Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke.

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.

Fasciola hepatica: localisation of glutathione S-transferase isoenzymes in adult and juvenile liver fluke.

Four cDNA clones (GST-1, -7, -47, and -51) encoding isoenzymes of the detoxification enzyme glutathione S-transferase (GST) have previously been identified and characterised from Fasciola hepatica. In the present study, antisera were generated to synthetic peptides of regions unique to each of the four GST proteins predicted by the cDNAs. The antisera were characterised, and two were found to distinguish GST-1 from GST-7, GST-47, and GST-51 as a group. These two antisera were used to localise different GSTs in adult and newly excysted juvenile F. hepatica. The antiserum to GST-1 was specific and localised GST-1 to the parenchyma of adult fluke but not to the lamellae of the intestinal caeca. The antiserum to a GST-51 peptide, which cross-reacted with GST-7 and GST-47 but not GST-1, localised the other GSTs not only to the parenchyma but also to the intestinal lamellae of adult fluke. This appears to be the first evidence of tissue-specific expression of GST isoenzymes in trematodes. In contrast to adult fluke, immunolocalisation of the GSTs in juvenile F. hepatica revealed the binding of both the GST-1 and GST-51 antisera to the parenchymal cytoplasm, to cytoplasmic extensions of the parenchyma cells in the subtegumental area, as well as the excretory ducts. No labeling was observed in the intestinal epithelium of the juvenile fluke. These results demonstrate that adult F. hepatica, in contrast to juvenile flukes, contain a GST, which is not GST-1, associated with the lamellae of the gut and suggest that GSTs in adult fluke may play a role in the absorptive function of the adult gut.

The secreted cathepsin L-like proteinases of the trematode, Fasciola hepatica, contain 3-hydroxyproline residues.

The cysteine proteinases synthesized by the adult stage of the trematode Fasciola hepatica were found to be a very heterogeneous group of proteins as demonstrated by one- and two-dimensional gel analyses. N-terminal amino acid sequencing indicated the presence of at least two distinct gene products among the secreted cysteine proteinases. Enzymic studies and peptide sequence analysis of the excreted/secreted cysteine proteinases suggested a close relationship to the plant thiol cathepsins and the mammalian cathepsin L subfamily. The cloning of a representative cDNA for a putative Fasciola cathepsin confirmed similarities to the cathepsin L subfamily but revealed low identity with the cathepsin-like proteinases of the related trematode, Schistosoma, nematode cathepsins and the mammalian cathepsin B subfamily. Furthermore, peptide and protein sequencing revealed the modification of certain highly conserved prolines to unusual 3-hydroxyproline derivatives. This is the first report of modified prolines in any proteinase. This finding, as well as the high activities of these cathepsins at neutral to alkaline pH values, raises a number of questions as to the physiological function of these thiol cathepsins and their interaction with host tissues.

Vaccination of sheep with purified cysteine proteinases of Fasciola hepatica decreases worm fecundity.

There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infection. A cysteine proteinase complex identified in the regurgitant of adult F. hepatica was examined in this context. The thiol-cathepsin-related proteinases of M(r) 28,000 were purified and tested in vaccine trials of sheep infected with liver fluke. Ten animals were immunised with the purified proteinases and developed antibodies to the cysteine proteinases prior to challenge with F. hepatica metacercariae. Infection appeared to cause a boost in antibody response by Week 4 into infection, and antibody levels were generally sustained throughout infection. The cysteine proteinases are not novel antigens, since low-level antibody titres were also detected in nonimmunised controls by late infection. On completion of the trial, there was no difference in worm burden between the two groups. However, faecal egg counts and therefore worm fecundity were significantly decreased.

Vaccination of sheep against Fasciola hepatica with glutathione S-transferase. Identification and mapping of antibody epitopes on a three-dimensional model of the antigen.

The glutathione S-transferases (FhGST) of the liver fluke Fasciola hepatica have been identified as novel vaccine candidates that protect sheep against a fluke infection. With the use of overlapping peptides covering the predicted amino acid sequences of four FhGST cDNAs, we have defined the linear epitopes recognized by polyclonal antibody from sheep vaccinated with FhGST. Dominant and minor epitopes were found to be present on all four of the sequences although some epitopes were shown to be specific to particular FhGST. A high percentage of the FhGST peptides were found to be antigenic although considerable variability in response to the peptides was observed among the animals. This analysis was extended to the IgG1 and IgG2 response at the peptide level. Based on the recently solved crystal structure of the rat mu-class GST 3-3, a three-dimensional model of one of the FhGST sequences was generated that allowed the predicted spatial localization of defined epitopes. Most epitopes were localized on regions of high flexibility and accessibility. A comparison of epitopes on FhGST with the B cell epitopes on Sm28, a 28-kDa GST from Schistosoma mansoni, has found few similarities. There was no correlation between an antibody response to linear peptide epitopes and the level of protection induced in sheep by vaccination with FhGST.

Primary sequence heterogeneity and tissue expression of glutathione S-transferases of Fasciola hepatica.

Glutathione S-transferases (GSTs) from Fasciola hepatica have been purified by glutathione affinity chromatography. Two closely migrating species of Mr 26,000 and 26,500 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several species resolved by two-dimensional gel analysis, indicating substantial heterogeneity among the GSTs. N-terminal amino acid sequencing revealed one core sequence containing three polymorphisms, whereas the sequence of GST peptides implied a minimum of three different GSTs. The amino acid sequence data assigned the F. hepatica GSTs to the mu class of GSTs with high similarities to these proteins in other helminths and mammals. The native GSTs of F. hepatica appeared to behave as dimers as determined by molecular sieving chromatography. The observation that the GSTs of F. hepatica are heterogeneous in sequence and behave as dimers in the native state suggest that these isoenzymes may exhibit considerable functional heterogeneity which may be of importance to the parasite. Immunocytochemical studies suggest that the main source of GST in F. hepatica are the parenchymal cells and peripheral tissues of the parasite. Some extracellular GST is associated with the lamellae of the intestinal epithelium. The identification of an intestinal GST is unique among trematodes studied to date.

Characterization of a 95,000 molecule on sheep leucocytes homologous to murine Pgp-1 and human CD44.

The phagocyte glycoprotein-1 (Pgp-1) antigen of mice is a 94,000 MW molecule with a wide tissue distribution, but no attributed function. We produced a monoclonal antibody (mAb) termed 25-32 which recognizes the Pgp-1 molecule of numerous mammalian species, including humans and sheep. Preclearing experiments with I42/5, a rat anti-mouse Pgp-1 mAb that cross-reacts with human Pgp-1, established the specificity of 25-32 for human and sheep Pgp-1. Moreover, an antibody recognizing human CD44, termed F10-44-2, also reacted with the same molecule as that recognized by 25-32 and I42/5, so establishing the co-identity of CD44 and Pgp-1. Within the sheep thymus, Pgp-1 was expressed most strongly by medullary thymocytes and stromal cells, and by small numbers of cells at the subcapsular cortex. Pgp-1 was expressed early in thymic ontogeny; all 35-40-day gestation fetal sheep thymocytes were intensely Pgp-1+, but by 80 days the number of reactive thymocytes had decreased to adult levels. The expression of Pgp-1 on lymphocytes was markedly increased after stimulation with mitogens, or with phorbol esters and ionomycin. The highly conserved nature of Pgp-1 through evolution, its expression on virtually all cell types within the body, and its increased expression on rapidly dividing cells indicate that this molecule mediates an important function, possibly serving as a hormone or metabolite receptor.

Genetic and biochemical characterization of antigens encoded by the Ly-24 (Pgp-1) locus.

Three allele-specific monoclonal antibodies to Pgp-1 (Ly-24) were used to biochemically characterize the cell surface structures with which they reacted and to map the gene(s) coding for these antigens. The targets of these three monoclonal antibodies (mAb) were shown to be encoded by a gene situated on chromosome 2 close to beta 2m [gene order (Pgp-1-beta 2m-a)] and no recombination between the loci detected by the three antibodies was revealed by genetic analysis. The genetic mapping of loci and tissue distribution of these antigens suggested that they might all correspond to a particular allelic form of the mouse phagocyte glycoprotein-1 (Pgp-1) antigen. Biochemical and serological analysis confirmed that this was indeed the case and revealed that all three mAbs were directed to one epitope. It is surprising that the tissue distribution defined by one mAb (Ly-24A) was different from that for the two other (Ly-24B) antibodies, despite the serological and biochemical identity of their respective targets. The possible reason for this unusual finding is discussed.