In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Nitration of cytoskeletal proteins in the chicken embryo chorioallantoic membrane

Protein tyrosine nitration is one of the post-translational modifications that alter the biological function of proteins. Two important mechanisms are involved: peroxynitrite formation and myeloperoxidase or eosinophil peroxidase (EPO) activity. In the present work we studied the nitration of proteins in the in vivo system of chicken embryo chorioallantoic membrane (CAM). 3-Nitrotyrosine was detected only in the insoluble fraction of the CAM homogenate. By immunoprecipitation, Western blot analysis, and double immunofluorescence, we identified two major polypeptides that were nitrated: actin and alpha-tubulin. Quantification of actin and alpha-tubulin nitration revealed that they are differentially nitrated during normal development of the chicken embryo CAM. After irradiation, although they were both increased, they required different time periods to return to the physiological levels of nitration. It seems that both peroxynitrite formation and EPO activity are involved in the in vivo tyrosine nitration of cytoskeletal proteins. These data suggest that tyrosine nitration of cytoskeletal proteins has a physiological role in vivo, which depends on the protein involved and is differentially regulated.     

Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin

The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.     

Ribonuclease activity associated with human eosinophil-derived neurotoxin and eosinophil cationic protein

The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.     

Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.     

Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microscopic technique

An immunoelectron microscopic technique using protein A-gold as a specific marker was used for precise intracellular localization of eosinophil granule proteins. Eosinophils from healthy individuals were isolated in metrizamide gradients. Eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were clearly located in the matrix of the large crystalloid-containing granules. In addition, ECP was probably present in the small granules of eosinophils. Major basic protein (MBP) was present in the crystalloid structure of specific granules. This method can be applied in studies of eosinophil degranulation to trace the release of biological effector molecules.     

Exposed tyrosine residues of lambda cro repressor protein evidenced by nitration and photo CIDNP experiments

The tyrosine residues of lambda cro repressor were partially nitrated with tetranitromethane under mild conditions. After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis. Tyr 26 was most easily nitrated and Tyr 51 followed it. Tyr 10 was resistant to nitration. By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues. The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization).     

Comparative toxicity of purified human eosinophil granule proteins for newborn larvae of Trichinella spiralis

Eosinophils have been implicated in both in vivo and in vitro destruction of helminths. One approach toward elucidating the role of the eosinophil in parasite killing has been to test the toxicity of purified eosinophil granule proteins for parasites in vitro. Previously, major basic protein (MBP) and eosinophil cationic protein (ECP) were shown to be toxic for schistosomules of Schistosoma mansoni, while eosinophil-derived neurotoxin (EDN) was only marginally so. We tested the toxicity of MBP, ECP, and EDN over a range of concentrations (0.006-5 X 10(-4) M) for newborn larvae of Trichinella spiralis. Our observations confirm previous reports of toxicity of mildly reduced and alkylated (R & A) MBP. At concentrations of 5 X 10(-5) M and above, R & A MBP killed 75% or more of the larvae within the first hour of culture. ECP was an effective toxin for these larvae after 3 hr of culture, and by 12 hr, dose-related toxicity was evident. After 24 hr, 100% of the larvae were killed at 5 X 10(-5) M ECP. EDN was much less toxic; after 12 hr, 90% of the larvae survived at concentrations of 1 X 10(4) M, while 5 X 10(-4) M EDN killed all the larvae. At the optimal toxic concentrations of 5 X 10(-5) M ECP and 5 X 10(-4) M EDN, kinetics of killing by these 2 proteins were essentially the same. Thus, on a molecular basis, both MBP and ECP appear to be potent helminthotoxins whereas EDN is much less so.     

Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein

Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are proteins of the ribonuclease A (RNase A) superfamily that have developed biological properties related to the function of eosinophils. ECP is a potent cytotoxic molecule, and although the mechanism is still unknown this cytotoxic activity has been associated with its highly cationic character. Using liposome vesicles as a model, we have demonstrated that ECP tends to disrupt preferentially acidic membranes. On the basis of structure analysis, ECP variants modified at basic and hydrophobic residues have been constructed. Changes in the leakage of liposome vesicles by these ECP variants have indicated the role of both aromatic and basic specific amino acids in cellular membrane disruption. This is the case with the two tryptophans at positions 10 and 35, but not phenylalanine 76, and the two arginines 101 and 104. The bactericidal activity of both native ECP and point-mutated variants, tested against Escherichia coli and Staphylococcus aureus, suggests that basic amino acids play, in addition to the effect on the disruption of the cellular membrane, other roles such as specific binding on the surface of the bacteria cell.     

Functional Characterization of ECP-Heparin Interaction: A Novel Molecular Model

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]₃, and residues Gln⁴⁰, His⁶⁴ and Arg¹⁰⁵ were indicated as major contributions for the interaction. Interestingly, Gln⁴⁰ and His⁶⁴ on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln⁴⁰-His⁶⁴ clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.     

Regulatory effect of cytokines on eosinophil degranulation

We tested the effects of different cytokines on IgA- and IgG-induced eosinophil degranulation in vitro to determine the potential interaction between eosinophils and mononuclear cells. Purified normodense eosinophils were incubated with cytokines (including rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, rIL-6, IFN-gamma, granulocyte-macrophage CSF stimulating factor (GM-CSF), and TNF) for 1 to 3 h after which Ig-coupled Sepharose 4B beads were added as targets and the mixtures were incubated with the eosinophils at 37 degrees C for 4 h. The Ig used were secretory IgA (sIgA), serum IgA and IgG, and myeloma IgA and IgG. The release of eosinophil-derived neurotoxin (EDN) was measured by RIA as an index of degranulation. rIL-5 was the most potent enhancer of Ig-induced degranulation and increased EDN release by 48% for sIgA and 136% for IgG. The effect of rIL-5 appeared as quickly as 15 min after incubation of eosinophils, sIgA beads and IL-5. GM-CSF and rIL-3 also enhanced Ig-induced EDN release but less potently than rIL-5. GM-CSF and rIL-5 by themselves induced a small but significant release of EDN from eosinophils in the absence of Ig-coated beads; rIL-3 did not. However, IFN-gamma suppressed sIgA-induced EDN release by 23%. The other cytokines did not have any effect on eosinophil degranulation. These results suggest that cytokines which induce eosinophil differentiation and proliferation during hematopoiesis also enhance the effector function of mature eosinophils and that IFN-gamma partially down-regulates eosinophil degranulation.     

Nitration of the tyrosyl radical in ribonucleotide reductase by nitrogen dioxide: a gamma radiolysis study

Nitrogen dioxide is a product of peroxynitrite homolysis and peroxidase-catalyzed oxidation of nitrite. It is of great importance in protein tyrosine nitration because most nitration pathways end with the addition of *NO2 to a one-electron-oxidized tyrosine. The rate constant of this radical addition reaction is high with free tyrosine-derived radicals. However, little is known of tyrosine radicals in proteins. In this paper, we have used *NO2 generated by gamma radiolysis to study the nitration of the R2 subunit of ribonucleotide reductase, which contains a long-lived tyrosyl radical on Tyr122. Most of the nitration occurred on Tyr122, but nonradical tyrosines were also modified. In addition, peptidic bonds close to nitrated Tyr122 could be broken. Nitration at Tyr122 was not observed with a radical-free metR2 protein. The estimated rate constant of the Tyr122 radical reaction with *NO2 was of 3 x 10(4) M(-1) s(-1), thus several orders of magnitude lower than that of a radical on free tyrosine. Nitration rate of other tyrosine residues in R2 was even lower, with an estimated value of 900 M(-1) s(-1). This study shows that protein environment can significantly reduce the reactivity of a tyrosyl radical. In ribonucleotide reductase, the catalytically active radical residue is very efficiently protected against nitrogen oxide attack and subsequent nitration.     

Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.     

Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases

Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.     

Peptides of major basic protein and eosinophil cationic protein activate human mast cells

The eosinophil granule proteins, major basic protein (MBP) and eosinophil cationic protein (ECP), activate mast cells during inflammation; however the mechanism responsible for this activity is poorly understood. We found that some theoretical tryptase-digested fragments of MBP and ECP induced degranulation of human cord blood-derived mast cells (HCMCs). The spectrum of activities of these peptides in HCMCs coincided with intracellular Ca²⁺ mobilization activities in Mas-related G-protein coupled receptor family member X2 (MRGPRX2)-expressing HEK293 cells. Two peptides corresponding to MBP residues 99–110 (MBP (99–110)) and ECP residues 29–45 (ECP (29–45)), respectively, induced degranulation of HCMCs and intracellular Ca²⁺ mobilization in MRGPRX2-expressing HEK293 cells in a concentration-dependent manner. Stimulation with MBP (99–110) or ECP (29–45) induced the production of prostaglandin D2 by HCMCs. The activities of MBP (99–110) and ECP (29–45) in both HCMCs and MRGPRX2-expressing HEK293 cells were inhibited by MRGPRX2-specific antagonists. In conclusion, these results indicated that MBP and ECP fragments activate HCMCs, and it may occur via MRGPRX2. Our findings suggest that tryptase-digested fragments of eosinophil cationic proteins acting via the MRGPRX2 pathway may further our understanding of mast cell/eosinophil communication.     

Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis

Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues.     

Role of pertussis toxin-sensitive G proteins in stimulus-dependent human eosinophil degranulation.

Stimulation of human normodense eosinophils with immobilized secretory IgA (sIgA) or IgG, or with the soluble stimulus, FMLP, triggers the exocytotic release of the granule protein, eosinophil-derived neurotoxin (EDN). In this report, we demonstrate that these stimuli also provoke an increase in phospholipase C-mediated phosphoinositide breakdown in eosinophils. Pretreatment of eosinophils with pertussis toxin (PTX) for 2 h irreversibly abolished the increases in phospholipase C activity and EDN release induced by immobilized sIgA or FMLP. In contrast, PTX treatment only transiently inhibited eosinophil activation induced by immobilized IgG. Maximal inhibition of IgG-stimulated phosphoinositide hydrolysis and EDN release occurred after 2 h of PTX pretreatment with PTX, followed by a gradual recovery of cellular responsiveness to immobilized IgG as the duration of PTX pretreatment was extended to 16 h. Activated PTX catalyzed the in vitro ADP-ribosylation of 41- and 44-kDa proteins in eosinophil membranes. A 2-h pretreatment of intact cells with PTX markedly reduced the pools of unmodified 41- and 44-kDa substrates available for subsequent ADP-ribosylation in vitro, suggesting that both proteins were substrates for PTX in intact eosinophils. Continuous exposure of eosinophils to PTX for times ranging from 2 to 15 h resulted in the gradual reappearance of unmodified 44-kDa protein, whereas the levels of unmodified 41-kDa protein were persistently reduced in PTX-treated cells. The time course of the decline and reappearance of unmodified 44-kDa substrate in PTX-treated eosinophils closely paralleled the changes in the responsiveness of these cells to immobilized IgG. These results suggest that the receptors for sIgA, FMLP, or IgG transduce activating signals for eosinophil degranulation through differential coupling to at least two PTX-sensitive G proteins.     

Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation

Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.     

Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit

Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.