Laura K. Nelson

ANALYSIS OF ACTIVATION-INDUCED CONFORMATIONAL CHANGES IN P47PHOX USING TRYPTOPHAN FLUORESCENCE SPECTROSCOPY

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47 phox , p67 phox , and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47 phox undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47 phox using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47 phox with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47 phox , whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47 phox fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47 phox . Protein kinase C phosphorylation of p47 phox in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47 phox was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47 phox . Thus, our studies provide direct evidence linking conformational changes in p47 phox to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47 phox that are similar to those mediated by phosphorylation in vivo.

Priming of human neutrophils by peroxynitrite: potential role in enhancement of the local inflammatory response.

Peroxynitrite is a potent oxidant generated from the reaction of nitric oxide (NO) and superoxide anion (O2−), both of which can be produced in inflammatory tissues. In these studies, we analyzed what direct effect peroxynitrite had on neutrophil (PMN) function. We found that peroxynitrite was an effective priming agent for PMNs, as demonstrated by enhanced O2− production on subsequent activation with low doses of PMA or N‐formyl‐methionine‐leucine‐phenylalanine (fMLF), changes in the expression of PMN surface markers (L‐selectin, Mac‐1, flavocytochrome b, and fMLF receptor), and increased intracellular calcium levels. Analysis of the mechanism of PMN priming by peroxynitrite demonstrated that peroxynitrite resulted in minimal oxidation of protein sulfhydryl groups and subsequent protein cross‐linking. In contrast, treatment of PMNs with peroxynitrite resulted in significant nitration of tyrosine residues on neutrophil proteins. In addition, inhibition of tyrosine nitration with a pyrrolopyrimidine antioxidant blocked the majority of peroxynitrite‐induced priming effects, further suggesting that PMN priming was mediated primarily by nitration of tyrosine residues on PMN proteins. The ability of peroxynitrite to serve as an effective priming agent for PMNs at sites of inflammation may play a key role in modulating the host‐defense process. J. Leukoc. Biol. 65: 59–70; 1999.

U-101033E (2,4-diaminopyrrolopyrimidine), a potent inhibitor of membrane lipid peroxidation as assessed by the production of 4-hydroxynonenal, malondialdehyde, and 4-hydroxynonenal--protein adducts.

4-Hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) are major lipid peroxidation products generated by free radical attack on membranes and appear to contribute to the cytotoxic effects of oxidative stress by a mechanism involving adduct formation with cellular proteins. In the present studies, we investigated the relationship between lipid peroxidation and eventual inactivation of plasma membrane proteins using a model system consisting of purified red blood cell membranes and Fe2+/EDTA. Using this system, we also analyzed the ability of a novel antioxidant, U-101033E (2,4-diaminopyrrolopyrimidine), to inhibit lipid peroxidation and associated protein damage. Our results demonstrated that significant levels of MDA and 4-HNE are generated in this model system, and that both aldehydes are capable of cross-linking membrane proteins. In addition, we used a monoclonal antibody to demonstrate the presence of 4-HNE-protein adducts in this system. The generation of 4-HNE-protein adducts closely paralleled the time course of lipid peroxidation and membrane protein cross-linking, suggesting that 4-HNE may contribute to membrane protein cross-linking. Analysis of U-101033E in this system showed that this antioxidant inhibited lipid peroxidation, prevented the appearance of 4-HNE-protein adducts, and strongly reduced membrane protein cross-linking, with an EC50 of 0.5 microM. We also show that these antioxidant effects were not due to the scavenging of superoxide anion. Thus, these studies demonstrate the potential usefulness of U-101033E for treating certain disease processes where lipid peroxidation plays a role in disease pathogenesis.

Platelet-activating factor induces a concentration-dependent spectrum of functional responses in bovine neutrophils.

We characterized the dose response of bovine neutrophils to platelet‐activating factor (PAF) with respect to the following functions: calcium flux and membrane potential changes, actin polymerization, degranulation, and the production and/or priming of the oxidative burst. PAF at very low concentrations (10 and 10 M) caused changes in intracellular calcium and membrane potential in bovine neutrophils, whereas moderate PAF concentrations (≥10–7 M) resulted in increased actin polymerization. Degranulation responses to PAF were more complex: low concentrations (10–9 M) caused secretory granule degranulation, moderate doses (≥ 10–7 M) caused specific granule degranulation, whereas azurophil degranulation only occurred at high (10–5 M) PAF concentrations. Treatment of bovine neutrophils with PAF at concentrations ≥10–7 M also caused up‐regulation of the adhesion molecules Mac‐1 and L‐selectin. PAF stimulation resulted in a very weak [compared to phorbol myristate acetate (PMA)] oxidative burst in bovine neutrophils, and only at high (10–6 M) concentrations. Unlike human neutrophils, bovine neutrophils were poorly primed by PAF treatment. Only high concentrations of PAF (10–5 M) caused an increased rate of PMA‐stimulated superoxide production, although lower doses of PAF did reduce the lag time preceding the PMA‐induced oxidative burst. The overall pattern that can be inferred is that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation, up‐regulation of adhesion molecules, and increased actin polymerization. In contrast, higher PAF concentrations can promote, albeit weakly, more direct bactericidal responses, such as the release of reactive oxygen species and granule enzymes. The ability of PAF to modulate a graded response in bovine neutrophils would allow the cell to respond proportionally to the severity of a stimulus. J. Leukoc. Biol. 64: 817–827; 1998.

Isolation of bovine neutrophils with biomagnetic beads: comparison with standard Percoll density gradient isolation methods.

A prerequisite for studies on bovine neutrophils is a reliable method of neutrophil isolation from blood to obtain highly purified cell populations that are functionally active. Since current techniques of neutrophil isolation fall short of these requirements, we have developed a newer and more effective technique for isolation of bovine neutrophils that utilizes biomagnetic beads coated with a monoclonal antibody that recognizes an abundant surface antigen on bovine neutrophils to purify these cells. Comparison of the purity and viability of bovine neutrophils isolated by a conventional method (continuous Percoll density gradient) with this new method showed that neutrophils isolated with biomagnetic beads were higher in purity and had an increased yield. In addition, cells isolated with biomagnetic beads demonstrated normal or even improved function in assays of chemotaxis, phagocytosis, degranulation, and respiratory burst activity. Finally, bovine neutrophils isolated using this method showed an overall lower level of spontaneous apoptosis, which correlates well with the high level of viability observed in the purified cell preparations. Thus, this method represents a significant advance over current methods for isolating bovine neutrophils and would be widely applicable to labs studying the biochemistry and signal transduction pathways in these cells.

The nos (nitrous oxide reductase) gene cluster from the soil bacterium Achromobacter cycloclastes: Cloning, sequence analysis, and expression

The nitrous oxide (N2O) reductase (nos) gene cluster from Achromobacter cycloclastes has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ (structural N2O reductase gene), nosD, nosF, nosY, nosL, and nosX are detected, indicating a genetic organization similar to that of Rhizobium meliloti. To aid homology studies, nosR from R. meliloti has also been sequenced. Comparison of the deduced amino acid sequences with corresponding sequences from other organisms has also allowed structural and functional inferences to be made. The heterologous expression of NosD, NosZ (N2O reductase), and NosL is also reported. A model of the CuA site in N2O reductase, based on the crystal structure of this site in bovine heart cytochrome c oxidase, is presented. The model suggests that a His residue of the CuA domain may be a ligand to the catalytic CuZ site. In addition, the origin of the spectroscopically-observed Cys coordination to CuZ is discussed in terms of the sequence alignment of seven N2O reductases.

Inhibition of the Neutrophil NADPH Oxidase by Adenosine Is Associated with Increased Movement of Flavocytochrome b Between Subcellular Fractions

Adenosine is a potent inhibitor of reactive oxygen species (ROS) production by the NADPH oxidase in fMLF-stimulated neutrophils. Although much is known about the pharamacology and signal transduction of this effect, it is not known how adenosine affects assembly and localization of the NADPH oxidase components within the neutrophil. We report here that adenosine pretreatment of fMLF-stimulated neutrophils results in decreased plasma membrane/secretory granule content of the flavocytochrome b components (p22phox and gp91phox) of the NADPH oxidase, which correlates with inhibition of ROS production. Adenosine treatment did not affect upregulation of secretory and specific granule surface markers, confirming that degranulation was not impaired by adenosine. However, adenosine treatment did result in increased movement of cell-surface flavocytochrome b to heavy granule fractions in fMLF-stimulated neutrophils. These data suggest that adenosine-mediated effects on neutrophil ROS production are due, in part to endocytosis and/or redistribution of flavocytochrome b between various subcellular compartments.

Cloning and sequencing of rabbit leukocyte NADPH oxidase genes reveals a unique p67phox homolog

The NADPH oxidase plays an important role in immune and nonimmune cell functions. Because rabbits represent an established model for studying a number of important disease processes that involve NADPH oxidase activity, we carried out studies to clone and sequence all five rabbit leukocyte NADPH oxidase genes. Comparison of the rabbit sequences with those of other species showed that, with the exception of p67phox, the rabbit phox proteins were highly conserved. In contrast, rabbit p67phox had a very divergent C‐terminus and was 17 amino acids longer than any other known p67phox homolog. This was surprising, given the high degree of conservation among all of the phox proteins sequenced previously. To evaluate the functional consequences of this difference, wild‐type rabbit p67phox and a mutated rabbit p67phox missing the C‐terminal 17 amino acids were expressed and analyzed in a cell‐free assay. Our results show that the full‐length and truncated rabbit p67phox proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full‐length p67phox. These studies contribute to our understanding of the nature of the leukocyte NADPH oxidase in different species and will be valuable in future research using the rabbit model.

Inhibition of GTP binding to Rac2 by peroxynitrite: potential role for tyrosine modification

Peroxynitrite is a potent oxidant generated by the reaction of nitric oxide (*NO) and superoxide anion (O2*-), and both can be produced in inflammatory tissues. In the present studies, we analyzed the effects of peroxynitrite treatment on the GTP-binding activity of Rac2, a low molecular weight GTP-binding protein important in regulating a number of cellular functions. Using a fluorescent analog of GTP (methylanthraniloyl guanosine triphosphate or mant-GTP) as a reporter group, we found that treatment of Rac2 with peroxynitrite inhibited the binding of mant-GTP to Rac2 in a dose-dependent manner. Peroxynitrite was also able to react directly with free mant-GTP, resulting in a significant decrease in mant-GTP fluorescence; however, the mechanism of peroxynitrite-mediated damage to mant-GTP was different than with Rac2. In the case of mant-GTP, protection from peroxynitrite-mediated oxidation was observed in the presence of the free radical scavengers, mannitol and DMTU. In contrast, DMTU was unable to prevent peroxynitrite-mediated inhibition of mant-GTP binding to Rac2. Instead, our data demonstrates a role for peroxynitrite-mediated tyrosine modification in the inhibition of mant-GTP binding to Rac2, and we were able to demonstrate the formation of a significant level of nitrotyrosine formation in Rac2 exposed to peroxynitrite. Thus, our studies support the premise that oxidative modification of key cellular proteins, such as Rac2, plays an important role in the cytotoxic effects observed for peroxynitrite and other reactive oxidants.

Molecular analysis of the bovine anaphylatoxin C5a receptor

Recruitment of phagocytes to inflammatory sites involves the coordinated action of several chemoattractants, including the anaphylatoxin C5a. While the C5a receptor (C5aR) has been well characterized in humans and rodents, little is known about the bovine C5aR. Here, we report cloning of bovine C5R1, the gene encoding bovine C5aR. We also analyzed genomic sequence upstream of the C5R1 translation start site. Although the bovine C5aR amino acid sequence was well conserved among species, significant differences in conserved features were found, including major differences in the N terminus, intracellular loop 3, and transmembrane domain VII. Analysis of C5aR expression by flow cytometry and confocal microscopy demonstrated high levels of C5aR on all bovine neutrophils and a subset of bovine monocytes. C5aR was not expressed on resting or activated bovine lymphocytes, although C5aR message was present in these cells. C5aR was also expressed on a small subset of bovine mammary epithelial cells. Pharmacological analysis of bovine C5aR‐mediated responses showed that bovine C5a and C5adesArg both induced dose‐dependent calcium fluxes and chemotaxis in bovine neutrophils, with similar efficacy for both agonists. Treatment of bovine neutrophils with C5a or C5adesArg resulted in homologous desensitization of bovine C5aR and cross‐desensitization to interleukin 8 (IL‐8) and platelet‐activating factor (PAF); whereas, treatment with IL‐8 or PAF did not cross‐desensitize the cells to C5a or C5adesArg. Overall, these studies provide important information regarding distinct structural and functional features that may contribute to the unique pharmacological properties of bovine C5aR.