Purification and characterisation of a cysteine ...

1 downloads 0 Views 211KB Size Report
The parasitic trematode Fasciola- burrows through the gut wall of its mammalian host and spends seven weeks migrating within the liver mass before taking up ...
86s Biochemical Society Transactions ( 1 99 1) 20 Purification and characterisation of a cysteine proteinase secreted by Fascioia hggmlhx. Andrew J. Dowd, Angela M. Smith, Carlos Carmona, Colin Robertson' and John P. Dalton. School of Biological Sciences, Dublin City University, Dublin 9,Eire.' Dept. Zoology, Univ. of Glasgow, U.K. The parasitic trematode Fasciolaburrows through the gut wall of its mammalian host and spends seven weeks migrating within the liver mass before taking up residence in the immunologically safe environment of the bile duct. While migrating the liver fluke is in direct contact with the host's circulation and therefore with its immune system. Many mechanisms of evasion of the host's immune system have been proposed including the production of proteinases which cleave host IgG in a Cathepsin-B or papain-like manner releasing Fab and Fc fragments [1,2]. In our laboratory a . E m cysteine proteinase was partially purified by HPLC size exclusion chromatography and was classified as a Cathepsin-B on the basis of its substrate specificity and pH optimum [2].ln this work we report (a) the purification of the€ heDatica proteinase, (b) the characterisation of this enzyme by using fluorogenic substrates and (c) the production of polyclonal antisera to the purified proteinase and the utilisation of immunofluorescence to locate the proteinase in the tissues of adult fluke. E m parasites were obtained from a local abbattoir and maintained in RPMl medium overnight [3].The culture medium was removed and centrifuged at 10,000 x g and the supernatant was stored at -20 C.Fifty ml of the stored supernatant was dialysed against 0.1 M Tris-HCI pH 7 at 4C and was subsequently applied to a 50 ml QAE-Sephadex column equlibrated in 0.1M Tris-HCI pH 7. The runthrough fraction from the QAE-Sephadex column was applied to a 120 ml Sephacryl S-200 column and monitored for Cathepsin activity using the fluorogenic substrate Z-Phe-Arg-AMC. The fractions containing ZPhe-Arg-AMC activity were pooled and analysed by gelatin substrate SDS PAGE [3]. Fluorogenic substrate analysis in SDS PAGE gels were carried out as described by Robertson at aL [4]. The fluorogenic substrates used included Z-Phe-Arg-AMC, H-Pro-PheArg-AMC, H-Phe-Val-AMC, Suc-Ala-Phe-Lys-AMC,ZArg-Arg-AMC and Z-Arg-AMC. Antisera to the cysteine proteinase were prepared by applying culture media from an overnight incubation of parasites to a SDS PAGE gel. A gelatin substrate SDS PAGE gel was run under identical conditions to allow the identification of the separated proteinase bands. The bands containing the proteinase were excised and used to raise antibodies in rats [5]. lmmunolocalisation studies were carried out on JB-4 plastic embedded adult fluke sections as outlined in Aronstein elal.[6]. Gelatin substrate SDS PAGE of the QAE-Sephadex run-through fraction showed that the proteolytic bands were identical to those obtained by HPLC purification of a 100 ug of crude culture medium (21. Contaminating proteins present after QAE-Sephadex chromatography were removed by gel filtration on a Sephacryl S-200 column. The capacity of the QAE-Sephadex column was 3.25 mg of protein which allows the purifcation of

the cysteine proteinase in a 30-fold greater quantity than the HPLC method reported previously [2]. To determine if the proteinase isolated had Cathepsin-like activity fluorogenic substrate SDS PAGE was carried out on the purified protein. The results from this method demonstrated that the substrate Z-Phe-Arg-AMC was cleaved while the substrate Z-Arg-Arg-AMC was not cleaved by the proteinase. This result classifies the proteinase as a Cathepsin-L rather than a CathepsinB. Another observation is the preference of the proteinase for the hydrophobic amino acid phenylalanine in the P2 position on the substrate which is typical of Cathepsin-L proteinases.

Fig. 1.lmmunolocalisation studies on plastic embedded adult liver fluke sections. Antibodies prepared against the Cathepsin 1 proteinase excised from PAGE were used to locate the enzyme to vesicles in the gut epithelial cells. GE, gut epithelium; parenchyma; T; tegument.

lmmunolocalisation studies using antisera prepared from the Cathepsin-L excised from SDS PAGE gels show a distinctive labelling pattern. Labelling was confined to vesicles which seemed to be pinched off from gut epithelial cells (Fig.1.). This demonstrates that Cathepsin-L proteinases are packaged and secreted from vesicles within the gut epithelial cells. The results presented in this report demonstrate that the antibody cleaving enzyme as reported earlier [2] is in fact a Cathepsin-L and can be purified in large quantities using a combination of QAE-Sephadex chromatography and gel filtration. Results from immunolocalisation studies show that the Cathepsin-L enzyme is located in vesicles in the gut which may be lysosomes modified for secretion of proteinase in sufficiently high concentrations in order to evade the host immune system. 1. Chapman, C.B. & Mitchell, G.F. (1982) Vet. Parasitol. 11,165-1 78. 2. Heffernan, M., Smith, A., Curtin, D., Mc Donnell, S . , Ryan, J., & Dalton, J.P. (1990) Biochem. SOC.Trans. 636 th meeting Dublin. 3. Dalton, J.P., & Heffernan, M. (1989) Mol. Biochem. Parasit.35, 161-166. 4. Robertson,C.D., North, N.J., Lockwood, B.C., & Coombs,G.H. (1990) J. Gen. Micro. 136,921-925. 5. Heffernan, M., Smith, A,, Dowd, A., Robertson, C., & Dalton,J.P. (1991) Mol.Biochem.Parasit.(submitted). 6. Aronstein, W.S., Dalton, J.P., i 3 Strand, M. (1985) Am. J.Trop. Med. Hyg. 34, 889-897. We acknowledge the technical assistance of C. Wilson.