Introduction: Production of reactive oxygen species (ROS) through the respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of an active NADPH oxidase enzyme system. This system has several components including gp91phox and p22phox comprising the membrane associated cytochrome b558 and the cytosolic components p47phox, p67phox, p40phox and rac2. Recently, we identified a novel protein with a molecular weight of 29 kDa which binds p67phox by immunoprecipitation and yeast two-hybrid analysis (

Leavey et al J Biol Chem 2002;277:45181–7
). This protein is classified as peroxiredoxin (Prx) by its amino acid sequence and enzyme activity. Prxs oxidize H2O2 through conserved cysteines. The neutrophil p29 Prx has a cysteine at amino acid residue 47 flanked by a consensus sequence shared by other 1-cys Prx proteins and a second cysteine at residue 91. We have also shown that neutrophil p29 Prx translocates to the cell membrane during oxidase activation and enhances oxidase activity in a cell-free assay with recombinant phox cytosolic components and neutrophil plasma membrane. The current studies were undertaken to evaluate requirements of cysteine residue(s) for the effect on the oxidase.

Methods: cDNA for p29 Prx was cloned into pBlueBacHis vector and a recombinant protein was expressed in a baculovirus expression system as previously described. Two additional recombinant proteins with ser substituted for cys at residue 47 and 91 were generated using the PCR overlap technique and also expressed in Sf9 cells. The wild type (WT), C47S, and C91S recombinant proteins were purified by affinity chromatography. The effect of these proteins on oxidase activity was determined in a SDS cell-free system of oxidase activity using plasma membrane (1 μg), p47phox (100 nM), p67phox (100 nM), a constitutively active Rac and 1 mM NADPH. O2− production was measured as SOD inhibitable cytochrome c reduction.

Results:

O2− Production (nmol/min)
Concentration (μM)WTC47SC91S
* Numbers are mean ± SEM, n=3 ** Significantly different from no addition, p<0.05 
0 7.15 ± 0.46* 6.38 ± 0.81 7.11 ± 0.10 
0.55 11.86 ± 1.33** 5.42 ± 0.73 6.57 ± 0.54 
0.83 14.58 ± 2.09** 7.32 ± 1.02 7.29 ± 6.70 
1.10 17.66 ± 2.50** 7.41 ± 1.19 7.64 ± 0.76 
1.65 19.93 ± 2.82** 8.37 ± 1.37 8.14 ± 1.23 
O2− Production (nmol/min)
Concentration (μM)WTC47SC91S
* Numbers are mean ± SEM, n=3 ** Significantly different from no addition, p<0.05 
0 7.15 ± 0.46* 6.38 ± 0.81 7.11 ± 0.10 
0.55 11.86 ± 1.33** 5.42 ± 0.73 6.57 ± 0.54 
0.83 14.58 ± 2.09** 7.32 ± 1.02 7.29 ± 6.70 
1.10 17.66 ± 2.50** 7.41 ± 1.19 7.64 ± 0.76 
1.65 19.93 ± 2.82** 8.37 ± 1.37 8.14 ± 1.23 
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WT p29 Prx increased oxidase activity in a concentration dependent manner. Neither the C47S nor C91S mutants altered oxidase activity to a significant degree. Additional studies varying concentration of NADPH at several concentrations of p29 Prx demonstrated that p29 Prx increased the Vmax without changing the km for NADPH. At a fixed concentration of NADPH, the increase in rate of O2− generation was dependent upon p29 Prx concentration demonstrating saturable kinetics with a km for p29 Prx of 1.72 ± 0.65 μM and a Vmax (increase in velocity) of 15.4 ± 3.0 nmol/min (mean ± SEM for four separate studies). Conclusion: Neutrophil p29 Prx enhances oxidase enzyme activity. The enhancement is specific, exhibiting a saturable effect dependent on the concentration of p29 Prx. However, replacement of either conserved or non-conserved cysteine residues with serines resulted in an inactive protein. Cysteine residues which are required for Prx activity are also critical to the effects of neutrophil p29 Prx on the oxidase.

Topics:
cysteine, neutrophils, oxidase, nadp, recombinant proteins, mechlorethamine, amino acids, chromatography, affinity, cytochrome c, cytochromes b

Author notes

Corresponding author

2005, The American Society of Hematology
2004
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