Sj-FABPc fatty-acid-binding protein of the human blood fluke Schistosoma japonicum: structural and functional characterization and unusual solvent exposure of a portal-proximal tryptophan residue.

Sj-FABPc of the blood fluke of humans, Schistosoma japonicum, is a member of the FABP/P2/CRBP/CRABP family of beta-barrel cytosolic fatty-acid-binding and retinoid-binding proteins. Sj-FABPc has at least eight different variants encoded by a single-copy polymorphic gene. In fluorescence-based assays, recombinant Sj-FABPc was found to bind 11-(dansylamino)undecanoic acid (DAUDA), inducing a shift in peak fluorescence emission from 543 to 493 nm. A similar spectral change was observed in dansyl-amino-octanoic acid (in which the dansyl fluorophore is attached at the alpha-carbon rather than the omega-carbon of DAUDA), indicating that the ligand enters entirely into the binding site. Sj-FABPc also bound the naturally fluorescent cis-parinaric acid, as well as oleic acid and arachidonic acid, by competition, but not all-trans-retinol. Dissociation constants were, for cis-parinaric acid, K(d)=2.5+/-0.1 microM (mean+/-S.E.M.) and an apparent stoichiometry consistent with one binding site per molecule of Sj-FABPc and, for oleic acid, K(i) approximately 80 nM. A deletion mutant from which alpha-II was absent failed to bind ligand. Sj-FABPc modelled well to known structures of the protein family; an unusually solvent-exposed Trp side chain was evident adjacent to the presumptive portal through which ligand is thought to enter and leave. Intrinsic fluorescence analyses of Sj-FABPc and of the deletion mutant (from which Trp-27 is absent) confirmed the unusual disposition of this side chain. Virtually all members of the FABP/P2/CRBP/CRABP protein family have prominent hydrophobic side chains in this position, with the exception of liver FABP and ileal FABP, which instead have charged side chains. Liver FABP is known to be distinct from other members of the protein family in that it does not seem to contact membranes to collect and deposit its ligand. It is therefore postulated that the unusually positioned apolar side chains in Sj-FABPc and others in the family are important in interactions with membranes or other cellular components.

B-cell epitopes recognized by Chinese water buffaloes (Bos buffelus) on the 22 kDa tegumental membrane-associated antigen (Sj-22) of the Asiatic bloodfluke, Schistosoma japonicum.

The 22.6 kDa tegumental membrane-associated antigen of schistosomes is of recognized importance in immunity to schistosomiasis. In China, bovines are known to play an important role in the transmission of Schistosoma japonicum. Ten buffaloes (Bos buffelus) were vaccinated with a recombinant form (reSj-22) of the S. japonicum 22.6 kDa tegumental antigen (Sj-22) and the sera were used to identify and map possible linear B-cell epitopes on this molecule using a series of 18 overlapping synthetic peptides (P1-P18). Sera from all of the ten vaccinated buffaloes reacted strongly with Sj-22 in western blots and in ELISA, while sera from a further ten adjuvant (Quil A) control buffaloes did not. Four peptides (P3, P8, P9 and P10) were predominantly recognized by at least 90% of the buffalo sera. This pattern of recognition is similar to that obtained in a previous study we undertook in mice immunized with the same antigen whereby peptides 3, 8, 9 and 10 were recognized by over 80% of CBA strain mice. The peptide most frequently recognized by mice (peptide 6), and mapping to an EF-hand calcium binding domain, was recognized by six of the ten vaccinated buffaloes. The major difference between buffaloes and mice occurred with peptide 1 which was recognized very frequently by all three strains of mice tested but was only weakly recognized by three of the ten buffaloes. This study provides a valuable reference for further study on the immunity stimulated by the 22.6 kDa tegumental antigen in the murine model and a natural bovine host of Schistosomiasis japonica.

Molecular characterization of a calponin-like protein from Schistosoma japonicum.

The gene for a Schistosoma japonicum (Philippine strain origin) (Sjp) calponin-like protein has been cloned and characterised. The clone, designated P14, was isolated from a Sjp adult worm lambda ZAP cDNA library by immunoscreening, and was shown to contain a full-length cDNA encoding a 38.3 kDa protein that shared significant sequence similarity to a number of previously reported calponins and 22 kDa smooth-muscle proteins. Northern analysis indicated the P14 transcript was approximately 2.2 kb in both Sjp and Chinese strain S. japonicum (Sjc) adult worms. Southern blot analysis of genomic DNA suggested that several copies of the P14 gene are present in the Sjc and Sjp genomes but only one copy was evident in the S. mansoni (Sm) genome. Western blot analysis indicated that the product of P14 occurs as a 38 kDa protein in adult Sjp worms and homologues are present in adult worms of Sjc and Sm. At least six isoforms, all with a similar molecular size of approximately 38 kDa and isoelectric points ranging from 8.1 to 9.5, were present in adult Sjc worms. The protein was immunolocalized to the muscle of male and female Sjc adult worms. Recombinant protein was expressed in E. coli and purified under denaturing conditions, and in yeast to produce a soluble protein in purified form. The availability of purified, correctly folded protein will allow investigations into its biological functions and potential involvement in host 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.

Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPC) from the human blood fluke Schistosoma japonicum.

We report the gene cloning, molecular characterisation and purification of a 14.7-kDa functionally active recombinant (re) cytoplasmic fatty acid-binding protein (Sj-FABPC) from the Chinese strain of the human bloodfluke Schistosoma japonicum (Sj). As schistosomes are unable to synthesise long chain fatty acids and sterols de novo and must, therefore, take up these lipids from the host, Sj-FABPC is an attractive vaccine and/or drug target. Clone 39 (C39), which contains the entire Sj-FABPC gene, was isolated from a Sj lambda ZAPII cDNA expression library immunoscreened with hyperimmune rabbit serum (HRS) raised against soluble adult Sj proteins. The complete ORF (open reading frame) of Sj-FABPC encodes a protein of 132 amino acids (aa) of 14.7 kDa. The aa sequence of Sj-FABPC exhibits 91% identity to a FABP of S. mansoni (Sm14) and 45% identity to a FABP of Fasciola hepatica (Fh15), putative vaccine candidates for schistosomiasis. Sj-FABPC was subcloned into the QIAexpress vector, pQE-10, and subsequently expressed in Escherichia coli. The re-Sj-FABPC, purified under non-denaturing conditions, was recognized by sera from patients with acute and chronic schistosomiasis japonica. The purified re-Sj-FABPC was also shown to bind to palmitic acid with high affinity. The functional expression of Sj-FABPC will facilitate studies on re-Sj-FABPC to assess its potential as a drug and/or vaccine candidate.