Schistosoma mansoni: Molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages

Abstract

Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase (SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composed of 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPI anchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blot experiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalization analysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of this enzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.

Immunization with tegument nucleotidases associated with a subcurative praziquantel treatment reduces worm burden followingSchistosoma mansonichallenge

“…The recombinant tegument nucleotidases have been previously characterized, expressed and purified, therefore we used the hexahistidine-tag expression vectors in which the respective cDNA sequences were directionally cloned: pAE-6His vector for SmAP and SmNPP-5 (Rofatto et al, 2009; Araujo-Montoya et al, 2011) and pET 21-b vector (Novagen) for SmNTPDase (DeMarco et al, 2003). These plasmids were used to transform E. coli BL21 Star (DE3) pLys (Invitrogen), which were grown in LB medium plus ampicillin (100 µg/mL) until reaching OD 600 0.6.…”

“…The recombinant proteins were then purified by immobilized metal affinity chromatography using the Äkta Prime system (GE Healthcare) under denaturing conditions, as previously described with slight modifications (DeMarco et al, 2003; Rofatto et al, 2009; Araujo-Montoya et al, 2011). Briefly, the samples were loaded onto a 5 mL bed volume Ni 2+ -NTA column (GE Healthcare) pre-equilibrated with the same buffer.…”

“…In spite of the fact that SmAP has long been used as a marker for tegument membranes (Roberts et al, 1983), characterization of Alkaline Phosphatase from S. mansoni at the molecular level was performed only recently (Araujo-Montoya et al, 2011; Bhardwaj & Skelly, 2011). Both studies concluded that the enzyme was expressed throughout the parasite life cycle and showed a widespread distribution in adult worms.…”

“…Both studies concluded that the enzyme was expressed throughout the parasite life cycle and showed a widespread distribution in adult worms. One study determined the surface activity of the enzyme, which was not inhibited by antibodies (Araujo-Montoya et al, 2011). The other study knocked down its expression by RNAi, which did not alter the parasite’s morphology or behavior (Bhardwaj & Skelly, 2011).…”

The cell biology of schistosomes: a window on the evolution of the early metazoa

“…Following the initial proteomic studies, individual protein constituents have been characterised in more detail. These include aquaporin Faghiri and Skelly 2009), phosphodiesterase Rofatto et al 2009), alkaline phosphatase (Araujo-Montoya et al 2011; and annexin 2 (Tararam et al 2010). In several of these studies, RNAi has been used successfully to reduce expression of both the gene and its encoded tegument surface protein Faghiri et al 2010;KrautzPeterson et al 2010) and so to alter both worm physiology and in some cases survival in vitro or in vivo.…”