Setaria cervi dual specific phosphatase: characterization and its effect on eosinophil degranulation.
Setaria cervi, a bovine filarial parasite contains significant acid phosphatase (AcP) activity in its various life stages. Two forms of AcP were separated from somatic extract of adult female parasite using cation exchange, gel filtration and concavalin affinity chromatography. One form having a molecular mass of 79 kDa was characterized as dual specific protein tyrosine phosphatase (ScDSP) based on substrate specificity and inhibition studies. With various substrates tested, it showed significant activity in the order of phospho-L-tyrosine>pNPP>ADP>phospho-L-serine. Inhibition by orthovanadate, fluoride, molybdate, and zinc ions further confirms protein tyrosine phosphatase nature of the enzyme. Km and Vmax determined with various substrates were found to be 16.66 mM, 25.0 microM/ml/min with pNPP; 20.0 mM, 40.0 microM/ml/min with phospho-L-tyrosine and 27.0 mM, 25.0 microM/ml/min with phospho-L-serine. KI with pNPP and sodium orthovanadate (IC50 33.0 microM) was calculated to be 50.0 mM. Inhibition with pHMB, silver nitrate, DEPC and EDAC suggested the presence of cysteine, histidine and carboxylate residues at its active site. Cross-reactivity with W. bancrofti-infected sera was demonstrated by Western blotting. ScDSP showed elevated levels of IgE in chronic filarial sera using ELISA. Under in vitro conditions, ScDSP resulted in increased effector function of human eosinophils when stimulated by IgG, which showed a further decrease with increasing enzyme concentration. Results presented here suggest that S. cervi DSP should be further studied to determine its role in pathogenesis and the persistence of filarial parasite.
Rathaur
S
S
Department of Biochemistry, Banaras Hindu University, Varanasi-221005, India. surathaur@redifffmail.com
Rai
R
R
Srikanth
E
E
Srivastava
S
S
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D002020:Buffaloes / Q000469:parasitology*; D020134:Catalytic Domain; D015550:Cell Degranulation; D003429:Cross Reactions; D004804:Eosinophils / Q000502:physiology*; D005260:Female; D005368:Filariasis / Q000662:veterinary; D005370:Filarioidea / Q000201:enzymology* / Q000502:physiology; D006790:Host-Parasite Interactions; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D007074:Immunoglobulin G / Q000276:immunology; D008297:Male; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity; D014958:Wuchereria bancrofti / Q000276:immunology
D000909:Antibodies, Helminth; D007074:Immunoglobulin G; D007073:Immunoglobulin E; D017027:Protein Tyrosine Phosphatases