The involvement of proteases in the intra-molluscan stage of parasite development has been well documented (Yoshino et al., 1993
). Although investigations showed that several aspects of parasite development, e.g., snail penetration, nutrient acquisition, and suppression of the snail defense system, rely on the release of proteolytic enzymes (including cysteine proteases) in excretory-secretory products, reciprocal studies to determine whether the presence of similar enzymes in the snail can interfere with the progression of parasite development have not been as intensely investigated. This study shows that higher levels of cysteine protease activity occurs in parasite resistant B. glabrata
than in susceptible snails. It is possible, therefore, that these enzymes may be important in determining the outcome of the S. mansoni
interaction. Although previous studies (Bahgat et al., 2002
; Mitta et al., 2005
) described the presence of several proteolytic enzymes in the snail (aminopeptidase, hydrolase, lysozymes, and genes encoding serine proteases, cathepsin L, and metalloproteases), none described differences (qualitative and quantitative) in activity of these enzymes between parasite resistant and susceptible snails as shown here for cysteine proteinases.
Proteolytic enzymes have been detected in both the humoral and cellular components of the snail's innate defense system, the hemolymph and hemocytes, respectively, with levels changing relative to either bacteria or schistosome infections (Cheng et al., 1977
; Kassim and Richards, 1978
). Granulocytes, a type of hemocyte involved in the cellular encapsulation reaction typically seen in the nonself reaction against incompatible parasites, were shown to express high levels of acid phosphatase activity in a resistant snail upon exposure to S. mansoni
miracidia, and they were thus hypothezied by Cheng and Garrabrant (1977)
to contribute to parasite destruction mediated by these cells. Other snail tissues, including the headfoot, hepatopancreas, and visceral mass (Cheng, 1978
; Cheng and Rodrick, 1980
), were also shown to express high levels of proteolytic enzymes. In the present study, although cysteine protease activity was detected in the hemolymph and ovotestis (data not shown), most of the enzyme activity was present in the hepatopancreas. Insignificant proteolytic activity was detected in the headfoot, and the activity in hemocytes remains to be tested. Experiments to test for enzyme activity in these cells by methods described here were hampered by the difficulty in isolating large numbers of hemocytes. However, in a recent study using the SSH approach, Bouchut et al. (2007)
were able to show the up-regulation of cathepsin L-like transcripts in these cells from an unrelated resistant B. glabrata
snail after exposure to the trematode Echinostoma caproni
. Because it is thought that schistosome sporocysts are not easily destroyed by toxic material present in snail plasma (Bayne and Yoshino, 1989
), we can only speculate that our results showing higher (qualitatively and quantitatively) activity of cysteine proteases in resistant compared with susceptible snails may indicate that these enzymes could be indirectly rather than directly involved in mechanisms relating to the processing of molecules that are directly toxic for sporocysts. The presence of higher enzyme activity in the hepatopancreas (in both resistant and susceptible snails) relative to other tissues, also suggests that these enzymes may play a significant role in the snail's digestive process. Natural subtrates of these cysteine proteases in the snail remain unknown.
Because of results showing elevated activity of these enzymes in the hepatopancreas, a cDNA library was constructed from this tissue and ESTs generated. As expected, several clones corresponding to B. glabrata
hydrolytic enzymes (cellulase, elastase, disintegrin and metalloprotease, lysozyme, α-L
-fucosidase, and serine protease), including cysteine proteases (cathepsin B and L, and legumain), were isolated. One of the ESTs (accession ES491472) encoding the full-length coding sequence of cathepsin B was sequenced in its entirety (accession EU035711). The snail cysteine protease cathepsin B encoding 333 amino acids has all the hallmark domains that are needed for a functional peptidase. Cysteine proteases have characteristic molecular topologies both in their 2- and 3-dimensional structures where the nucleophile is the sulfhydryl group of a cysteine residue. In addition, they are also divided into clans that are evolutionarily related, and further into families on the basis of the architecture of their catalytic dyad or triad (Barrett and Rawlings, 2001
). Based on the above-mentioned criteria, the snail cathepsin B belongs to the MEROPS (accession MER00647; Rawlings and Barrett, 1993
) cysteine peptidase family C1 and subfamily C1A similar to papain. The catalytic residues of family C1 have been identified as Cys and His, forming the catalytic dyad (Cys 115 and His 281). Two other active site residues are found, a Gln residue preceding the catalytic Cys and an Asn residue following the catalytic His (Gln 109 and Asn 301). The C1A cathepsin B family may contain both endo- and exopeptidase activities, which allows it to make internal cleavages and also remove the C-terminal dipeptide units from the substrate. E-64 is an irreversible inhibitor of peptidases in family C1 (Barrett et al., 1982
). In cathepsin B, the presence of an approximately 20-residue “occluding loop” that carries the histidine residues is important for peptidyl-dipeptidase (exopeptidase) activity, and it is inserted between the catalytic Cys and His residues (Illy et al., 1997
). Although we do not know the localization of the snail cathepsin B, the presence of the hyrophobic signal peptide at the amino terminus (residues 1–19) of the preprocathepsin B shows it may be a secreted molecule. In addition, results of proteolysis in the gel zymograhs coinciding with a complex high-molecular-weight smear (220 to 66 kDa) is considerably higher than the expected size of cysteine proteases (approximately 30–36 kDa). The snail recombinant cathepsin B that has been deduced from the translated sequence has a potential N
-glycosylation site (n = 1), protein kinase C (n = 5), casein kinase II (n = 5) phosphorylation sites, and N
-myristoylation (n = 13) sites. It is, therefore, possible that posttranslational modifications accounts for the discrepancies in the sizes of the native enzyme and the deduced amino acid sequence.
The biological role of cathepsins in mechanism(s) relating to the antiparasite function of the snail innate defense system, especially encapsulation, remains unknown. With the availability of several cloned transcripts encoding B. glabrata cysteine pro-teases and at least 1 full-length cathepsin B, characterization of various activities of this enzyme at both biochemical and molecular levels can be achieved with the expression of the recombinant protein. We hope to express the full-length recombinant enzyme in a prokaryotic expression system to raise polyclonal antisera that will be used to purify the native snail cathepsin B by affinity column chromatography. Future physical and biochemical characterization of the purified enzyme should help to resolve the discrepancy between the sizes of the deduced translated sequence and the native enzyme. Antibodies against the recombinant protein will also be useful in the identification of homologs of cathepsin B from hemocytes and other tissues that are not easy to obtain in large quantities, but that are considered important regarding mechanisms involved in snail/parasite interactions, e.g., cerebral ganglia.
Aside from the identification of several ESTs encoding proteolytic enzymes from a hepatopancreas cDNA library (), transcripts encoding a natural inhibitor (Kazal-like serine protease inhibitor) were also isolated. Previously, we identified the gene encoding cystatin, a known inhibitor of cysteine pro-tease from a resistant (BS-90) snail cDNA library (Knight et al., 1998
). Several recent studies have now shown the quantitative increase of the cystatin transcript after trematode infection of B. glabrata
snails (Guillou et al., 2007
; Lockyer et al., 2007
). The occurrence of the proteinase inhibitor α-macroglobulin has also been shown in the snail hemolymph (Bender and Bayne, 1996
; Fryer et al., 1996
In other studies where the effects of parasite infection on hydrolytic enzyme activity have been investigated, levels of glycosidases were shown to correlate with the progress of infection in schistosome infection of B. glabrata
snails (Zelck, 1999
). Likewise, in the American oyster, Crassostrea virginica
, a significant increase in protease activity was observed after infection with the parasite Perkinsus marinus
(Munoz et al., 2003
). Together, it is clear that future investigations of the possible cytotoxicity of proteases and their natural inhibitors toward warding off trematode infection in the snail host are warranted. Results from our studies using real-time quantitative RTPCR showing a higher -fold increase of the corresponding cathepsin B transcript in resistant compared with susceptible snails upon parasite exposure is further evidence that proteolytic enzymes play a significant role in the host–parasite relationship.
In summary, qualitative and quantitative differences in the levels of protease activity have been shown to occur between snail stocks that are either resistant or susceptible to S. mansoni infection, with resistant snails consistently expressing higher protease activity than susceptible snails. The majority of enzyme activity detected corresponded to the presence of cysteine proteases in the hepatopancreas. With the availability of cloned cathepsin B and other cysteine proteases from B. glabrata, we anticipate that the molecular and biochemical pathways involving cysteine proteases in killing of schistosomes in the snail host will soon be unravelled.