Cell-free Oxidase System Research Articles

Neutrophil p29 Peroxiredoxin 6/aiPLA2 (p29 Prdx6) Enhances Superoxide Anion (O2-) Production by Nox2: Role for the PLA2 Active Site

AbstractAbstract 926Introduction: An intact respiratory burst is critical to the microbial activity of neutrophils. The respiratory burst is initiated by assembly and activation of the NADPH oxidase, Nox2. We have identified a 29 kDa protein from human neutrophils (p29) which binds to the oxidase component p67phox and enhances superoxide anion (O2-) production in a cell-free, NADPH activity oxidase system. Identified as Prdx6 by sequence and was found to have Prdx activity by the recombinant protein. p29 Prdx6 also has a calcium-independent PLA2 activity that is optimal at low pH (aiPLA2). Although Prdx6 is found in many tissues, its function in myeloid cells is not well established. p29 Prdx6 was knocked down in a myeloid cell line (PLB-985) and the effect on the Nox2 activity of theses cells was investigated. In addition, wild type p29 Prdx6 or mutants with altered Prdx and PLA2 active sites were re-expressed in the knockdown cells, to explore the possibility that at least one of the catalytic activities is involved in enhancing the oxidase. Methods: PLB-985 cells were transfected with a plasmid encoding a p29 Prdx6 targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx6 was confirmed by Western blot, but no change in the other known oxidase components was detected. cDNAs for wild type (wt) p29 Prdx6 and known mutants for the Prdx activity site (C47S) and PLA2 active site (H26A and S32A) were cloned into pcDNA3.1 producing plasmids generating mRNAs resistant to shRNA. Knockdown cells transfected with these plasmids were cultured with both puromycin and G418 with re-expression of wt or mutant Prdx6 to comparable levels in PLB985 cells, documented by Western blot. After maturation of the knockdown, Prdx re-expressing, and control cells with DMSO for 4 days, fMLP (1μM) stimulated O2- production was measured by luminescence of Diogenes probe. Results: Using stable expression of shRNA, p29 Prdx6 protein was reduced to 31±8% (mean ± SEM, n=5) of that in non-knockdown control PLB985 cells. O2- production in response to fMLP was reduced (Student's t-test, p<0.05) to 51±4% (mean ± SEM, n=25). Re-expression of wt p29 Prdx6 reestablished full Nox2 activity (100±5%, n=6). For comparison, empty re-expression vector control was 52±6%, n=22). When the Prdx activity (C47S) mutant was expressed, Nox2 activity was slightly increased (133±5%, n=6). In contrast, expression of mutants at PLA2 active site had no effect on recovery of Nox2 activity (H26A, 60±2%, n=5; S32A, 58±9%, n=10, different from non-knockdown and C47S, p<0.05). Conclusion: These results indicate that p29 Prdx VI is required for optimal O2- production in intact myeloid cells confirming our data from the cell-free oxidase system. Furthermore, studies evaluating expression of p29 Prdx protein mutated at sites associated with Prdx and PLA2 suggest the latter is responsible for Nox2 activity in intact cells. These studies document a specific biochemical role and mechanism for p29 Prdx6 in the neutrophil respiratory burst and associated microbicidal activity and define a molecular target for modulating Nox2 activity. Disclosures:No relevant conflicts of interest to declare.

Neutrophil p29 Peroxiredoxin VI/aiPLA2 (p29 Prdx VI) Enhances Superoxide Production by the NADPH Oxidase in a Manner Dependent On the PLA2 Active Site.

Abstract 3592Poster Board III-529 IntroductionAn intact respiratory burst is critical to the microbial activity of neutrophils. The respiratory burst is initiated by assembly and activation of the NADPH oxidase. We have identified a 29 kDa protein from human neutrophils (p29) which binds to the NADPH oxidase component p67phox and enhances superoxide anion (O2−) production in a cell-free, NADPH activity oxidase system. The protein was identified as Prdx VI by sequence and the recombinant molecule was found to have Prdx activity. Interestingly, Prdx VI also has a calcium-independent PLA2 activity that is optimal at low pH (aiPLA2). Although Prdx VI is found in many tissues, its function in myeloid cells is not well established. p29 Prdx VI was knocked down in a myeloid cell line (PLB-985) and the effect on the respiratory burst of theses cells was investigated to determine its role in the respiratory burst. In addition, mutant or wild type p29 Prdx were introduced into the cell free system, to explore the possibility that at least one of the catalytic activities is involved in enhancing the oxidase. MethodsPLB-985 cells were transfected with a plasmid encoding a p29 Prdx VI targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx VI was confirmed by Western blot, but no change in the other known oxidase components was detected. After maturation of the knockdown and control cells by DMSO for 4 days, they were stimulated with fMLP (1μM) and O2− production was measured by luminescence of Diogenes probe. In separate experiments, neutrophil plasma membranes, recombinant p47phox, p67phox and constitutively active Rac1 were added to an assay mix. Addition of SDS assembled of the complex in this cell free system, NADPH was added and O2− production was measured as SOD inhibitable, cytochrome C reduction. O2− production when recombinant wild type p29 Prdx VI was added to the system was compared to activity caused by p29 Prdx VI mutated in the peroxiredoxin active site (C47S) or the aiPLA2 active site (D140A). Wild type and mutant p29 Prdx VI were expressed with tags in sf9 insect cells and purified by Ni2+ chelation chromatography. ResultsUsing stable expression of shRNA, p29 Prdx VI protein was reduced to 31+/−18% (SD) of that in non-knockdown control cells but the levels of the other known oxidase components were unchanged. O2− production in response to fMLP was reduced to 51%+/−12% (SD). When added to the cell-free oxidase system, wild type p29 Prdx VI and p29 Prdx VI mutated in the peroxiredoxin active site increased the O2− production rate by similar amounts (to 323+/−94% and 417+/−125% of the assay rate in the absence of p29 Prdx VI respectively; numbers are mean ± SD for three separate experiments). Recombinant p29 mutated in the PLA2 active site however increased O2− production to only 178+/−33%. ConclusionThese results indicate that p29 Prdx VI is required for optimal O2− production in intact myeloid cells. Data from the cell-free oxidase system suggest that the PLA2 activity of p29 Prdx VI, rather than its peroxiredoxin activity, account for its ability to stimulate O2- production. These studies document a specific biochemical role for p29 Prdx VI in the neutrophil respiratory burst and associated microbicidal activity. Furthermore, lipid modification by p29 Prdx VI may thus be a key step in maximal activation of the NADPH oxidase. Disclosures:No relevant conflicts of interest to declare.

Antagonistic Cross-talk between Rac and Cdc42 GTPases Regulates Generation of Reactive Oxygen Species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.

Translocation of Recombinant p47 phox Cytosolic Component of the Phagocyte Oxidase by in Vitro Phosphorylation

Activation of superoxide-generating NADPH oxidase system of human neutrophils involves phosphorylation-dependent translocation of p47 phox and other cytosolic components to the plasma membrane. In contrast to the stimulation of the NADPH oxidase in intact cells, however, the activation of cell-free system requires the addition of anionic amphiphiles such as sodium dodecyl sulfate (SDS) and arachidonate. In this system, translocation of p47 phox is also an essential step for activation, but phosphorylation is not required. The basis of this difference in oxidase activation is not yet clear. We now report that in a cell-free oxidase system, phosphorylated recombinant p47 phox can be translocated to the membrane in the absence of SDS or arachidonate. These findings suggest that both phosphorylation and SDS could cause a common change in conformation or charge of p47 phox that may result in the association of p47 phox with the plasma membrane.

Low NADPH oxidase activity in Epstein-Barr-virus-immortalized B-lymphocytes is due to a post-transcriptional block in expression of cytochrome b558

The NADPH oxidase of phagocytes is known to be expressed in Epstein-Barr-virus-transformed B-lymphocytes, albeit at levels only approx. 5% of those found in neutrophils. We have investigated the basis of this low level of expression and find that all four specific components of the NADPH oxidase are expressed in B-lymphocytes, but only p47-phox protein attains levels equivalent with those found in neutrophils. This component was shown to phosphorylate and translocate to the membrane normally on activation. The other cytosolic component, p67-phox, did show a deficit, and by supplementing a B-cell cytosol extract with recombinant p67-phox, this was shown to account for the somewhat reduced activity of B-cell cytosol in a cell-free oxidase system. The cell-free analysis also clearly located the major deficiency in superoxide-generating capacity of B-lymphocytes to the membrane. Western blotting of membrane proteins revealed major reductions in the amount of cytochrome b558. Analysis of the levels of mRNA for both subunits of cytochrome b558, however, showed levels greater than expected. Significantly more mRNA for gp91-phox was present in B-cells than in undifferentiated HL60 cells, although it was not quite as abundant as in differentiated HL60 cells, which are capable of producing large amounts of superoxide. We conclude that the failure of B-lymphocytes to generate amounts of superoxide equivalent to those generated by neutrophils is primarily due to a post-transcriptionally determined block to the accumulation of cytochrome b558.

Autosomal recessive chronic granulomatous disease with absence of the 67-kD cytosolic NADPH oxidase component: identification of mutation and detection of carriers

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to kill certain bacteria and fungi. This is caused by deficiencies in one of the components of NADPH oxidase, the enzyme in phagocytic leukocytes that generates superoxide. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of NADPH oxidase (p67-phox) is missing. Until now, mutations in the gene coding for this protein have not been identified. We now report on a 10-year- old girl with lymph node and liver abscesses who was recognized as an A67(0) CGD patient by lack of NADPH oxidase activity in her granulocytes, a cytosolic defect in a cell-free oxidase system, and lack of immunoreactive material with an antiserum against the p67-phox protein. mRNA for this protein was present in normal amounts in her monocytes. This p67-phox mRNA was reverse-transcribed, and the coding region was amplified by polymerase chain reaction in six overlapping fragments and was sequenced. The patient appeared to be homozygous for a G-233--&gt;A mutation, resulting in a nonconservative amino acid change (78Gly--&gt;Glu). This mutation was also found in the genomic DNA of this patient but not in that of 38 normal donors. Both parents and a sister proved to be carriers of the disease, as deduced from the mutation in only one allele. The carrier state was also manifested by intermediate superoxide production by their intact granulocytes and in the cell-free system.

Autosomal Recessive Chronic Granulomatous Disease With Absence of the 67-kD Cytosolic NADPH Oxidase Component: Identification of Mutation and Detection of Carriers

AbstractChronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to kill certain bacteria and fungi. This is caused by deficiencies in one of the components of NADPH oxidase, the enzyme in phagocytic leukocytes that generates superoxide. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of NADPH oxidase (p67-phox) is missing. Until now, mutations in the gene coding for this protein have not been identified. We now report on a 10-year-old girl with lymph node and liver abscesses who was recognized as an A670 CGD patient by lack of NADPH oxidase activity in her granulocytes, a cytosolic defect in a cell-free oxidase system, and lack of immunoreactive material with an antiserum against the p67-phox protein. mRNA for this protein was present in normal amounts in her monocytes. This p67-phox mRNA was reverse-transcribed, and the coding region was amplified by polymerase chain reaction in six overlapping fragments and was sequenced. The patient appeared to be homozygous for a G-233 → A mutation, resulting in a nonconservative amino acid change (78Gly → Glu). This mutation was also found in the genomic DNA of this patient but not in that of 38 normal donors. Both parents and a sister proved to be carriers of the disease, as deduced from the mutation in only one allele. The carrier state was also manifested by intermediate superoxide production by their intact granulocytes and in the cell-free system.

Functional domain in an arginine-rich carboxyl-terminal region of p47phox.

Activation of the neutrophil respiratory burst oxidase involves phosphorylation-dependent translocation of the cytosolic proteins p47phox and p67phox to the plasma membrane, a process in intact cells that is inhibited by staurosporine. We now report that in a cell-free oxidase system, staurosporine and protein kinase C pseudosubstrate PKC(19-36) both inhibited p47phox phosphorylation but had no effect on superoxide generation. In contrast, p47phox phosphorylation, translocation, and superoxide generation were inhibited by a peptide, p47phox(323-332) (AYRRNSVRFL), based on a putative serine phosphorylation domain. This effect was specific for the 323-332 peptide, as it was not observed with two peptides based on other p47phox phosphorylation domains. All three peptides served as substrates for phosphorylation, but the extent of peptide phosphorylation did not correlate with inhibition of oxidase function. p47phox(325-330), which represents the serine phosphorylation motif of p47phox(323-332), did not inhibit translocation or superoxide production despite its ability to block phosphorylation of p47phox. These data indicate the presence of functionally important sites within the p47phox(323-332) peptide. Although serine 328 is in a consensus phosphorylation motif, the lack of correlation in the cell-free system between p47phox phosphorylation and either protein translocation or superoxide formation suggests that a phosphorylation-independent function resides in the 323-332 segment of p47phox.

Immunochemical and Electrophoretic Analyses of Phosphorylated Native and Recombinant Neutrophil Oxidase Component p47-Phox

AbstractHuman polymorphonuclear neutrophils (PMNs) possess a potent oxygen-dependent microbicidal system that depends on the activity of a stimulus-activated multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Patients with chronic granulomatous disease (CGD) lack activity of this oxidase and consequently suffer severe and frequent infections. Components of the oxidase include both membrane-bound factors (most notably, cytochrome b559, which is absent in the X-linked form of CGD) and at least two cytosolic factors, one or the other of which is absent in autosomal CGD. Patients with CGD, particularly the autosomal type, have defective phosphorylation of proteins in the 44 to 48 Kd range. A polyclonal antiserum (B-1) that recognizes cytosolic oxidase components of 47 and 67 Kd was used to identify phosphoproteins in a cell-free oxidase system. Two-dimensional gel electrophoresis showed the identity of the 47-Kd cytosolic protein (p47-phox) recognized by B-1 and the cationic 47-Kd protein that is phosphorylated in normal but not p47-phox-deficient CGD cytosol during activation of the NADPH-dependent oxidase. All full-length and C-terminal recombinant p47-phox proteins augmented the superoxide-generating capacity of the cell-free system and were phosphorylated when added to cytosol from normal subjects or from a patient with p47-deficient autosomal CGD. These studies provide compelling evidence that the 47-Kd cationic protein that is a substrate for phosphorylation during the activation of PMNs is, in fact, p47-phox, a cytosolic protein previously shown to be critical for normal activity of the NADPH-dependent oxidase of PMNs.

Immunochemical and electrophoretic analyses of phosphorylated native and recombinant neutrophil oxidase component p47-phox

Human polymorphonuclear neutrophils (PMNs) possess a potent oxygen- dependent microbicidal system that depends on the activity of a stimulus-activated multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Patients with chronic granulomatous disease (CGD) lack activity of this oxidase and consequently suffer severe and frequent infections. Components of the oxidase include both membrane- bound factors (most notably, cytochrome b559, which is absent in the X- linked form of CGD) and at least two cytosolic factors, one or the other of which is absent in autosomal CGD. Patients with CGD, particularly the autosomal type, have defective phosphorylation of proteins in the 44 to 48 Kd range. A polyclonal antiserum (B-1) that recognizes cytosolic oxidase components of 47 and 67 Kd was used to identify phosphoproteins in a cell-free oxidase system. Two-dimensional gel electrophoresis showed the identity of the 47-Kd cytosolic protein (p47-phox) recognized by B-1 and the cationic 47-Kd protein that is phosphorylated in normal but not p47-phox-deficient CGD cytosol during activation of the NADPH-dependent oxidase. All full-length and C- terminal recombinant p47-phox proteins augmented the superoxide- generating capacity of the cell-free system and were phosphorylated when added to cytosol from normal subjects or from a patient with p47- deficient autosomal CGD. These studies provide compelling evidence that the 47-Kd cationic protein that is a substrate for phosphorylation during the activation of PMNs is, in fact, p47-phox, a cytosolic protein previously shown to be critical for normal activity of the NADPH-dependent oxidase of PMNs.

Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation.

The superoxide-forming respiratory burst oxidase of human neutrophils is composed of membrane-associated catalytic components and cytosolic constituents required for oxidase activation. This study concerns the hypothesis that cytosolic oxidase components translocate to a membrane fraction when neutrophils are stimulated and the oxidase is activated. A polyclonal antiserum that recognizes two discrete cytosolic oxidase components of 47 and 67 kD was used to probe transfer blots of electrophoresed membrane and cytosol fractions of resting and stimulated neutrophils. In contrast to their strictly cytosolic localization in unstimulated cells, both proteins were detected in membrane fractions of neutrophils activated by phorbol esters and other stimuli. This translocation event was a function of stimulus concentration as well as time and temperature of exposure to the stimulus. It was inhibited by concentrations of N-ethylmaleimide that blocked superoxide formation but was unaffected by 2-deoxyglucose. There was a correlation between translocation of the cytosolic proteins and activation of the oxidase as determined by superoxide formation. Quantitative analyses suggested that approximately 10% of total cellular p47 and p67 became membrane-associated during phorbol ester activation of the oxidase. Analysis of Percoll density gradient fractions indicated that the target membrane for translocation of both proteins was the plasma membrane rather than membranes of either specific or azurophilic granules. In the cell-free oxidase system arachidonate-dependent but membrane-independent precipitation of the cytosolic oxidase proteins was demonstrated. The data show that activation of the respiratory burst oxidase in stimulated human neutrophils is closely associated with translocation of the 47- and 67-kD cytosolic oxidase components to the plasma membrane. We suggest that this translocation event is important in oxidase activation.

Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system.

Chronic granulomatous disease, a syndrome of recurrent infections and failure of oxidative microbicidal activity in phagocytes, results from defects in the gene for one of several components of an oxidase system that can undergo activation. To determine the relative prevalence of certain of the genetic variants of this disorder, we used immunoblotting to detect two specific neutrophil cytosolic proteins of 47 and 67 kd recently shown to be required for oxidase activation. Chronic granulomatous disease is usually an X-linked disorder associated with the absence of membrane cytochrome b558. Of our 94 patients with chronic granulomatous disease, however, 36 had a phenotype characterized by autosomal inheritance, normal membrane oxidase components (including cytochrome b558), and functionally defective cytosolic activity in a cell-free oxidase system. We studied 25 of these 36 patients and found that 22 lacked the 47-kd cytosolic protein, and the remaining 3 were missing the 67-kd component. Patients with chronic granulomatous disease whose functional defect was localized to the neutrophil membrane (classic X-linked cytochrome b-negative type and two other rare variants) had normal amounts of both cytosolic components. We estimate that approximately 33 percent of all patients with chronic granulomatous disease are missing the 47-kd cytosolic oxidase component and about 5 percent of patients are missing the 67-kd component. Chronic granulomatous disease caused by a defect in any cytosolic factors other than the 47-kd and 67-kd proteins, if it exists, is apparently rare.