Electrophoretic detection of Trypanosoma cruzi peptides

Electrophoretic detection of Trypanosoma cruzi peptides
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  Molecular and Biochemical Parasitology, 39 (1990) 31-38 31 Elsevier MOLBIO 01272 Electrophoretic detection of rypanosoma cruzi peptidases Stephen Greig and Frank shall Department of Pure and Applied Biology, Imperial College of Science, Technology and Medicine, London, U.K. (Received 24 August 1989; accepted 21 September 1989) Peptidases of Trypanosoma cruzi epimastigotes were examined by polyacrylamide gel electrophoresis n gels containing gelatin as peptidase substrate. Mini-gels were far superior to large gels in their sensitivity of peptidase detection. Patterns of peptidases were similar between different strains of T. cruzi, although some inter-strain heterogeneity was found. In strain Y, at least five peptidases were detected: four of these enzymes were shown to be cysteine-type peptidases with acidic pH optima. The other peptidase was a 60-kDa membrane-associated peptidase that was sensitive o o-phenanthroline; it was tentatively characterised as a metallopeptidase, and was optimally active at alkaline pH. This membrane-associated peptidase was conserved between strains of T. cruzi. Key words: Trypanosoma cruzi; Peptidase; Membrane-associated peptidase ntroduction A number of peptidases (nomenclature of Bar- rett and McDonald, ref. 1) have been detected in Trypanosoma cruzi, the protozoan parasite that causes South American trypanosomiasis (Chagas' disease), and some of these enzymes have been purified and characterised [2-9]. However, the functions of these peptidases are not known, al- though Cazzulo and his coworkers have charac- terized a cysteine peptidase of To cruzi with an apparently lysosomal location and amino acid se- quence homology to the papain family of pepti- dases, and particularly to cathepsin L. [7-9]. We have characterised a peptidase with unusual in- hibitor sensitivity that we have classified as an al- Correspondence address: Frank Ashall, Dept. of Pure and Applied Biology, Imperial College of Science, Technology nd Medicine, Prince Consort Road, South Kensington, London, SW7 2BB, U.K. Abbreviations: E-64, trans-epoxysuccinyl-L-leucylamido- 4- guanidino) butane; Hepes, 4-(2-hydroxyethyl)-l-piperazine- ethanesulphonic acid; PMSF, phenyimethylsulphonyl luor- ide; SDS, sodium dodecyl sulphate; TLCK, N-et-p-tosyl-L-lys- inechloromethyl ketone; TPCK, N-et-p-tosyl-L-phenylalani- nechloromethyl ketone. kaline cysteine peptidase [10]. This enzyme is the major peptidase in extracts of T. cruzi epimasti- gotes that cleaves benzoyl-arginine-p-nitroanilide at pH 8.0, and we have presented evidence that the peptidase occurs in other species of trypano- somatids [10]. Peptidases of various parasites have been char- acterised using polyacrylamide gels containing proteinaceous substrates: peptidase bands appear as clear areas on a background of stained, non- digested protein substrate [11-14]. This method is presumably suitable only for the detection of peptidases that extensively digest protein sub- strates, because peptide products must diffuse out of the gels in order to produce a clear band of proteolysis. Indeed, we have detected at least four peptidases of T. cruzi that do not show up fol- lowing electrophoresis in gelatin-containing po- lyacrylamide gels (unpublished data). Here, we examine epimastigotes of T. cruzi for peptidases that produce extensive proteolysis in protein-con- taining gels. Materials and Methods Parasites and chemicals. T. cruzi epimastigotes were grown at 28°C axenically in RPMI-1640 me- 0166-6851/90/ 03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)  32 dium supplemented with 10 (v/v) foetal calf serum (heat-treated at 45°C for 20 min), 0.2 mM L-glutamine, 25 mg ml -~ trypticase, 25 txg ml -~ haemin, 20 mM Hepes and 0.2 mM sodium py- ruvate. Strains used were: Dog Y, Armadillo, Opossum (each from domestic or wild animals in the United States), California (from a human pa- tient in the United States), Vinc C68, Esmeraldo, Sylvio X-10 and Y (all from South America: see ref. 15 for sources). All peptidase inhibitors were obtained from Sigma Chemical Company. Parasite extracts About 108 epimastigotes were centrifuged at 3000 x g at room temperature for 10 rain from a culture containing about 5 x 106 parasites m1-1. The cell pellet was washed once with 20 ml of 150 mM NaC1/20 mM sodium phos- phate, pH 7.4, then suspended at about 109 epi- mastigotes ml -l in 0.75 (v/v) Nonidet P-40/150 mM NaC1/25 mM Tris-HC1, pH 7.5. The suspen- sion was vortexed rapidly for 15 s and centrifuged at 12 000 × g for 3 rain at room temperature. The resultant supernatant was taken as the parasite extract and mixed with an equal volume of 0.4 M glycine/2 (w/v) SDS/0.1 (w/v) bromophenol blue. About 15 txl (25-50 txg protein) of this mix- ture was loaded into each track prior to electro- phoresis in mini-gels; in large gels, about 75 ILl of sample was used for each track. Separation of parasite extracts into membrane- associated and aqueous phases using the deter- gent Triton X-114 was carried out essentially as described by Bordier [16]. Between 25 and 50 ixg of protein from each phase was electrophoresed in each gel track. Electrophoresis Polyacrylamide gels (10 acry- lamide/0.27 bisacrylamide) containing 0.2 (w/v) gelatin were prepared as described previ- ously [11]. Samples were electrophoresed for 2--4h at 50 V (mini-gels) or for 16 h at 60 V (large gels). For mini-gels, electrophoresis was carried out us- ing an LKB Midget gel system; gel dimensions were 0.75 mm thick, 7.5 cm wide and 5.0 cm long. Large gels were 1.5 mm thick, 15 cm wide and 12.5 cm long. After electrophoresis, gels were shaken in 200 ml of 2 (v/v) Triton X-100 for 30-60 rain at room temperature, then incubated at 37°C for 16-36 h in the appropriate digestion buffer (see below). Gels were then stained for 1-24 h in 0,2 (w/v) Coomassie Brilliant Blue R/45 (v/v) methanol/45 (v/v) water/10 (v/v) glacial acetic acid, and destained with 10 gla- cial acetic acid/45 water/45 methanol. Digestion buffers For all proteolytic steps involv- ing incubation of gels in buffers at 37°C, the buffer used contained 50 mM sodium acetate, 50 mM Hepes, 50 mM sodium phosphate and 50 mM so- dium borate, and was adjusted to the pH re- quired. Effects of peptidase inhibitors were ex- amined by addition of the inhibitors to this buffer and incubating gels for 24 h at 37°C in their pres- ence. Radioiodination Intact, viable T cruzi epimas- tigotes were radioiodinated using a Bio-Rad En- zymobead radioiodination kit. Reaction mixtures consisted of 25 txl of parasite cell suspension (containing 2 x 107 epimastigotes), 50 Ixl of 0.2 M sodium phosphate, pH 7.2, 25 Ixl of 125I- (20 mCi ml-l; Amersham), 25 Ixl of 1 (w/v) [3-D- glucose and 50 Ixl of rehydrated Enzymobead re- agent. The mixture was incubated at room tem- perature for 10 min, then centrifuged at 12 000 x g for 3 min. The pellet (epimastigotes and En- zymobeads) was washed twice with 1 ml of 150 mM NaCI/20 mM sodium phosphate, pH 7.4, then extracted with 0.75 Nonidet P-40 (see 'parasite extracts'). About half of the radioiodinated ma- terial was loaded onto one gel track for electro- phoresis. After electrophoresis, gels were dried down and exposed to XAR-5 film (Kodak) for 1 to 7 days at -70°C using a photographic enhanc- ing screen. esults Initially we examined the pattern of peptidases produced by electrophoresis of T cruzi epimas- tigotes in large gels (see Materials and Methods for dimensions) containing 0.2 (w/v) gelatin as substrate. With strain Y of the parasite, a single band of proteolysis was observed (50 kDa), and a band of the same size occurred in all other strains tested (Fig. 1A). Some strains produced two peptidase bands in large gels. The Y strain  peptidase was strongly inhibited by E-64, TLCK and leupeptin, but was not affected by PMSF, trypsin inhibitor, Pepstatin, o-phenanthroline, EDTA, chymostatin or TPCK. Hence, this pep- tidase belongs to the cysteine peptidase family (see Fig. 1B); its pH optimum was between pH 5 and pH 7, with little activity at alkaline pH. When the same parasite extracts were elec- trophoresed in mini-gels containing 0.2 gelatin (see Materials and Methods for dimensions), many more bands of proteolysis were observed than with the large gel system. Thus, whereas Y A 2 3 4 Sr kD) 2 94 67 45 3 2 1 2 3 4 5 6 33 7 xr ~) 94 --67 ~45 ~30 Fig. 2. Peptidases of T cruzi detected in polyacrylam- ide/gelatin mini-gels. Lane 1, strain Dog Y; lane 2, Opossum; lane 3, California; lane 4, Vinc C68; lane 5, Esmeraldo; lane 6, Armadillo; lane 7, strain Y. Digestions were at pH 6.5. strain epimastigotes produced a single band in large gels, at least four peptidases were present in the mini-gel system (Fig. 2). Indeed, all strains of T cruzi tested had multiple peptidase bands in mini-gels, and patterns were quite similar be- tween different strains, although some strain dif- ferences did occur. The reason for the superior sensitivity of mini- gels over large gels for detection of peptidases is not clear, although it might be due to the fact that clear bands of proteolysis are more readily pro- duced in mini-gels because there is a smaller area, and therefore less substrate per unit enzyme ac- tivity, to digest. All subsequent analyses were carried out using the mini-gel system. Addition- ally, we found gelatin gels superior to casein or B I 2 3 4 5 Fig. 1. Peptidases of T cruzi epimastigotes detected in large gels containing gelatin as substrate. A) Different strains of T cruzi: lane 1, strain Y; lane 2, Esmeraldo; lane 3, Sylvio X-10; lane 4, Vinc C68. B) Inhibitor profile of Y strain pep- tidase: lane 1, no inhibitor; lane 2, 1 mM o-phenanthroline; lane 3, 25 I~M leupeptin; lane 4, 100 I~M TLCK; lane 5, 0.5 mM E°64, Digestions were at pH 5.5. 2 3 4 5 6 7 ~r ira) 94 67 45 3 2 Fig. 3. pH profiles of T cruzi Y strain peptidases in gelatin- containing gels. Gel strips were incubated during proteolysis at the following pH: lane 1, pH 4; lane 2, pH 5; lane 3, pH 6; lane 4, pH 7; lane 5, pH 8; lane 6, pH 9; lane 7, pH 10, as described in Materials and Methods.  34 haemoglobin gels for detection of T. cruzi pep- tidases, and we therefore used gelatin routinely as substrate not shown). pff profiles. Over the pH range 4-10, at least five bands of proteolysis were detected in T. cruzi Y strain epimastigotes Fig. 3). Four of these bands had apparent sizes of 50 kDa, 55 kDa, 120 kDa and >200 kDa. Each was more active at pH 4-5 than at any other pH, and little or no activity of these enzymes was seen above pH 7.0. The other peptidase, of 60 kDa, was optimally active at pH 10 and showed little activity below pH 7.0. It should be mentioned, however, that all these en- zymes were visible at pH 7.0 if a sufficient amount of extract was loaded onto the gels, and we be- lieve that the amounts of extract loaded need to be carefully controlled in order to avoid hetero- geneity in banding patterns between one experi- ment and another. Effects of peptidase inhibitors. All four pepti- dases with acidic pH optima were highly sensitive 1 2 3 4 -94 - 67 - 45 -30 Fig. 4. Effects of peptidase inhibitors on Y strain epimasti- gote peptidases. Lane 1, non-treated; lane 2, + 0.2 mM E-64; lane 3, + 1 mM PMSF; lane 4, + 50 kg/ml Pepstatin A. Digestions were at pH 5.5. The extra band at 65 kDa was not reproducibly present in the extract. Fig. 5 ” 45 T. cruzi Y strain peptidases following phase separa- tion ot extracts in Triton X-114. peptidases of aqueous phase lanes 3 and 4) and detergent phase lanes 1 and 2) were treated without inhibitor lanes 2 and 4) and with 1 mM o- phenanthroline lanes 1 and 3) during the proteolytic step. Digestions were at pH 7.5. Lane 5, radioiodinated surface proteins of Y strain epimastigotes electrophoresed under the - ORIGIN z- \- - 94- 67 same conditions as samples in lanes 1-4. to E-64, TLCK, leupeptin and Hg2’, but were in- sensitive to 1 mM PMSF, 100 PM Pepstatin A, 0.1 mg ml-’ trypsin inhibitor soy bean), 1 mM o-phenanthroline, 5 mM EDTA and 100 p,M TPCK see Fig. 4). Hence, all these enzymes are cysteine-type peptidases. The 50-kDa peptidase was generally the most intense band in mini-gels and was thought, therefore, to be identical to the single 50-kDa peptidase produced in large gels by Y-strain epimastigote extracts, since this band was also a cysteine-type peptidase Fig. 1). The alkaline peptidase 60 kDa) was not inhib- ited by any of the serine-, cysteine- or aspartic- type peptidase inhibitors that we tested, but it was strongly inhibited by 1 mM o-phenanthroline see Fig. 5). Hence, we tentatively characterise this enzyme as a metallopeptidase. Phase separation experiments. Separation of Tri- ton X-114 extracts of Y strain epimastigotes into aqueous and detergent phase membrane-associ- ated) proteins resulted in the 60-kDa alkaline peptidase being partitioned into the detergent  A 1 2 3 4 5 6 r 94 67 45 30 20 el 2 3 4 5 6 ~ ~) 94 67 30 20 Fig. 6. Peptidases of aqueous and membrane-bound material following Triton X-114 phase separation of T cruzi epimas- tigote extracts. Gel A was incubated in the absence of added inhibitors, gel B in the presence of 1 mM o-phenanthroline. Lane 1, strain Y aqueous phase); lane 2, strain Y detergent phase); lane 3, strain Sylvio X-10 aqueous phase); lane 4, strain Sylvio X-10 detergent phase); lane 5, strain California aqueous phase); lane 6 detergent phase). phase; the other enzymes appeared in the aqueous phase Fig. 5). However, this o-phenanthroline- sensitive, membrane-associated peptidase was not a major iodinatable surface protein Fig. 5), which contrasts with the o-phenanthroline-sensitive peptidase gp63) on the surface of Leishmania promastigotes [17,18]. Additionally, we failed to release the peptidase from its membrane-associ- ation using Bacillus cereus phospholipase C not shown). Examination of six different strains of T. cruzi 35 for membrane-associated peptidases using the Triton X-114 phase separation procedure showed that all strains expressed an o-phenanthroline- sensitive peptidase that was associated with membranes and had a size of about 60 kD: the results obtained with three strains are shown in Fig. 6. The 60-kDa membrane-associated pepti- dase was the only detectable membrane pepti- dase in some strains, although other strains ap- parently had several membrane peptidases Fig. 6). A greater degree of inter-strain heterogeneity was also discernible when extracts were sepa- rated into detergent and aqueous phases. Hence, the membrane-associated, o-phenan- throline-sensitive 60-kDa peptidase appears to be conserved amongst different strains of T. cruzi. It was routinely noticed during these studies that o-phenanthroline enhanced the activities of cysteine peptidases in gelatin gels see Figs. 5 and 6). We believe this enhancement is due to che- lation of heavy metal ions from our buffers by o- phenanthroline. Low levels of heavy metal ions contaminating the water or buffer salts might in- hibit the cysteine peptidases and o-phenanthro- line would then de-inhibit the enzymes. Evidence in favour of this explanation was obtained when it was shown that EDTA has a similar enhancing effect on cysteine peptidase bands not shown). iscussion We recommend, based on the data shown in Figs. 1 and 2, that mini-gels containing gelatin as substrate be used to examine T. cruzi and per- haps other parasite, peptidases. Since the major peptidase detected in mini-gels 50 kDa) was, in fact, the only peptidase in the Y strain that was detected on large gels, mini-gels appear to be much more sensitive for this method of peptidase detection than large gels. Total detergent extracts of epimastigotes showed very similar peptidase patterns amongst different strains of T. cruzi suggesting that many of these enzymes are conserved between strains. Indeed, at least three 50 kDa, 60 kDa and >200 kDa) of the peptidases of strain Y appeared to be present in all other strains examined. However, a greater degree of heterogeneity was observed when extracts were separated into aqueous and
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