Fig 1.
Identification of linear B-epitopes on SjSAP4.
(A) Peptide design for epitope mapping. SjSAP4 without the signal peptide (179-AA in length) was divided into 17 peptides, including 16 peptides (P1 –P16) 20-AA in length and another peptide, P17, 19-AA in length. Ten AA were overlapped between any two adjacent peptides. (B) Western blot analysis showed that P15 was recognized by sera obtained from three out of five confirmed schistosomiasis japonica patients. M, Pre-stained protein ladder. Lane 1–17, GST-peptide fusion proteins GST-P1 –GST-P17, respectively. Recombinant SjSAP4 protein was used as a positive control. The serum samples from KK-positive individuals were pre-incubated with purified GST protein.
Fig 2.
Identification of the core epitope on P15.
(A) A panel of truncated peptides spanning the P15 region were expressed as GST fusion proteins. (B) Western blotting and (C) relative quantification analysis determined the recognition of the ten truncated peptides (P15-T1 – P15-T10) by pooled patient sera. Data are represented as the mean ± SD from three different assays (* P < 0.05 when compared with P15-T1 using the student’s t-test). (D) Western blot assay and (E) relative quantification analysis revealed a significant impaired binding of P15-T11 and P15-T12 when compared with P15-T1 using the student’s t-test. Data are represented as the mean ± SD from three different assays (* P < 0.05).
Fig 3.
Multiple sequence alignment of the Sj SapB domains and epitope localization within the Sj SapB domains.
(A) Structure-based sequence alignments of saposin domains of SjSAP4, SjSP-13 variant 1 (SjSP-13V1) and SjSP-13 variant 2 (SjSP-13V2). Secondary structure elements referring to the structure of human saposin A (PDB code 2DOB) are indicated on the top of the alignment. (B) Visualization of immunogenic epitopes in the 3D structure of the SapB domain of SjSAP4 (SjP) and SjSP-13V2 (SjC). Both the core B-cell epitopes (-S163QCSLVGDIFVDKYLD178- on SjSAP4 and -K80CLDVTDNLPE90- on SjSP-13V2) (indicated in magenta), are located in the third α-helix of the SapB domains.
Fig 4.
The three-dimensional structure of SjSAP4 and surface electrostatic contact potential analysis.
(A) The predicted 3D structure of SjSAP4 (without signal peptide) colored in rainbow colors, shows an α-helix enriched region at the N-terminus, a disordered loop in the middle region, and a Sap-B domain at the C-terminus. The pink sticks indicate disulfide bonds formed between cysteine residues. (B) Electrostatic contact potential analysis showing the surface of the core epitope of SjSAP4 presents as a wavy plane with two hydrophobic AA in the center surrounded by polar and charged AA. Positively charged and negatively charged residues are colored in blue and red, respectively, whereas neutral residues are colored in white.
Fig 5.
Diagnostic performance of the SjSAP4-, SjSAP4-Peptide- and SjSP-13V2-Peptide-ELISA.
(A-C) Scatter plots showing the IgG responses to SjSAP4, SjSAP4-Peptide and SjSP-13V2-Peptide, respectively, in healthy controls (n = 35) and KK-positives (n = 50). Data were analyzed using a Mann Whitney U-test. (D-F) Receiver operating characteristic curve (ROC) analysis was performed for the SjSAP4-, SjSAP4-Peptide- and SjSP-13V2-Peptide-ELISA, respectively.
Fig 6.
Complementary recognition of B-cell epitopes and diagnostic performance of the SjSAP4-Peptide + SjSP-13V2-Peptide-ELISA.
(A) Complementary recognition of the SjSAP4-Peptide and SjSP-13V2-Peptide was observed in a subset of KK-positives (n = 25) by ELISA. (B) The specific IgG levels to the dual peptides (SjSAP4-Peptide + SjSP-13V2-Peptide) in healthy controls (n = 35) and KK-positives (n = 50) were evaluated by ELISA. Data were analyzed using a Mann Whiten U-test. (C) ROC analysis was performed for the SjSAP4-Peptide + SjSP-13V2-Peptide-ELISA.