immediately for electrophoretic analysis.
Proteinase assays and inhibition studies Muscle-stage larvae were homogenised in 0.2 M phosphate buffer (pH 7.2 ) and centrifuged at 100 0 0 0 × g for 30 rain. The supernatant was recovered
PROTEOLYTIC ENZYMES FROM TRICHINELLA SPIRALIS LARVAE
135
and recentrifuged. The supernatant protein content was determined according to Lowry et al. ( 1951 ) and the protein concentration in the crude extract was adjusted to 6 mg ml-~ by appropriate dilution with homogenisation buffer. Crude extracts were used subsequently or frozen at -70°C. Proteinase assays were colorimetric measurements of hydrolysis of azo-dye-coupledhide powder (azocoll) as described by Monroy et al. ( 1989 ). Incubation time was 24 h in assays. The following buffers were used: 0.1 M citric acid/sodium citrate, pH 3, 4 and 5, 0.1 M phosphate-buffered saline (PBS), pH 6, 7 and 8. Proteolytic activity was also measured in the presence of the following inhibitors: phenylmethylsulphonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), chymostatin, sodium dodecyl sulphate (SDS), soybean trypsin inhibitor (STI), MgC12, leupeptin and pepstatin A. All of these inhibitors were dissolved in water except PMSF and pepstatin A, which were solubilised in dimethyl sulphoxide (DMSO).
Substrate gel electrophores& In a first series of experiments, we tried an electrophoretic analysis of T.
spiralis larvae extracts or ES products in gelatin-containing polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-gelatin-PAGE) as described by Sakanari and McKerrow (1990). Detection of proteolytic activity in these conditions was difficult, as weak protease bands were observed in some gels and others showed no activity at all. Suspecting possible SDS inhibition ofproteolytic activity, we changed electrophoresis conditions, using no SDS or reducing the SDS concentration (in gel and electrophoresis buffer) to 0.01% in order to facilitate the detection of proteolytic bands in gels. Electrophoretic analysis was conducted in polyacrylamide gels copolymerisedwith a gelatin substrate (the final gelatin concentration in gels was 0.15%). Samples were diluted 3:1 in sample buffer and electrophoresed in a Bio-Rad Mini Protean apparatus at 50 V per gel. Afterwards, gels were washed in 2.5% Triton X-100 for 1 h to remove SDS, incubated in 0.1 M citrate buffer, pH 5, 2 mM CaCI2 for 24 h and stained with Coomasie blue. After destaining, proteases were located as clear bands in a blue gel.
Chemicals Azocoll, gelatin, proteinase inhibitors and electrophoresis calibration kits were purchased from Sigma (St. Louis, MO). All other chemicals were of analytical grade.
136
A. CRIADO-FORNELIOET AL.
RESULTS
The colorimetric assay using azocoll as substrate has been used for detection of proteinases in T. spiralis crude extracts. Azocoll hydrolysis was found over a relatively broad pH range (Fig. 1 ). Maximal activity was obtained at pH 5. Intact larvae incubated in an azocoll-containing m e d i u m at pH 5 showed a strong azocollytic activity, degrading 35.1% of the azocoll present in the m e d i u m after 20 h of incubation. Electrophoresis of T. spiralis crude extracts and ES products under non-
100
3am
~-.
50
,q
I,-,.,.i
0
I
I
2
4
I
i
6
I
I
8
pH
Fig. l. pH activity curves of T. spiralis larvae proteases on azocoll (measured in crude extracts). Each point represents the mean of three experiments. The point of maximal azocoll hydrolysis was taken as 100%. TABLE1 The effect of inhibitors on T. spiralis proteases measured in crude extracts. Assays were conducted at pH 5, except in SDS experiments, performed at pH 3. All of the experiments were repeated at least three times. Results are expressed as a percentage of reduction in absorbanee compared with an inhibitor-free control Inhibitor
Concentration
Inhibition (%)
SDS Leupeptin Pepstatin A PMSF EDTA MgC12 STI Chymostatin DMSO
0.1% 100/zM 1 mM 5 mM 5 mM 10 mM 50/lg m l - l 500/gg m l - ~ 0.5%
100 5.6 12.9 60.1 39.5 34.0 3.1 68.5 53.1
Abbreviations: SDS, sodium dodecyl sulphate; PMSF, phenylmethylsulphonyl fluoride; EDTA, ethylenediaminetetraacetic acid; STI, soybean trypsin inhibitor; DMSO, dimethyl sulphoxide.
137
PROTEOLYTIC ENZYMES FROM TRICHINELLA SPIRALIS LARVAE
MW kDa
2A
MW kDa
2B
27266-
66-
45-
45-
29-
Fig. 2. Electrophoretic analysis of T. spiralis proteases in polyacrylamide gels. Gels were incubated for 24 h at 37 °C. Molecular weight markers are indicated. (A) Worm extract sample (50 #g of protein) run in a 7.5% gel containing gelatin. No SDS was present in gel or electrophoresis buffer. (B) ES products sample (60 #g of protein) run in an 11% gel containing gelatin. SDS concentration in gel and electrophoresisbuffer was 0.01%.
reducing conditions and low SDS concentration in buffer and gel (0.01%) revealed several proteinases. Crude extract proteinases showed apparent molecular weights ( M W ) o f 48, 54 and 62 kDa (Fig. 2A). In ES products we found three proteases at M W 33, 62 and 230 kDa (Fig. 2B). No additional bands could be observed by ovedoading the gels with samples or by changes in the acrylamide percentage o f the gels. Trials for detection o f proteolytic activity in electrophoresis performed with 0.1% SDS in buffer and gels led to poor results, as proteases generally showed low or no activity at all, even when gels were overloaded with samples. These data suggested that proteases were not totally resistant to SDS. Studies on the inhibition o f azocoll degradation by SDS and other inhibitors were carried out using w o r m crude extracts. The results obtained are shown in Table 1. In standard assays at pH 5, protease
138
A. CRIADO-FORNELIOET AL.
activity of T. spiralis extracts was decreased by DMSO, EDTA, MgClz, PMSF, chymostatin and pepstatin A. In inhibition assays at pH 3 with SDS (0.1% w / v ) a strong inhibition ofproteolytic activity was observed. DISCUSSION
Significant proteolytic activity was observed in T. spiralis extracts at pH 3, 4 and 5. As azocoll assay measures net proteolytic activity of the extract, this result may reflect the presence of multiple proteases, each with its own optimum pH. Trichinella spiralis proteases have an optimum pH of 5, which is similar to that of the proximal portion of the small intestine, where most of the adults have been found in experimental infections in mice (De ArmasSerra et al., 1987). This fact, together with the finding that several proteases are excreted by the larvae, and the absence of an oral styler (Bruce, 1970) may be indicative of their function as lytic enzymes used to facilitate larval penetration into host enterocytes. Niimura et al. ( 1988 ) pointed out that a 160 kDa molecule obtained from T. spiralis extracts was active as a proteinase at pH 4, but further details have not been published. The present data did not show any proteolytic enzyme at that molecular weight, but this fact may be explained by the incubation of gels at pH 5 in our experiments instead of pH 4. Another possibility is the existence of biochemical differences between Trichinella isolates (RodriguezCaabeiro et al., 1985; La Rosa et al., 1991 ). The azocoll degradation assay demonstrated that T. spiralis larvae secreted proteases and subsequent electrophoretic analysis demonstrated their presence in ES products and worm extracts. It is interesting to note that two proteases of MW 230 and 33 kDa could only be detected in ES products. The reasons for this are unknown, but possible speculative explanations are that these enzymes are associated with the moulting process and start to be synthesised after decapsulation. Therefore, they are only detectable in ES products when they are accumulated in the medium, or the proteases are found within the worm cells as inactive forms and are "activated" when secreted out of the cells. The 48 and 54 kDa proteases, which seem to be present only in worm extracts, could merely be enzymes used for digestive functions inside the worm. Furher investigation is needed to confirm this assumption. Finally the 62 kDa enzyme, which is common to ES products and worm extracts is likely to be an enzyme used for penetration into host enterocytes, as it does not need to be activated or synthesised after decapsulation and might be quickly used for lysis of host cells membranes. Further studies will be conducted to ascertain these speculative hypotheses. In studies on the inhibition of azocollytic activity, partial inhibition was noted with DMSO, EDTA, PMSF, chymostatin, pepstatin A and MgCla, in assays conducted at pH 5. A strong inhibition was found for SDS at pH 3.
PROTEOLYTIC ENZYMES FROM TRICHINELLA SP1RALIS LARVAE
139
DMSO is a solvent widely used to test the effect of insoluble inhibitors on proteolytic activity (Dresden et al., 1984; Monroy et al., 1989). Unfortunately, DMSO showed some inhibitory action on T. spiralis proteases, so that the effects reported here for PMSF and pepstatin A (tested as a solution in DMSO) could have been obscured by interference of the solvent. Likewise, the effect of 0.1% SDS on T. spiralis proteases was difficult to assess at pH 4 and 5, because significant degradation of azocoll was observed in SDS controls without homogenate at these pHs. However, SDS did not degrade azocoll at pH 3 and, at least at this pH, our results seem to confirm that this detergent is a strong inhibitor of proteolytic activity in T. spiralis larvae. The sensitivity to the inhibitors mentioned above indicated that certain enzymes were metaUoproteases and serine proteases. The presence of serine proteases seems to be a very common feature in parasitic nematodes. They have been found in Dirofilaria immitis (Tamashiro et al., 1987), Nematospiroides dubius (Monroy et al., 1989), Onchocerca spp. (Lackey et al., 1989) and Anisakis simplex (Sakanari and McKerrow, 1990). The present studies on T. spiralis proteases have provided us with new data about the biochemical basis for penetration in their hosts. Besides their activity in penetration, however, another possible role of proteolytic enzymes is as a means of escaping the host's immune response. Dirofilaria immitis proteases have been found to cleave IgG (Tamashiro et al., 1987). Additional studies on T. spiralis proteases are underway to ascertain if proteolytic enzymes are used in worm digestion and evasion of the immune response. Finally, further investigations on inhibition of protease activity by chemicals or antibodies would be, at least theoretically, an effective trend in anthelmintic therapy. ACKNOWLEDGEMENTS This investigation received financial support from the Scientific and Technical Assessor Committee (Ministry of Education and Science, Spain), No. PM 89-0211 and from the Vicerrectorado de Investigaci6n (University of Alcal~t de Henares, Spain), No. 91 A/22. REFERENCES Brand, T., Weistein, P., Mehlan, B. and Weinbach, E., 1952. Observation on the metabolism of bacteria-free larvae of Trichinella spiralis. Exp. Parasitol., 1: 245-255. Bruce, R.G., 1970. Structure of the esophagus of the infective juvenile and adult Trichinella spiralis. J. Parasitol., 56: 540-549. De Armas-Serra, C., Martinez-Fernandez, A.R., Bolas-Fernandez, F. and Gomez-Barrio, A., 1987. A comparative study of the endogenous biological behaviour of six Trichinella isolates (in Spanish, abstract in English). Rev. Ib6r. Parasitol., Vol. extra: 153-157.
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