Extracellular loop of the candidate proteins contain SEA-domains.

<p>(<b>A</b>) Modeled molecular structures of the extracellular domains with striking similarity with SEA-domain. Also shown for comparison is the SEA-domain of mouse TMPRSS2. Typical of SEA-domains, the secondary structure components showed an antiparallel arrangement of β-sheets. A summary of structural models of the entire transcripts in this gene family is shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002644#pntd.0002644.s008" target="_blank">Table S1</a>. (<b>B</b>) Rigid body superposition of SjP3842 (blue) over the highest scoring template, PDB: 2e7v (olive). The graph is the Ramachandran plot (φ/ψ) showing conformational angles distribution of the residues. Over 98% of residues were in the favored regions while less than 2% were in the outlier region. (<b>C</b>) Alignments of SjCP3842 with two well defined SEA-domains (human MUC1 and mouse TMPRSS2). Putative SEA-domain consensus cleavage site (red arrow) was identified between β2 and β3.</p

Protein expression and antigenicity of the candidate proteins.

<p>(<b>A</b>) SDS-PAGE of recombinant <i>E. coli</i> lysates (lane L) and purified protein without inclusion bodies (lane P). Arrows indicate expected molecular weights of oligomers. Other bands are expected SEA-domain cleavage products. (<b>B</b>) Western blots of recombinant protein expression as in (B), probed with anti-HisG antibody. (<b>C</b>) Anti-HisG probed western blots showing oligomerization of proteins with multiple bands of additive ∼30 kDa subunits, and tetramer as the most stable state. (<b>D</b>) Size exclusion gel filtration chromatography of SjCP3842 showed multiple elution peaks, another evidence of oligomerization. (<b>E</b>) Immunoblots showing reactivity of parasite crude antigen preparations (SEA and SWA) with immune sera. (<b>F</b>) The candidate proteins specifically reacted with infected miniature pig sera in IgG ELISA, indicating potential antigenicity during schistosomiasis. Bars represent standard deviation (SD). * = p<0.05, ** = p<0.01. n = 4 for each group.</p

Heme-binding pocket of SjCP3842.

<p>(<b>A</b>) Heme-binding mode of SjCP3842 showing the hydrophobic vinyl end of the protoporphyrin heme inserted into a hydrophobic cavity, while hydrophilic propionate end of points away from the pocket. Heme is represented using spheres model colored by atoms (C: green, N: blue, O: red, Fe: brown). The protein is shown using cartoon model. (<b>B</b>) Heme-binding site showing the Connolly surface of the binding pocket (dots). (<b>C</b>) Heme iron (brown sphere) hexa-coordinated with His-149 and His-147 as axial ligands.</p

<i>S. japonicum</i> SEA-domain proteins are heme-binding proteins.

<p>(<b>A</b>) Hemin-agarose binding assay showing potential of SjCP3842 to bind heme on hemin-agarose beads. (<b>B</b>) Hemin-agarose binding assay confirmed by immunoblotting using three candidates. ‘U’: unbound, ‘W’: last wash, ‘E’: eluates. (<b>C</b>) Identification of SjCP3842 in heme-binding protein fractions from parasite crude extracts (SWA). (<b>D</b>) Estimation of the amount of heme bound using peroxidase activity of bound heme. Standard curve (linear graph) of peroxidase activity of known concentrations of hemin was used to estimate the amount of bound heme. (<b>E</b>) Differential spectral titration of protein-heme interaction using 10 µM of heme and increasing concentrations of the protein (0 to 28 µM). Soret peak was red shifted from 388 nm to 412 nm, and absorption maximum increased with increasing accumulation of protein-heme complex. The inset is the heme-binding curve constructed by plotting ΔA₄₁₂ versus protein concentration, showing 1∶1 stoichiometry.</p

Developmental stage specific expression profiles of the candidate genes.

<p>Developmental stage specific expression of the candidate genes presented as copy number per nanogram of cDNA. The full data statistics is shown in a supplementary table (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002644#pntd.0002644.s009" target="_blank">Table S2</a>). There was differential expression of the three characterized genes among developmental stages of the parasite, with SjCP3842 expressed at higher levels relative to the other two candidates. (<b>A</b>) SjCP3842 was overtly expressed at the adult stage especially in female adult worm. The expression level at the snail intermediate sporocyst and cercaria stages were also relatively high as compared to schistosomula and egg stage. SjCP3842 was expressed at the minimal level at the egg stage. (B) Conversely, SjCP1084 was mainly expressed at the egg stage in relation to other stages. (C) The expression levels of SjCP1531 in all stages of the parasite were relatively low and mainly expressed at the egg and adult stages. Bars represent standard deviation (SD). * = p<0.05, ** = p<0.01. n = 4 for each group.</p

Immunolocalization on the teguments and gastrodermis.

<p>Tissue localization of native SjCP3842 was shown using immunofluorescence (A–F) and immunoperoxidase (G–L) methods. (<b>A</b>) Bright field image of cross section of adult worm pair. (<b>B</b>) Bright field image of cross section of female adult worm showing the ovary. (C) Bright field image of a cross section of adult worm pair for control IFA. (<b>D</b>) IFA on a cross section of adult worm pair using monoclonal antibody showed that the native SjCP3842 was localized on the adult worm tegument (t) and gastrodermis (yellow arrows) of the parasites gut (g). Scale bar = 50 µm. (<b>E</b>) IFA on a cross section of a female worm showing FITC staining of SjCP3842 on adult worm teguments but not in the content of the ovary. DAPI staining of nuclei was detected in the tissue and ovary. (<b>F</b>) IFA using pre-immune serum as negative control. (<b>G</b> and <b>H</b>) Immunoperoxidase detection (brown) of SjCP3842 in a section of adult worm pair showed localization on adult worm tegument. (<b>I</b>) Immunoperoxidase localization of SjCP3842 on juvenile schistosomula showed localization on the tegument. Negative immunoperoxidase detection was observed in sections of adult worm (<b>J</b> and <b>K</b>) and schistosomulae (<b>L</b>) probed with pre-immune serum.</p

<i>S. japonicum</i> SEA-domains mediate binding to glycosaminoglycans (GAGs).

<p>Interactions between glycans and SEA-domain proteins were analyzed using array type sugar chip in SPR system. Shown here are the SPR imaging and SPR signals (RU), which is proportional to the amount of proteins bound to glycans immobilized on sensor chips in an array format. There was high-affinity binding to chondroitin sulfate, heparin, dextran sulfate and other sulfated GAGs. The binding kinetics is shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002644#pntd.0002644.s006" target="_blank">Figure S6</a>.</p