Saposin-like proteins are expressed in the gastrodermis of Schistosoma mansoni and are immunogenic in natural infections

Multiple sequence alignment of translated Schistosoma expressed sequence tags (ESTs), which belong to the saposin-like protein (SAPLIP) family. AF521090 corresponds to Sm-SLP-1 and AI977047 corresponds to Sm-SLP-2 before full-length sequence was obtained. GenBank accession numbers are listed following schistosome species: Sm, Schistosoma mansoni; Sj, Schistosoma japonicum (exceptions are Sm01689 which is GeneDB ID and C605193.1 which is a contig ID). D1 and D2 refer to SAPLIP domains 1 and 2 of AI977047, respectively. The conserved cysteine residues are boxed in gray and the histidine thought to be involved in dimerization is shaded pink. Conserved tyrosines found in saposins are shaded in blue. Gaps have been introduced to maximize amino acid alignments.

Comparison of Schistosoma mansoni SLP-1 and SLP-2 predicted proteins with other members of the saposin-like (SAPLIP) family of proteins. (A) Schematic representation of the general architecture of members of the SAPLIP family. Features highlighted include signal peptides (SP), the six-cysteine containing domain, and predicted N-glycosylation sites (N). (B) Multiple sequence alignment of Sm-SLP-1 and SLP-2 with other SAPLIPs using the SAPLIP domain between the first and sixth cysteines only. Sj_BU712004, Schistosoma japonicum (GenBank accession number BU712004); EhA, Entamoeba histolytica amoebapore A (M83945); EhB, E. histolytica amoebapore B (CAA54226); Ac-PFP-1 (Ac-SLP-1), Ancylostoma caninum saposin-like protein (DQ855414); Ce_spp4, Caenorhabditis elegans SAPLIP protein family 4 (AAA81416); Sap_A_slimemould, Dictyostelium discoideum saposin A (BAA32237); Sap_B_human, Homo sapiens saposin B (NP_001035930); Sap_C_bovine, Bos taurus saposin C (P26779); SPB_human, H. sapiens pulmonary surfactant B (P07988); clonorin, Clonorchis sinensis clonorin (AF421960); FhSAP2, Fasciola hepatica SAP2 (AF286903); NK_Lysin, Sus scrofa NK-lysin (CAA59720); Aoah_human, H. sapiens acyloxyacyl hydrolase (BAD97196); countin, D. discoideum AX4 countin (XP_643887); Ce_ZK455.4, C. elegans hypothetical protein (CAA91493); Asm_human, H. sapiens acid sphingomyelin phosphodiesterase 1 (BAD93012). The six conserved cysteine residues are boxed in gray with predicted disulfide bonding patterns shown underneath (1–6, 2–5, 3–4). Hydrophobic residues are boxed in blue; yellow denotes conserved proline in saposins or alanine in SAPLIPs and green highlights the conserved tyrosine (or phenylalanine) in saposins. The histidine predicted to be involved in dimerization of amoebapores is shaded in pink. Predicted alpha-helices of amoebapore A are shown as solid lines above the alignment, and putative N-linked glycosylation sites are shown in bold font. Gaps have been introduced to maximize the alignment.

Expression and purification of recombinant Sm-SLP-1 (rSLP-1) proteins in different expression vectors recombined with baculovirus. (A) Coomassie Brilliant Blue-stained SDS-PAGE gel of purified recombinant Sm-SLP-1 highlighting the size difference due to the different C-terminal purification tags. Lane 1, rSLP-1; lane 2, rSLP-1–pHW. (B) Western blots showing recognition of recombinant proteins by homologous mouse antiserum. Lanes 1 and 3, rSLP-1; lanes 2 and 4, rSLP-1–pHW. Lanes 1–2 probed with normal mouse serum; lanes 3–4 probed with mouse anti-Sm-SLP-1. Molecular weight markers are shown in kDa on the left.

Immunostaining of adult mixed sex Schistosoma mansoni sections with mouse anti-Sm-SLP1 serum followed by anti-mouse-Cy3 (red fluorescence) in all panels and DAPI (blue fluorescence to visualize nuclei) in panels A and B only. All images are shown with corresponding bright field images on the left and fluorescent images on the right. Boxed regions are magnified on the far right of each panel. (A) Section of a male worm probed with pre-immune serum (NMS). (B) Section of two male worms probed with anti-Sm-SLP-1 serum. (C) Section of paired male and female worms probed with anti-Sm-SLP-1 serum. Native Sm-SLP-1 was localized to the gastrodermis (gas) of both male and female adult worms.

(A) The relationship between classes of infection intensity of Schistosoma mansoni and mean IgG against recombinant Sm-SLP-1. Bars represent standard error of the mean; epg, eggs per gram of feces. Classes of infection intensity are set forth by Montresor et al.²⁵ PR=putative resistant (see reference 22). (B) The relationship between infection status of endemic-negative, endemic-positive, and PR individuals and IgG reactivity to recombinant Sm-SLP-1 and crude schistosome adult worm (SmTX) and egg (SEA) antigens. The endemic-positive group was generated from a pool of the three groups of infected people (light, moderate, and heavy) shown in panel A. Bars represent standard errors of the mean.