Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni

Pujol, Flor H.; Liprandi, Ferdinando; Rodríguez, M.; Cesari, Italo M.

doi:10.1016/0166-6851(90)90078-z

Cited by 13 publications

(4 citation statements)

References 36 publications

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“…These enzymes, however, do not seem to be restricted to the double surface membrane: Ca2'-ATPase has been histochemically shown to be located in the cytoplasmic leaflet of both the surface and the basal tegumental membranes (6). AP, although certainly restricted to the tegument, as confirmed by the immunoreactivity of a monoclonal antibody against this enzyme (22). seems to be present in the double surface membrane and also in small intrategumental cytoplasmic inclusions called discoid bodies (5, 16, 23).…”

Section: Discussionmentioning

confidence: 86%

Schistosoma mansoni: Surface Membrane Isolation with Lectin-Coated Beads

Pujol

Cesari

1993

Membrane Biochemistry

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Lectins from Lens culinaris and Arachis hypogaea immobilized on polyacrylamide beads were used for selective isolation of glycosylated surface membrane domains of adult Schistosoma mansoni worms, and the method was compared with the membrane isolation procedure developed with polycationic (Affi-Gel) beads. The lentil lectin proved to be suitable for interaction with surface membrane components: an increment in the specific activities of tegumental phosphohydrolases was observed in the bound fraction with respect to that observed in a total worm homogenate. A characteristic polypeptide pattern on gel electrophoresis was also seen, more restricted than that obtained with the bound Affi-Gel fraction. Immobilized peanut lectin was not successful as a method for isolating membrane material from the tegument of adult worms. Solubilization and dissociation of the lentil lectin-bound enzyme markers was achieved after addition of detergent and competing sugars. Glycosylation of the solubilized enzymes was further confirmed by affinity chromatography with fresh lentil lectin-coated beads. These results, together with histochemical evidences, suggest that the active sites of some of these enzymes are located within or close to the cytoplasmic leaflet of the surface tegumental membranes, and allow us to propose a model for the double surface membrane complex where some proteins may be crossing the two bilayers.

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Section: Discussionmentioning

confidence: 86%

Schistosoma mansoni: Surface Membrane Isolation with Lectin-Coated Beads

Pujol

Cesari

1993

Membrane Biochemistry

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“…The cDNA encoding SmAP was recently cloned and characterized (Bhardwaj & Skelly, 2011). SmAP is a ∼62 kDa glycosylphosphatidylinositol (GPI) anchored protein that is expressed in the tegument and internal tissues of the adult worms (Bhardwaj & Skelly, 2011; Cesari, 1974; Dusanic, 1959; Levi-Schaffer et al, 1984; Morris & Threadgold, 1968; Pujol et al, 1990). The protein can be cleaved from cultured schistosomula (Espinoza et al, 1988) and from adult worms (Castro-Borges et al, 2011) by the phosphatidylinositol-cleaving enzyme—phosphatidylinositol-specific phospholipase C. Tegumental proteomic analysis confirms that SmAP is found in the schistosome surface membranes (Braschi et al, 2006; van Balkom et al, 2005) and is available for surface biotinylation (Braschi & Wilson, 2006).…”

Section: Introductionmentioning

confidence: 99%

Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides

Da’dara

Bhardwaj

Ali

et al. 2014

PeerJ

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Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs) and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP). Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5) and ATP diphosphohydrolase (SmATPDase1). In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms’ ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ± 0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals like ATP, and pro-thrombotic signals like ADP, these parasite enzymes may minimize host immune responses, inhibit blood coagulation and promote schistosome survival.

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“…The cDNA encoding SmAP was recently cloned and characterized . SmAP is a ~62 kDa glycosylphosphatidylinositol (GPI) anchored protein that is expressed in the tegument and internal tissues of the adult worms Cesari 1974;Dusanic 1959;Levi-Schaffer et al 1984;Morris & Threadgold 1968;Pujol et al 1990). The protein can be cleaved from cultured schistosomula (Espinoza et al 1988) and from adult worms (Castro-Borges et al 2011) by the phosphatidylinositol-cleaving enzyme -phosphatidylinositol-specific phospholipase C. Tegumental proteomic analysis confirms that SmAP is found in the schistosome surface membranes van Balkom et al 2005) and is available for surface biotinylation .…”

Section: Introductionmentioning

confidence: 99%

Peer Review #1 of "Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides (v0.1)"

Davies¹

2014

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Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs) and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP). Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5) and ATP diphosphohydrolase (SmATPDase1). In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP.. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms' ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, fulllength recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The K m of SmATPDase1 for ATP is 0.4 ±0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals like ATP, and pro-thrombotic signals like ADP, these parasite enzymes may minimize host immune

show abstract

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni

Cited by 13 publications

References 36 publications

Schistosoma mansoni: Surface Membrane Isolation with Lectin-Coated Beads

Schistosoma mansoni: Surface Membrane Isolation with Lectin-Coated Beads

Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides

Peer Review #1 of "Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides (v0.1)"

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