Isolation and partial characterization of a Taenia taeniaeformis metacestode proteinase inhibitor

Interncllional Journalfor Parwsifology Vol. 14,No. 2,pp. 165-172, 1984. Printed

0020-7519/64$3.00+0.00 Pergamon Pra Ltd.

in Great Britain. 0 1964 A&r&an

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for Paradto/ogy

ISOLATION AND PARTIAL CHARACTERIZATION OF A TAENIA TAENIAEFORMIS METACESTODE PROTEINASE INHIBITOR C. SUQUBT,* C. GREEN-EDwARDs+ and R. WES LEID*+ *Department of Veterinary Microbiology/Pathology, Washington State University, Pulhnan, WA 99164, U.S.A. and j-formerly of the Department of Pathology, Michigan State University, East Lansing, MI 48824, U.S.A. (Received 24 May 1983)

Abstract-&p_rET C., GREEN-EDWARDS C. and WES LEIDR. 1984. Isolation and partial characterization of a Taenia taeniaeformis metacestode proteinase inhibitor. International Journal for Parasitology 14: 165-172. A proteinase inhibitor from the metacestode of Tueniu tueniueformis was purified 136.fold to apparent homogeneity as evidenced by one Coomassie Blue protein staining band on 10% SDS slab gels under both reducing and non-reducing conditions. The apparent molecular weight under dissociating conditions was 19,500. This parasite protein inhibited esterolysis of TAME and BTEE by bovine pancreatic trypsin and chymotrypsin respectively in a time and dose-dependent manner. Proteolysis of casein by both enzymes was also inhibited in a time and dose-dependent manner. The parasite inhibitor was stable from pH 2.2 to 10.5 and was fully active after heating at 56°C for 3 h. The proteases pronase and thermolysin, at concentrations of 1 mg ml-t, completely inactivated the metacestode inhibitor. Two sulfhydryl proteases, papain and chymopapain, used at concentrations of 1 mg ml-’ were without effect. The irreversible proteinase inhibitors effect on the parasite inhibitor.

TLCK,

TPCK

and PMSF at concentrations

up to 10 mM had no

INDEX KEY WORDS: Taeniu tueniaeformis; metacestode; proteinase inhibitor; larval cestodes; longterm survival; trypsin inhibition; chymotrypsin inhibition; esterolysis; proteolysis.

INTRODUCTION

of laboratory rats with the larval stage of the cat-rat cestode Taenia taeniaeformis leads to a pronounced resistance to reinfection (Leid, 1977). The vexing question of this host-parasite relationship is how the existing worm burden in the liver can survive in an immunocompetent animal while the host is resistant to challenge infections. Recent investigations, therefore, have sought to define mechanisms which permit this parasite to evade the effector arms of the host immune response. Hammerberg, Musoke & Williams (1977) and Hammerberg & Williams (1978a, b) have shown very clearly that taeniid parasites possess an anticomplementary factor which brings about the consumption of C3 in the fluid phase away from the parasite membrane. This anti-complementary factor has been characterized as a glycosaminoglycan and is apparently secreted into the milieu around the living worm (Hammerberg & Williams, 1978a, b). The importance of the host complement system in resistance to this particular infection and in host resistance to infections in general (Leid, 1977; Leid & Williams, 1979; Fearon & Austen, 1980) has prompted us to search for a more specific class of INFECTION

*Author to whom all correspondence

is directed.

inhibitors for both the host complement system and other host inflammatory systems. Many species of parasitic worms possess trypsin and chymotrypsin inhibitors of broad specificity (Hajdu, Matskasi & Juhasz 1979; Willadsen, 1977; Matskasi & Nemeth, 1979; Pudles, Rola & Matida, 1967; Peanasky & Laskowski, 1960; Peanasky & Szucs, 1964; Kucich & Peanasky, 1970; Rhoades & Romanowski, 1974; Rhoades, Romanowski, Doherty & Stewart, 1978). Recently taeniid parasites other than T. taeniaeforrnis have been shown to possess proteinase inhibitors and in certain taeniid species the active principle has been partially characterized (Nemeth, Juhasz & Baintner, 1979; Nemeth & Juhasz, 1980; Nemeth & Juhasz, 1981). However, in no instance has the trypsin or chymotrypsin inhibitor been tested for inhibition of the trypsin-like enzymes of the complement cascade or the Hageman Factor dependent pathways to coagulation, kinin generation or fibrinolysis or indeed suppression of inflammatory cell activation including mast cells/basophils as well as lymphocyte proliferation. We present herein the isolation to homogeneity and partial characterization of a Tuenia tueniaeformis metacestode proteinase inhibitor which inhibits both chymotrypsin and trypsin. In other work (Leid, Suquet, 165

Blanchard,

& Bing, manuscript

C. SUQUET,C. GREEN-EDWARDS and R. WESLEID

166

in preparation),

this proteinase inhibitor has been shown to inhibit both the alternative and classical pathways of activation for human complement.

MATERIALS

AND METHODS

Materials. QAE cellulose, SDS molecular weight standards, protein assay kit, acrylamide, bis-acrylamide, TEMED, ammonium persulfate, Coomassie Blue R-250, dithiothreitol (DTT), Protease Detection Kit, (BioRad Laboratories, Richmond, California), Con-A Sepharose, Sephadex G-75, Gel Filtration Calibration Kit (Pharmacia Fine Chemical, Piscataway, New Jersey) Ultrogel ACA-54 (LKB Instruments, Rockville, Maryland), Barbital (sodium salt), barbituric acid, ethylene&amine tetracetic acid (EDTA), ethylenealvcol-bis-l-aminoethvl ether) tetracetate (EGTA); bovine pancreatic try&in (Ed 3,4~21*4,DPCC treated), bovine pancreatic a-chymotrypsin (EC 3.4.21.1, TLCK treated), tosyl-arginine methyl ester (TAME), benzoyl-tyrosine ethyl ester (BTEE), tosylamidephenylethyl chloromethyl ketone (TPCK), tosyl-lysine chloromethyl ketone (TLCK), phenylmethylsulfonyl fluoride (PMSF), papain, (EC 3+4*22+2), Protease X (thermolysin, EC 3,4.24.4) Protease XIV (pronase E, EC 3.4.21+4), chymopapain (EC 3.4~22~6) (Sigma Chemical Company, St. Louis, Missouri). SDS (Pierce Chemical Co.. Rockford, Illinois); YM-5 ‘and YM-10 ultrafiitratio~ membrane (Amicon Corporation, Lexington, Massachusetts), and dialysis tubing (3500 and 6000 mol. wt cut off) (American Scientific, Redmond, Washington) were purchased from the manufacturers as noted. All other chemicals and solvents used were reagent grade or better. Trypsin inhibition. The biologic activity of the T. tueniaeformis-derived inhibitor (T.t.in,,) was assayed by its ability to block esterolysis of TAME by bovine pancreatic trypsin (EC 3.4.21.4). TAME hydrolysis utilized the method of Hummel (1959). Aliquots of 100 ~1 of the column fractions or concentrated pools of inhibjtor were diluted with 2.7 ml of buffer (46 mM Tris adjusted to pH 8.1 with I M HCI, containing 11.5 mM CaCl,) and incubated for 15 min at room temperature with 2-O pg of bovine trypsin. At the end of the incubation, 200 ~1 of a TAME solution (10 mM) was added to each tube and all tubes were further incubated at 37OC for 8 min. The samples were immediately iced down and the absorbance at 247 nm of each determined. The degree of inhibition was determined by comparison to tubes incubated without inhibitor added. One unit of trypsin inhibition is defined as that quantity of T.t.inh. which gave 50% inhibition under the assay conditions described. u-Cbymotryps~n inhibition. The biologic activity of the T.t.inh was also assayed for its ability t,o block esterolysis of BTEE by bovine pancreatic cr-chymotrypsin (EC. 3.4.21 ,i). BTEE hydrolysis followed the method of Hummel (1959). Aliquots of concentrated pools of inhibitor were diluted with 1.4 ml of buffer (80 mM Tris, 100 mM CaCl,, pH 7.8) and 2 ng of a-chymotrypsin was added and the solution incubated for 15 min at room temperature. The reaction was developed by the addition of 1.4 ml of substrate (1.07 mt.r BTEE in 50% methanol) and the mixture incubated for 5-10 min at 37°C. Samples were iced down at the end of this time and the absorbance at 256 nm determined. The degree of inhibition was determined by comparison to tubes incubated with enzyme and substrate alone and to which no inhibitor was added. Inhibition of proteolysis. Proteolytic activities of trypsin

t.J.P.VOL.14. 1984

and ff-chymotrypsin were quantitated using the BioRad Protease Detection Kit. The substrate, casein, was sotubilized and plates poured according to the instructions of the manufacturer. After the gel had cooled, 3.0 mm diameter sample wells were removed from the gel and 12 4 of sample containing either trypsin or a-chymotrypsin, buffer or combinations of these enzymes with the parasite inhibitor was applied. Aliquots of the two enzymes (15 ~1 each) plus the inhibitor (15 ~1)were preincubated for 15 min at room temperature prior to removal of the 12 ~1 for addition to the casein plates. The plates were incubated at room temperature for 18 h and the diameters for the zones of hydrolysis quantitated. Inhibition of casein hydrolysis was determine by comparing the zones of hydrolysis for buffer treated enzyme controls with the enz~e-inhibitor mixture lytic areas. The areas of hydrolysis in the casein plates were linear with respect to both time and concentration over the enzyme concentrations tested. E;ffects of pH and heat on T.t.,, Aliquots of 50 pl of T&,,, were dialyzed against 10 mM T&s-HCI, ranging in pH from 2.2 until 10.5 for 2 h at 4’C. Although Tris does not buffer well in the lower pH ranges, replicate samples had the pH in each dialysis bag measured and the values were as indicated. After the fmt dialysis all samples were subsequently dialysed against 0.01~ Tris-HCl, pH 8.0 for a further 3 h. A 15 nl sample of each fraction was incubated with 15.~1of trypsin (20.0 ,ug ml-t) or u~hymot~psin (20.0 ug ml-r) for 15 min at 22°C. An aliquot of 12 nl from each mixture was then added to the casein plates. Aliquots of 50 ~1 of T.t.inh. were also heated at 56°C in 10 mM Tris-HCl buffer (pH 8.0) for periods of time from 30 min to 3 h. Aliquots of 15 ~1 of each fraction were subsequently incubated for 15 min at 22’C with i5 ~1 of trypsin (20 ng ml-t) or a-chymotrypsin (20 ug ml-r) after which time 12 pl was applied to the casein gel plates. Enzyme and peptide inactivetion of TX, , Papain (7.5 U mg“ protein), chymopapain (5.0 U rngeP protein) thermolysin (54 U mg-’ protein and pronase (5.4 U mgwi protein) were incubated with T.t.inh. for 60 min at 37°C. The samples were incubated for 15 min at 22°C with 2 c(g of trypsin. TAME was then added and the inhibition of trypsin esterolysis was determined. Papain and chymopapain proteolytic activities were assessed according to standard procedures (Worthington Biochemical Manual, 1972) in order to ensure that the enzyme preparations were active. The activities listed above are those values obtained. PMSF was preincubated with T.t.inh, for up to 2% h at both 37°C and 22°C at concentrations ranging from 10 nha to 10 mM. The samples were extensively dialyzed against 0.15~ PBS to remove free PMSF and the T.t.inh then tested in the standard trypsin inhibition assay. ‘ PMSF at a concentration of 10 mM resulted in complete inhibition of trypsin-mediated esterolysis under these conditions. Both TLCK and TPCK were preincubated with T.t.inh. for 1 h at 37°C and 22°C at concentrations up to 1 rnM and the samples extensively dialyzed against 0.15 M PBS to remove free TLCK and TPCK. The 1 mM concentration resulted in complete inhibition of trypsin (TLCK) and chymotrypsin (TPCK) mediated esterolysis under these conditions. Isolation of T. taeniaeformis protease inhibitor (T. t.jnh,). T. tueniueformis infections were maintained in cats and rats as described by Leid & Williams (1974a). Methods for extraction of the IO-12 month-old parasites were modified from previous work (Leid & Williams, 1974b) as the metacestodes were teased from infected rat livers, washed extensively with saline (150 mM NaCl) homogenized in a

I.J.P.VOL.

Taenia taeniaeformis proteinase inhibitor

14. 1984

Waring Blender with a l/4 (v/v) ratio of worms to 3 MKC1 and the mixture stirred overnight at 4OC. The extracts were centrifuged at 81,000 g for 60 min at 4°C (Beckman LS-50B, Beckman Inst. Co., Fullerton, California), dialyzed extensively against 100 mM Tris-HCl, pH 8.0, followed by 10 mM Tris-HCl, pH 8.0, all at 4°C. The dialyzed soln. was clarified in each case by centrifugation at 81,800 g for 60 min at 4°C. Protein concentrations were determined by the BioRad Protein Assay Kit using bovine plasma albumin as the standard. Ion exchange chromatography. Approximately 100 mg protein of the dialyzed parasite extract was applied to a QAE cellulose (1.5 cm x 24 cm column) equilibrated in 10 mM Tris-HCl (pH 8.0) and the column washed with 5 bed volumes of starting buffer. A linear salt gradient to 500 mM NaCl was then applied and every other fraction tested for inhibition of bovine pancreatic trypsin. Those fractions containing trypsin inhibitory activity were pooled and concentrated by ultrafiltration (YM-5) to a 5 ml volume. The pooled and concentrated trypsin inhibitor from ion exchange chromatography was applied directly to a Con-A Sepharose column (1.5 cm x 24 cm) equilibrated and run in 20 rnM Tris-HCl (nH 7.4) containing 500 mM NaCl. 1 mM Mn’+ and 1 mu Cai+. Fractions of 2 ml were collected and each tested for trypsin inhibitory activity. Dialysis of the ion exchange pool prior to application to the Con-A column did not improve binding of the inhibitor and in fact decreased the yield of the active principle (Suquet & Leid, unpublished observations). Fractions with trypsin inhibitory activity were pooled and concentrated by lyophilization as we found that this increased our yields rather markedly (Suquet & Leid, unpublished observations). The Iyouhilized T.t.,_, was resuspended in 2 ml of distilled water -and dialy% overnight against 3% ammonium carbonate using 3500 mw dialysis tubing. The sample was finally chromatographed on a ACA 54 column (1.5 cm x 40 cm or 1.5 cm x 90 cm) equilibrated in 3%

167

ammonium carbonate. The columns were calibrated with protein markers of known molecular weight prior to and after the T.t.inh, was run. Fractions of 2 ml were collected and each fraction assayed for trypsin inhibitory activity with the active fractions pooled and concentrated by lyophilization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) utilized a modified Laemmeli continuous buffer system on a 10% slab, according to the instructions of the manufacturer (Pharmacia Fine Chemicals Piscataway, N.J.). Protein samples were reduced in 10 mM sodium phosphate buffer (pH 7.0), containing 1% SDS, 100 mM dithiothreitol (DTT) and boiled for 5 min. Electrophoresis was carried out at 8OV until the sample had run into the gel and then the voltage was raised to 16OV for 2 h and 45 min. The gels were fixed in 10% sulfosalicylic acid for 1 h and stained overnight in 0.1% Coomassie Blue R-250 (45% ethanol, 45% acetic acid, 10% water, v/v). The gels were destained by a diffusion apparatus (BioRad Laboratories, Richmond, California) for 4-12 hat 22’C.

RESULTS Approximately 100 mg of parasite saline soluble protein was applied to QAE cellulose and a representative chromatogram is shown in Fig. 1. We

have repeated this particular step more than 70 times with no change in the protein or enzyme inhibitor profiles. The bulk of the protein comes through in the effluent while the bulk of the T.t.i,,r,. is eluted between a conductivity of 2-5 and 9 mS. The pooled and concentrated QAE T.t.i,,,, eluate was applied to Con-A-Sepharose and a representative chromatogram is shown in Fig. 2. This step has also been repeated more than 70 times with similar results

3.0 2.8

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0.8 0.0

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Tube number

Fro. 1. QAE cellulose chromatography of the Taenia taeniaeformis proteinase inhibitor. An aliquot of 100 mg of a 3 M KC1 parasite extract was applied to a 1.5 cm x 24 cm column of QAE cellulose equilibrated in 10 mM Tris (pH 8 ‘0) and a linear salt gradient developed (- - -). The optical density at 280 nm of the and the inhibition of pancreatic trypsin plotted (- . - -). 3 ml fractions was determined (-)

168

C. SUQUET,C. GREEN-EDWARDS and R. WESLEID

r

3.0

i

Tube

number

Con-A Sepharose chromatography of the Tueniu tue~i#for~~ proteinase inhibitor. The fractions from QAE cellulose with trypsin inhibitory activity were pooled, concentrated to 5 ml and applied to a Con-A Sepharose column (1.5 cm x24 cm) equilibrated and run in 20 mrd Tris (pH 7.4) and containing 500 mM NaCl, 1 mM MnZ+ and 1 mrd CaZ’ . The optical density at 280 nm of the 2 ml fractions was determined () and the inhibition of pancreatic trypsin plotted (- . - . -). FIG. 2.

obtained each time. The majority of the protein comes through in the effluent while the T.teinh. is retarded slightly and comes through after the major

protein peak. We have detected, on some separations, lo-30% of the total trypsin inhibiting activity in the major protein peak but have not as yet been able to purify this activity. On repeated Con-A Sepharose

I.J.P. VOL.14. 1984

rechromatography of this latter T.t.inh,, the trypsin i~ibitory activity has always been recovered in the major protein peak (Suquet & Leid, unpublished). The retarded T.t.i”h. fractions were pooled and lyophilized. T.t.inh. was then chromatographed on a calibrated ACA-54 column and a representative chromatogr~ is shown (Fig. 3). A slight protein peak was noted at the void volume of the column while all the T.t.in,,. activity eluted at an apparent mol. wt of 6000-8000 (Fig. 3) with only trace amounts of protein detectable at 280 nm. This column step has been repeated over 30 times with similar profiles observed each time. A typical purification is outlined in Table 1. We have obtained at least a 136fold purification of this parasite proteinase inhibitor with a removal of 99.96% of the starting protein content. We have also recovered between 10 and 20% of the original trypsin inhibitory activity and have obtained as high as 40% yields of the biologic activity with some purification runs. The increase in total T.&. activity observed after QAE cellulose most hkely represents the removal of parasite enzymes capable of hydrolysing TAME. A 15pg sample of T.t.i,t,, obtained after the ACA 54 chromatographic step was analyzed by SDSPAGE on 10% gels under both reducing and nonreducing conditions. Because there was no difference in the apparent molecular weights between the non-reduced and reduced samples only the reduced sample is shown in Fig. 4. A single stained protein band was observed after the ACA-54 purification step with a apparent mol. wt of 19,500. A typical of trypsindose-response curve for inhibition

- 0.6 E - 0.4

c

s

2

-0.2

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Tube number

3. ACA-54 gel filtration chromatography of the Tuenia taeniaeformir proteinase inhibitor. The fractions fron Con-A Sepharose with trypsin inhibitory activity were pooled, lyophilyzed, resuspended in distilled water and dialyzed against 3% ammonium carbonate prior to gel filtration on a ACA-54 column (1’ 5 cm x 40 cm) equilibrated in 3% ammonium carbonate. The optical density at 280 nm of the 2 ml fractions was determined (------) and the inhibition of pancreatic trypsin plotted (-). FXG.

I.J.P.

VOL. 14.

1984

Tueniu taeniueformis proteinase inhibitor TABLE ~-PURIFICATION OF

Taenia taeniaeformis

169

PROTEINASEINHIBITOR

(T.t.inh,)

Chromatographic

step

Crude extract QAE cellulose Con-A Sepharose ACA-54

Protein

Activity

Specific activity

287 mg 133 mg 2.86 mg 0.19Omg

1380 U* 1978 U 473 u 124 U

4.8 U mg-’ 14.8 U mg-’ 165 U mg-’ 652 U mg-’

Fold purification 1 3 34 136

*One unit of inhibitory activity is defined as that amount of parasite protein which results in 50% inhibition of 2 pg of trypsin when incubated for 5 min at 22°C prior to TAME addition.

mediated proteolysis is shown in Table 2 for the ACA purified inhibitor. The T.t.i,h, is stable over a broad pH range as well as resistant to heating at 56°C for periods of times -92.5 K hanging up to 3 h. Dialysis of the T.&, against -66.2 K solutions over the pH range from 2.2 to 10.5 gave no reduction in the T.t.i,,,, ability to inhibit both -45.0 K a-chymotrypsin and trypsin-mediated proteolysis of casein. In addition, heating at 56°C for periods of time up to 3 h resulted in no reduction of the T.t.i,,,, ability to inhibit both a-chymotrypsin and trypsin-31.0 K mediated proteolysis of casein. Dose-response curves and kinetic curves were constructed for a more dynamic appraisal of the inhibition observed for both trypsin and a-chymo-21.5 K trypsin esterolysis (Figs. 5, 6). In Figs. 5 and 6, dose-response and kinetic curves for ACA purified T.t.i,,,, were compared using both trypsin and chymo- 14.4 K respectively. Very rapid and marked trypsin, inhibition was noted almost as soon as the enzyme, and substrate were mixed with each other. Tet’inh. The ACA purified material used 5.4, 2.7, 1.35 and FIG. 4. SDS/ PAGE of the ACA-54 purified Taenia 0.67 trypsin inhibitory units as defined by the standard assay. The ACA-54 purified material taeniaeformis proteinase inhibitor on 10% slab gels. appeared to be more effective on a-chymotrypsin Molecular weight standards consisted of the following proteins: lysozyme, 14,400; soybean trypsin inhibitor, than on trypsin (Fig. 5 vs. 6) but was still highly 21,500; carbonic anhydrase, 31,000; ovalbumin, 45,GOO; effective at inhibiting trypsin when tested at the bovine serum albumin, 66,200; and phosphorylase B, 92,500 highest inhibitor input. and were used in Lanes A and D. Lane B contains Pretreatment of the T.t.i,h, with four broad 100 ~1 of a crude 3 M KC1 extract while Lane C contains spectrum proteases was not particularily illuminating 15 Hg of the ACA-54 purified proteinase inhibitor which as only preincubation with proteases X (thermolysin) had been reduced with dithiothreitol. There was no difference in apparent molecular weights between the reduced and XIV (pronase) reduced the trypsin inhibitory and non-reduced proteinase inhibitor so only the reduced activity (Table 3). The irreversible peptide protease sample is shown. The direction of electrophoretic migration inhibitors TLCK, TPCK and PMSF at concenwas from top to bottom on this slab. trations of 10 mM had no effect on the ability of

A

B

C

D

TABLE ~-DOSE-RESPONSE

CURVE FOR TRYPSIN INHIBITIONBY THE PROTEINASE INHIBITOR (T.t.inh,)

Taeniu

taeniueformis Concentration of T.Linh,

pg of protein

% Inhibition of casein hydrolysis

1:2 1:4 1:8 1 : 16

0.68 0.34 0.17 0.084

100 100 50 0

*The T.t.inh, was ACA-54 purified and had a protein content of 225 pg ml-‘. The dilutions indicated were preincubated (15 ~1) with 15 ~1 of trypsin (20-O pg ml-‘) for 15 min at 22°C prior to the addition of 12 ~1 to casein gel plates. The amount of hydrolysis was measured after an 18 h incubation at 22°C and compared to standard curve of trypsin without T.kinh..

170

C. SUQUET,

C.

and R. WESLEID

GREEN-EDWARDS

Control

-1.35u

I.J.P. VOL.14. 1984

DISCUSSION

2.7ou

g 5.4ou : 8

Time (minutes) FIO. 5. Kinetic and dose-response

inhibition of trypsin by the Taenia taeniaeformis proteinase inhibitor after gel filtration on ACA-54. Units of inhibition are relative to trypsin where 1 unit is equal to 50% inhibition of trypsin when the inhibitor was preincubated with 2.0 pg of trypsin for 15 min at 22°C prior to addition of substrate. The various concentrations of inhibitor depicted were chosen and added to cuvettes which contained 2.0 pg trypsin in 46 mM Tris (pH 8.1) and 11.5 mM Cacl,. Immediately thereafter substrate (TAME, 10 mM) was added and esterolysis followed over time in a Beckman Model 25

spectrophotometer and recorder at 22°C and 247 nm. A control cuvette contained only enzyme and substrate and had no added inhibitor. to inhibit trypsin even though these same T’t’inh. peptide inhibitors were able to inhibit trypsin, chymotrypsin and trypsin respectively by 100% at concentrations of 10 mM.

Proteinase inhibitors derived from a wide variety of parasites have been known for many years. In the case of the nematode Ascaris suum both a trypsin and chymotrypsin inhibitor have been purified and characterized (Pudles et al., 1967; Peanasky & Laskowski, 1960; Peanasky & Szucs, 1964; Kucich & Peanasky, 1970). In the majority of cases for the other parasite inhibitors only partial characterization has occurred except for the nematode Sfephanurus edenfatus (Rhoades & Romanowski, 1974; Rhoades et al., 1978). In the case of cestodes such as Taenia pisiformis, purification has been obtained but no critical criteria including SDS-PAGE of the isolated proteins have been provided (Nemeth et aI., 1979; Nemeth & Juhasz, 1980, 1981). The isolation procedure described herein for the cestode T. taeniaeformis and the proteinase inhibitor purified from the larval stage resulted in a single protein staining band on 10% SDS-PAGE with a recovery of between 10 and 20% of the biologic activity and a purification of 136-fold or greater. The molecule appears to be composed of a single polypeptide chain of an apparent mol. wt of 19,500. A more accurate analysis of the mol. wt must await amino acid and carbohydrate analysis. The discrepancy in apparent mol. wts between SDS-PAGE and gel filtration may well be due to carbohydrate interactions with the gel filtration resin used. The T.t.i,,,, has no detectable proteolytic activity against casein (Suquet & Leid, unpublished results) and is not affected by the irreversible proteolytic

2.7OU 5.4ou

2

4

6

8

10 Time

12

14

16

18

20

22

24

26

(minutes)

FIO. 6. Kinetic and dose-response inhibition of a-chymotrypsin by the Taenia taeniaeformisproteinase inhibitor after gel filtration on ACA-54. Units of inhibition are relative to trypsin where 1 unit is equal to 50% inhibition of trypsin when the inhibitor was preincubated with 2.0 c(g of trypsin for 15 min at 22°C prior to addition, of substrate. The various concentrations of inhibitor depicted were chosen and added to cuvettes which contained 2.0 c(g of a-chymotrypsin in 80 mM Tris (PH 7.8) and 100 mM CaCl,. Immediately thereafter, substrate (BTEE, 1.07 mM in 50% methanol) was added and esterolysis followed over time in a Beckman Model 25 spectrophotometer and recorder at 22°C and 256 nm. A control cuvette contained only enzyme and substrate and no added inhibitor.

I.J.P. VOL.14. 1984

Tuenia tueniaeformis proteinase inhibitor TABLE %-ENZYME

171

INACTIVATION OF THE Taenia tUenitZfOr??IiS INHIBITOR (T. Linh.) %

Enzyme Papain (E.C. 3.4.22.2) Ch~opap~n (E.C. 3.4.22.6) Protease X (E.C. 3.4.24.4) Protease XIV (E.C. 3.4.21.4) TLCK TPCK PMSF

Concentration 1 mg ml-’ (7.5 U rng-‘1 1 m$ ml-’ (5.0 U rng-‘j 1 mg ml-’ (5.4 U mg-‘) 1 mg ml-’ (5.4 U mg-‘) 1 x 10-s 1 x 10-3 1 x 10-Z

PROTEINASE

Inactivation of T.t.inh, 0 0 100 100 0 0 0

Enzymes of peptide inhibitors were preincubated with T.t.inh. for periods from 1 to 2% h at 37°C the peptide inhibitors dialysed away and the enzvme-inhibitor solutions assaved for inhibition for trypsin-mediated hy&olysis of TAME.

inhibitors TLCK, TPCK and PMSF suggesting that the T.t.i,h. is not a protease itself. The stability of the T.t.i,h. to both pH and heat indicates a very stable molecule which can remain active under adverse physiologic conditions. In this respect, the inhibitor is similar to that described for T. pisiformis (Nemeth et al., 1979; Nemeth & Juhasz, 1980, 1981). The destruction of the T.t.inh. with thermolysin (Protease X) and pronase (Protease XIV) would confirm the protein nature of the T.t.inh,. Chymotrypsin was inhibited by the T.t.i,h, and the kinetics of inhibition were quite similar to those seen with trypsin. It seems likely that the same molecule may serve to inhibit two distinctly different enzymes. These results would suggest a rather broad inhibitory profile heretofore appreciated only in the T. pisiformti-derived proteinase inhibitor of the parasite proteinase inhibitors described. Another interpretation of these results is obviously that a minor contaminant undetected by SDS-PAGE is responsible for the chymotrypsin inhibition observed. This latter interpretation is highly unlikely given to the data in Fig. 6 and assuming a 1 : 1 molar complex of T.t.i,,, and chymotrypsin. The correct answer however mist await further characterization. The T.&. has a mol. wt which is approximately 2.8 times that observed for the T. p~i~orrn~ molecule (Nemeth & Juhasz, 1981). However our mol. wt was determined by SDS-PAGE and not by gel filtration. If we calculate the mol. wt of the T.t.j,,, by our gel filtration ~hromatographi~ data, the apparent mol. wt is around 6-8000, a value compatible with the molecule described by Nemeth & Juhasz (1981) for T. pisiformis. Whether these are the same or similar molecules remains to be determined. A protective role for these parasite proteinase inhibitors has been postulated for some time (Fetteroli, 1907; Nemeth & Juhasz, 1981). Unfortunately, the evidence for such a conclusion has never been provided. In recent research (Leid, Suquet, Blanchard B Bing, manuscript in preparation) we have shown that the T.t.i,,,. will inhibit an important host defence system, the complement cascade,

including both the alternative and classical pathways of activation. In addition, in collaboration with Dr. Lance Perryman, (Leid, Suquet & Perryman, manuscript in prep~ation) we have also shown that the T.&,,, will block lectin-induced proliferation of rat spleen cells. We would postulate, therefore, that parasite proteinase inhibitors potentially may have rather marked and extensive dampening effects on host immunologic effector mechanisms. The spectrum and molecular mode of inhibition for other plasma and cell surface proteases remains to be elucidated. In addition, the physiologic importance of this proteinase inhibitor for parasite survival is suggested by our results in rat strains which can control and eventually eliminate the parasite. In these animals, less than 5% of the inhibitor is present when similar isolation protocols to those outlined in this paper are followed (Suquet & Leid, unpublished observations). We are presently examining whether or not antibody is made against the inhibitor in these nonsusceptible rat strains. The T.t.i,h. seems therefore to play a major protective role for the parasite and thusly permits the organisms to exist for prolonged periods of time in susceptible animals. Acknow~edgemenfs-This work was supported Grant NC. 17913 to R.W.L.

by NIH

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