Characterization and kinetic analysis of protein tyrosine phosphatase-H2 from Microplitis demolitor bracovirus.
The polydnavirus Microplitis demolitor bracovirus (MdBV) encodes 13 genes that share homology with classical protein tyrosine phosphatases (PTPs). Prior sequence analysis suggested that five members of the MdBV PTP gene family (ptp-H2, -H3, -H5, -N1 and -N2) encode PTPs, seven family members encode pseudophosphatases, and one family member is a pseudogene. Prior experimental studies further implicated PTP-H2 in disabling the function of host hemocytes following infection by MdBV. Here we report expression of PTP-H2 and selected mutants in Escherichia coli cells as non-fusion or thioredoxin-fusion proteins. Following purification by nickel affinity chromatography, the full-length and mutant proteins ran as single bands of predicted size on SDS-PAGE gels under reducing conditions. The non-fusion form of PTP-H2 exhibited classical Michaelis-Menten kinetics using the phosphopeptide END(pY)INASL and difluoro-4-methylumbiliferyl phosphate (DiFMUP) as substrates. As expected, the non-fusion mutant PTP-H2(C236S) had no enzymatic activity, while the thioredoxin-fusion form of PTP-H2 had low levels of activity. PTP-H2 exhibited optimal activity at pH 4.0 and 26 degrees C in sodium acetate buffer, and its activity was diminished by increasing buffer ionic strength. Activity was also greatly reduced by the presence of copper, heparin, and the classical PTP inhibitor vanadate. Using an anti-PTP-H2 antibody, immunoblotting and immunocytochemical studies only detected PTP-H2 in hemocytes from MdBV-infected Pseudoplusia includens. Overall, our results indicate that PTP-H2 is a functional tyrosine phosphatase that is specifically expressed in MdBV-infected hemocytes.
Eum
Jai-Hoon
JH
Department of Entomology, University of Georgia, Athens, GA 30602, USA.
Bottjen
Rachel C
RC
Pruijssers
Andrea J
AJ
Clark
Kevin D
KD
Strand
Michael R
MR
D003300:Copper / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018071:Polydnaviridae / Q000201:enzymology*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D014638:Vanadates; D013879:Thioredoxins; D003300:Copper; D006493:Heparin; D017027:Protein Tyrosine Phosphatases