Characterization of schistosome tegumental alkaline phosphatase (SmAP)

Abstract

Schistosomes are parasitic platyhelminths that currently infect over 200 million people globally. The parasites can live for years in a putatively hostile environment - the blood of vertebrates. We have hypothesized that the unusual schistosome tegument (outer-covering) plays a role in protecting parasites in the blood; by impeding host immunological signaling pathways we suggest that tegumental molecules help create an immunologically privileged environment for schistosomes. In this work, we clone and characterize a schistosome alkaline phosphatase (SmAP), a predicted ∼60 kDa glycoprotein that has high sequence conservation with members of the alkaline phosphatase protein family. The SmAP gene is most highly expressed in intravascular parasite life stages. Using immunofluorescence and immuno-electron microscopy, we confirm that SmAP is expressed at the host/parasite interface and in internal tissues. The ability of living parasites to cleave exogenous adenosine monophosphate (AMP) and generate adenosine is very largely abolished when SmAP gene expression is suppressed following RNAi treatment targeting the gene. These results lend support to the hypothesis that schistosome surface enzymes such as SmAP could dampen host immune responses against the parasites by generating immunosuppressants such as adenosine to promote their survival. This notion does not rule out other potential functions for the adenosine generated e.g. in parasite nutrition.

Figure 1. Alignment of SmAP predicted amino acid sequence with homologs.

Designations and GenBank accession numbers of these proteins are: SmAP, Schistosoma mansoni (EU040139); Hs, Homo sapiens (NP000469); Dm, Drosophila melanogaster (NP649315) and Sc, Saccharomyces cerevisiae (AAA34871). Identical residues are indicated by shading. Proposed active site residues are indicated by # and residues reported to be important in metal binding are indicated by *. A potential signal peptide sequence at the N-terminal end and a single predicted transmembrane domain at the C-terminus are both underlined. A potential site for GPI-linked modification is S⁴⁹², indicated in bold. Conserved cysteine residues are boxed. The peptide sequence from residue 44 to 65, used to generate anti-SmAP antibodies, is indicated in bold. Underlined asparagine (N) residues indicate several potential N-linked glycosylation sites.

Figure 2. SmAP detection and expression.

A. Expression of the SmAP gene in different schistosome developmental stages. Egg, sporocyst (Sp), cercaria (Cer), 24 h-cultured schistosomulum (som), adult female (F) and adult male (M), B. SmAP protein expression in membrane preparations from different schistosome developmental stages. Adult male (M), adult female (F), 24 h-cultured schistosomulum (Som), cercaria (Cer) and egg. The arrowhead indicates the position of the SmAP protein. Molecular mass markers are depicted in the right lane and sizes are indicated in kDa. C. Adult male and female worm extract was resolved in the presence (lane 1) or absence (lanes 2 and 3) of reducing agent by SDS-PAGE. Lanes 1 and 2 represent western blots of the extract probed with anti-SmAP antibodies. The arrowhead indicates the position of the SmAP monomeric protein (at ∼60 kDa) while the arrow indicates the proposed homodimeric form (running at ∼120 kDa). Lane 3 represents one tract of the gel after incubation with the alkaline phosphatase substrate BCIP. The arrow indicates a band of activity at ∼120 kDa.

Figure 3. Immunolocalization of SmAP in adult parasites.

A. Cross section through a male/female couple showing widespread immunofluorescent staining with anti-SmAP antibody. B. Higher magnification image of the peripheral tissue of an adult male. The arrow indicates the outer tegument. C. Electron micrograph of the adult tegument showing immunogold labeling of SmAP. Arrows indicate gold particles at the host-parasite interface. Numbers above scale bars represent microns.

Figure 4. Suppression of SmAP gene expression by RNAi.

A: Relative SmAP gene expression (mean±S.E.) in adult worms 10 days after treatment with SmAP siRNA or a control siRNA targeting another schistosome gene (Sm control) or an irrelevant control (irr control) siRNA. B: Detection by western blot of SmAP protein in adult parasite extracts 10 days after treatment with SmAP siRNA, a control siRNA targeting another schistosome gene (Sm control) or an irrelevant control (irr control) siRNA (top panel). The arrow indicates the greatly diminished level of SmAP protein seen in extracts of parasites targeted with SmAP siRNA (left lane). The bottom panel shows a strip of the gel stained with Coomassie blue to illustrate roughly equivalent protein loadings in each lane.

Figure 5. SmAP suppression phenotype.

A. Relative alkaline phosphatase activity (mean±S.E.) displayed by live adult parasites 8 days after treatment with SmAP, control or no siRNA. B. AMP remaining (µM, mean±S.E.) in culture medium containing adult parasites 8 days after treated with SmAP, control or no siRNA, 40 hrs after the addition of 500 µM AMP. C. Adenosine generated (µM, mean±S.E.) in culture medium containing parasites treated with SmAP, control or no siRNA, 40 hrs after the addition of 500 µM AMP.