Research on calpain of Schistosoma japonicum as a vaccine candidate

Vaccine development by the use of calpain of Schistosoma japonicum has been tried in our laboratory. We cloned cDNA encoding the heavy chain of S. japonicum calpain, and prepared recombinant molecule of a possible vaccine region of the heavy chain. When BALB/c mice were immunized with our recombinant calpain of S. japonicum with Freund's complete adjuvant, we observed significant reduction in worm burden (41.2% reduction, P<0.05), and also significant anti-fecundity effects. In this sense, calpain of S. japonicum seems to have infection control as well as anti-disease effects. Mechanisms of vaccine effects of calpain remain to be clarified, however, several effector mechanisms are suspected. In immunized mice, raised level of iNos expression was observed, while adhesion of peritoneal exudates cells were also observed in the presence of calpain-immunized sera, suggesting the possibilities of both cellular and humoral protective mechanisms. We examined tissue distribution of calpain in various developmental stages of S. japonicum. Strong signal was observed around excretory grand of cercariae, and they secreted calpain during their migratory movement tested in vitro. Together with the findings, calpain seems to induce larvicidal effects in the immunized mice. We observed time-course kinetics of antibody production against vaccine candidates in experimental S. japonicum infection in pigs. Although significant levels of antibody production were observed for paramyosin and GST, no significant antibody production was observed for calpain. This suggests that calpain is less immunogenic, and route of immunization and/or choice of adjuvant are important in future trials of calpain vaccine.     

Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3' and 5' ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coll.SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (positive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also suggested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.     

Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3′ and 5′ ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2 pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokary- otic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (posi- tive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also sug- gested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.     

Molecular cloning and functional expression of a cDNA encoding the major endoplasmic reticulum-associated calcium-binding protein, calreticulin, from Philippine strain Schistosoma japonicum

We describe the cloning of a full length calreticulin (CR)-encoding cDNA clone isolated by immunoscreening of a cDNA library prepared with mRNA from adult worms of the Philippine strain of Schistosoma japonicum, the cause of Asian schistosomiasis. The sequence of the cDNA is presented, and its molecular characterisation and functional expression as a Ca2+-binding protein described. The potential role of CR in inducing protective immunity in the schistosomes is discussed.     

Complete sequence and expression of a cDNA encoding a chicken 115-kDa melanosomal matrix protein

A full-length cDNA clone encoding a 115-kDa melanosomal matrix protein (MMP115) was isolated from a cDNA library constructed from poly(A)+ RNA of the chicken pigmented epithelial cells. Sequence analysis showed that the cDNA encoded a polypeptide of 762 amino acids, including a hydrophobic signal peptide. There are no membrane-spanning regions, but there are five N-linked glycosylation signals. A cysteine- and histidine-rich domain is present near the C-terminus. A sequence of 24 amino acids is repeated three times in the polypeptide. A database search for homologies yielded no sequence similarities in other proteins. A plasmid containing the full-length cDNA was transferred into mouse cell lines by transfection. The transfected cells produced a protein that had the same size, 115 kDa, as the mature MMP115. When B16 mouse melanoma cells were transfected, the chicken MMP115 was expressed in the melanosomes. The presence of a specific sorting signal was suggested for localization of melanosomal proteins. Southern blot analysis has revealed that the homologues of the chicken MMP115 gene are found in many vertebrate genomes.     

Characterisation and expression studies of a root cDNA encoding for ferredoxin-nitrite reductase from Lotus japonicus

A full-length cDNA encoding for ferredoxin-nitrite reductase (NiR, EC 1.7.7.1), has been isolated from a root cDNA library from the legume Lotus japonicus and characterised. The NiR gene ( Nii ) is present as a single copy in this plant, and encodes a protein of 582 amino acids. The Lotus NiR protein is synthesised as a precursor with an amino-terminal transit peptide consisting of 25 amino acid residues. Sequence comparisons with leaf NiRs from different plant species and with other related redox proteins identified in the root NiR the same highly conserved residues involved in the cofactor binding than previously reported for leaves. Besides, a putative binding site for ferredoxin was also found in the N-terminal region of the protein. The NiR gene is expressed in roots and leaves, although the level of expression is much higher in roots, in accordance with the fact that L. japonicus assimilates nitrate mainly in roots. NiR mRNA, protein and activity are induced by nitrate in roots and leaves, while ammonium-grown plants only showed basal levels. No oscillations of NiR mRNA, protein and activity were observed during the day/night cycle, neither in roots nor leaves, making an interesting difference with rhythms observed in other plant species.     

Schistosoma japonicum: localization of calpain in the penetration glands and secretions of cercariae

A monoclonal antibody was generated against the large subunit of Schistosoma japonicum calpain to study the localization and possible function of the molecule in vivo. Mice were immunized with recombinant S. japonicum calpain and polyclonal antisera and a monoclonal antibody specific to schistosome calpain was obtained. In immunohistochemistry, a monoclonal antibody against S. japonicum calpain, KG-2E11, bound weakly to calpain expressed at the surface of adult worm tegument, however, it bound strongly to the cercarial secretions ("footprints") of S. japonicum, emitted from the penetration glands. The present study indicates that calpain is multifunctional as it is expressed at various locations in different developmental stages. Calpain-based vaccines could thus possibly induce protective immunity against cercariae and the following early developing stages.     

Characterization of protective immunity induced against Schistosoma mansoni via DNA priming with the large subunit of calpain (Sm-p80) in the presence of genetic adjuvants

Despite advances in control via snail eradication and large-scale chemotherapy using praziquental, schistosomiasis continues to spread to new geographic areas particularly in sub-Saharan Africa. Presently, there is no vaccine for controlling this disease. We have concentrated on a functionally important schistosome antigen Sm-p80 as a possible vaccine candidate for schistosomiasis. Here we report the proliferation of spleen cells in response to the recombinant Sm-p80 protein and cytokine (IFN-gamma and IL-4) production by the splenocytes. These spleen cells were obtained from groups of mice that were vaccinated with a DNA vaccine formulation containing Sm-p80 and one of the Th-1 (IL-2 or IL-12) or Th-2 (GM-CSF, IL-4) enhancer cytokines. The splenocytes from the groups of mice vaccinated with Sm-p80 DNA in the presence of Th-2 enhancer cytokines showed moderate but detectable proliferation. The splenocytes obtained from mice vaccinated with Sm-p80 DNA with Th-1 enhancer cytokines IL-2 and IL-12 provided the highest proliferation. The IFN-gamma production by splenocytes was found to follow the similar pattern [(Sm-p80) < (Sm-p80 + IL-4) < (Sm-p80 + GMCSF) < (Sm-p80 + IL-12) < (Sm-p80 + IL-2)], as has been observed for the proliferation and protection data. However, the elevated IL-4 production was inversely correlated to Sm-p80-induced splenocyte proliferation or the protection. These results show again that protective immune response induced by Sm-p80 is of Th-1 type.     

Cloning of the calpain regulatory subunit cDNA from fish reveals a divergent domain-V

Calpains are Ca2+-dependent intracellular cysteine proteases, including the ubiquitously expressed micro- and m-calpains. Both are heterodimers, consisting of a distinct catalytic subunit and a common regulatory subunit. We describe cloning and sequencing of the calpain small (regulatory) subunit (cpns) cDNA from rainbow trout. This represents the first fish and lower vertebrate full cDNA of cpns. The rainbow trout cpns cDNA was used to retrieve the zebra fish and Japanese flounder homologues. We present evidence that fish cpns, unlike the conventional mammalian predominant isoform, cpnsl, is lacking the glycine-rich region of domain V. Because the glycine-rich region is known to play a role in membrane targeting, this divergent cpns suggests potentially different functional and activation mechanisms of the fish calpain system. A phylogenetic tree for the cpns gene superfamily has been constructed and the evolution of cpns considered.     

Autolysis of calpain large subunit inducing irreversible dissociation of stoichiometric heterodimer of calpain

Calpain, a calcium dependent cysteine protease, consists of a catalytic large subunit and a regulatory small subunit. Two models have been proposed to explain calpain activation: an autolysis model and a dissociation model. In the autolysis model, the autolyzed form is the active species, which is sensitized to Ca2+. In the dissociation model, dissociated large subunit is the active species. We have reported that the Ca2+ concentration regulates reversible dissociation of subunits. We found further that in chicken micro/m-calpain autolysis of the large subunit induces irreversible dissociation from the small subunit as well as activation. So we could propose a new mechanism for activation of the calpain by combining our findings. Our model insists that autolyzed large subunit remains dissociated from the small subunit even after the removal of Ca2+ to keep it sensitized to Ca2+. This model could be expanded to other calpains and give a new perspective on calpain activation.     

Characterisation of a family of Schistosoma japonicum proteins related to dynein light chains.

Dynein light chains (DLC) are components of dynein, an enzyme complex involved in various aspects of microtubule-based motility. We report here the molecular cloning and sequencing of cDNAs encoding a family of DLC-like polypeptides (SjcDLC1-5) from the human bloodfluke Schistosoma japonicum with open reading frames of 87-104 amino acids and deduced molecular masses ranging from 10.5 to 12.3 kDa. Two-dimensional Western blot analysis confirmed the presence of several S. japonicum DLC isoforms with differing pI values and molecular sizes. We also describe the molecular characterisation, genomic organisation and expression of clone SjcDLC1, and the immunological characterisation and localisation of its encoded protein. Northern blot analysis of adult worm RNA indicated SjcDLC1 is encoded by a single message of approximately 650 bp and Southern analysis suggested one SjcDLC1 gene exists in the S. japonicum genome. Immunolocalisation studies demonstrated that the SjcDLC1 protein is present in the tegument of the adult and cercarial stages of S. japonicum. SjcDLC1 and the other SjcDLC may function in the transport of specialised organelles, comprising membranous and discoid bodies, through the tegument to the schistosome-unique heptalaminate tegumental membrane at the external surface of the adult worm. As a consequence, they may provide novel targets for anti-schistosome vaccine and/or drug development.     

Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris

Novel affinity ligands, consisting of ATP-resembling part coupled with specificity determining peptide fragment, were proposed for purification of protein kinases. Following this approach affinity sorbents based on two closely similar ligands AdoC-Aoc-Arg4-Lys and AdoC-Aoc-Arg4-NH(CH2)6NH2, where AdoC stands for adenosine-5'-carboxylic acid and Aoc for amino-octanoic acid, were synthesized and tested for purification of recombinant protein kinase A catalytic subunit directly from crude cell extract. Elution of the enzyme with MgATP as well as L-arginine yielded homogeneous protein kinase A preparation in a single purification step. Also protein kinase A from pig heart homogenate was selectively isolated using MgATP as eluting agent. Protein kinase with acidic specificity determinant (CK2) as well as other proteins possessing nucleotide binding site (L-type pyruvate kinase) or sites for wide variety of different ligands (bovine serum albumin) did not bind to the column, pointing to high selectivity of the bi-functional binding mode of the affinity ligand.     

Characterisation of three cDNA clones encoding different mRNAs for the precursor to the small subunit of wheat ribulosebisphosphate carboxylase.

We have isolated and sequenced three cDNA clones for the nuclear-encoded precursor to the small subunit of the chloroplast enzyme, ribulose-1,5-bisphosphate carboxylase of wheat. The nucleotide sequences of these clones are different, indicating that they are probably derived from three different mRNAs. This finding is consistent with the proposal that this polypeptide is encoded by a multigene family in wheat, in support of similar data reported by Broglie et al. (Bio/Technology 1:55-61, 1983). We deduce that the mature small subunit polypeptide is comprised of 128 amino acids and that its precursor contains an N-terminal transit peptide sequence. The sequences of both the mature small subunit and its transit peptide differ at several positions from those determined by Broglie et al, (1983) from a different wheat cultivar. Different wheat cultivars might therefore contain different small subunit polypeptides. A comparison of nucleotide and amino acid sequences of the small subunit from wheat, pea, soybean and spinach shows that these sequences are not highly conserved, particularly between monocotyledon and dicotyledon species.