Immunoglobulins G from patients with ANCA-associated vasculitis are atypically glycosylated in both the Fc and Fab regions and the relation to disease activity.

BACKGROUND: Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are pathogenic in ANCA-associated vasculitis (AAV). The respective role of IgG Fc and Fab glycosylation in mediating ANCA pathogenicity is incompletely understood. Herein we investigate in detail the changes in Fc and Fab glycosylation in MPO-ANCA and Pr3-ANCA and examine the association of glycosylation aberrancies with disease activity. METHODOLOGY: Total IgG was isolated from serum or plasma of a cohort of 30 patients with AAV (14 MPO-ANCA; 16 PR3-ANCA), and 19 healthy control subjects. Anti-MPO specific IgG was affinity-purified from plasma of an additional cohort of 18 MPO-ANCA patients undergoing plasmapheresis. We used lectin binding assays, liquid chromatography, and mass spectrometry-based methods to analyze Fc and Fab glycosylation, the degree of sialylation of Fc and Fab fragments and to determine the exact localization of N-glycans on Fc and Fab fragments. PRINCIPAL FINDINGS: IgG1 Fc glycosylation of total IgG was significantly reduced in patients with active AAV compared to controls. Clinical remission was associated with complete glycan normalization for PR3-ANCA patients but not for MPO-ANCA patients. Fc-glycosylation of anti-MPO specific IgG was similar to total IgG purified from plasma. A major fraction of anti-MPO specific IgG harbor extensive glycosylation within the variable domain on the Fab portion. CONCLUSIONS/SIGNIFICANCE: Significant differences exist between MPO and PR3-ANCA regarding the changes in amounts and types of glycans on Fc fragment and the association with disease activity. These differences may contribute to significant clinical difference in the disease course observed between the two diseases.

Dysregulation of autoantigen genes in ANCA-associated vasculitis involves alternative transcripts and new protein synthesis.

Proteinase 3 (PR3) and myeloperoxidase (MPO) are two major autoantigens in patients with vasculitis with ANCA. The genes encoding these autoantigens are abnormally expressed in peripheral granulocytes of patients with active ANCA-associated vasculitis. This study provides evidence that this transcriptional dysregulation results in a variety of mRNA processing events from the PRTN3 gene locus. In addition to elevated levels of PR3 message, leukocyte RNA from patients contained PR3 transcripts with an alternative 3' untranslated region. Furthermore, we detected usage of an alternative transcription start site within intron 1 of the PRTN3 gene locus that coincided with active disease (odds ratio, 3.3; 95% confidence interval, 1.3 to 8.4; P=0.01). This promoter may be developmentally regulated, because it was active in normal human bone marrow, multiple leukemia cell lines, MCF-7 cells, and subjects after GM-CSF treatment but not subjects with a neutrophil left shift. This transcript, which lacks exon 1 of PRTN3, encodes a 24-kD protein (p24(PR3/MBN)) with a sequence similar to that previously described for myeloblastin. Notably, PR3, p24(PR3/MBN), and MPO were synthesized in cultured neutrophils from patients with active ANCA-associated vasculitis, indicating that increased transcription results in newly synthesized autoantigens in peripheral neutrophils of patients. The synthesis of p24(PR3/MBN) seems to expand the autoantigen repertoire, because immunoblots showed that sera from patients recognized p24(PR3/MBN). These findings emphasize the importance of transcriptional dysregulation of the autoantigen in autoimmune disease.

Free radical metabolism of methyleugenol and related compounds.

Methyleugenol, the methyl ether of eugenol, both of which are flavorant constituents of spices, has been listed by the National Toxicology Program's Report on Carcinogens as reasonably anticipated to be a human carcinogen. This finding is based on the observation of increased incidence of malignant tumors at multiple tissue sites in experimental animals of different species. By contrast, eugenol is not listed. In this study, we show that both methyleugenol and eugenol readily undergo peroxidative metabolism in vitro to form free radicals with large hyperfine interactions of the methylene allylic hydrogen atoms. These large hyperfine splittings indicate large electron densities adjacent to those hydrogen atoms. Methyleugenol undergoes autoxidation such that the commercial product contains 10-30 mg/L hydroperoxide and is capable of activating peroxidases without the presence of added hydrogen peroxide. Additionally, the hydroperoxide is not a good substrate for catalase, which demonstrates that these antioxidant defenses will not be effective in protecting against methyleugenol exposure.

Sulfite-mediated oxidation of myeloperoxidase to a free radical: immuno-spin trapping detection in human neutrophils.

Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of the reactive sulfur trioxide ((•)SO3(-)), peroxymonosulfate ((-)O3SOO(•)), and sulfate (SO4(•-)) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions, and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of this study was to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage, possibly leading to (bi)sulfite-exacerbated allergic reactions.

Biotinylated analogue of the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide for the detection of low-abundance protein radicals by mass spectrometry.

Protein radicals are implicated in oxidative stress and are associated with a wide range of diseases and disorders. In the present work, we describe the specific application of a newly synthesized nitrone spin trap, Bio-SS-DMPO, for the detection of these highly reactive species by mass spectrometry (MS). Bio-SS-DMPO is a biotinylated analogue of the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) that allows for specific capture of the protein(s)/peptide(s) labeled by the spin-trap on a (strept)avidin-bound solid matrix. The disulfide bond in the linker arm joining biotin to DMPO can be cleaved to release captured spin-adduct peptide from the solid matrix. This (strept)avidin-based affinity purification reduces the complexity of the samples prior to MS analyses, thereby facilitating the location of the sites of spin trap addition. In addition, the biotin moiety on the spin-trap can efficiently be probed with (strept)avidin-conjugated reporter. This offers an effective means to visualize the presence of DMPO-adducted proteins in intact cells.

Spin scavenging analysis of myoglobin protein-centered radicals using stable nitroxide radicals: characterization of oxoammonium cation-induced modifications.

Spin scavenging combined with chromatographic and mass spectrometric procedures can, in principle, be employed to detect and identify protein-based radicals within complex biological matrices. This approach is based on the well-known ability of stable synthetic nitroxide radicals to scavenge carbon-centered radicals, forming stable diamagnetic addition products. Hence, characterization of these addition products would allow for the identification of specific free radicals. In the present work, we have explored the use of the stable nitroxide radical 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) in scavenging protein-based radicals generated in a horse heart metmyoglobin/hydrogen peroxide (metMb/H(2)O(2)) system. Inclusion of a substoichiometric amount of TEMPOL in the metMb/H(2)O(2) system resulted in a complete loss of peroxyl and tyrosyl radical signals and effectively inhibited the formation of oxidatively damaged heme species, as monitored by electron paramagnetic resonance and reversed-phase liquid chromatography. Scavenging of globin radicals by TEMPOL did not lead to the formation of stable diamagnetic addition adducts; in fact, reversed-phase liquid chromatographic studies and oxygen electrode measurements indicated that TEMPOL acts as a catalyst and is recycled in this system. The oxoammonium cation generated in the course of this reaction initiated secondary reactions resulting in the formation of a free carbonyl on the N-terminal Gly-residue of the protein. This oxidative deamination was confirmed through the combined use of reversed-phase liquid chromatographic purification, tandem MS experiments, and chemical analysis (e.g., by use of 2,4-dinitrophenyl hydrazine). The results reveal the pitfalls inherent in using stable nitroxide radicals such as TEMPOL to identify sites of radical formation on hemoproteins.

Identification of protein radicals formed in the human neuroglobin-H2O2 reaction using immuno-spin trapping and mass spectrometry.

Neuroglobin (Ngb) is a recently discovered protein that shows only minor sequence similarity with myoglobin and hemoglobin but conforms to the typical 3-over-3 alpha-helical fold characteristic of vertebrate globins. An intriguing feature of Ngb is its heme hexacoordination in the absence of external ligands, observed both in the ferrous and in the ferric (met) forms. In Ngb, the imidazole of a histidine residue (His-64) in the distal position, above the heme plane, provides the sixth coordination bond. In this work, a valine residue was introduced at position 64 (H64V variant) to clarify the possible role(s) of the distal residue in protecting the heme iron of Ngb from attack by strong oxidants. SDS-PAGE analyses revealed that the oxidation of the H64V variant of metNgb by H 2O 2 resulted in the formation of dimeric and trimeric products in contrast to the native protein. Dityrosine cross-links were shown by their fluorescence to be present in the oligomeric products. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture, nitrone adducts were detected by immuno-spin trapping. The specific location of the DMPO adducts on the H64V variant protein was determined by a mass spectrometry method that combines off-line immuno-spin trapping and chromatographic procedures. This method revealed Tyr-88 to be the site of modification by DMPO. The presence of His-64 in the wild-type protein results in the nearly complete loss of detectable radical adducts. Together, the data support the argument that wild-type Ngb is protected from attack by H 2O 2 by the coordinated distal His.

Identifying the site of spin trapping in proteins by a combination of liquid chromatography, ELISA, and off-line tandem mass spectrometry.

An off-line mass spectrometry method that combines immuno-spin trapping and chromatographic procedures has been developed for selective detection of the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) covalently attached to proteins, an attachment which occurs only subsequent to DMPO trapping of free radicals. In this technique, the protein-DMPO nitrone adducts are digested to peptides with proteolytic agents, peptides from the enzymatic digest are separated by HPLC, and enzyme-linked immunosorbent assays (ELISA) using polyclonal anti-DMPO nitrone antiserum are used to detect the eluted HPLC fractions that contain DMPO nitrone adducts. The fractions showing positive ELISA signals are then concentrated and characterized by tandem mass spectrometry (MS/MS). This method, which constitutes the first liquid chromatography-ELISA-mass spectrometry (LC-ELISA-MS)-based strategy for selective identification of DMPO-trapped protein residues in complex peptide mixtures, facilitates location and preparative fractionation of DMPO nitrone adducts for further structural characterization. The strategy is demonstrated for human hemoglobin, horse heart myoglobin, and sperm whale myoglobin, three globin proteins known to form DMPO-trappable protein radicals on treatment with H(2)O(2). The results demonstrate the power of the new experimental strategy to select DMPO-labeled peptides and identify sites of DMPO covalent attachments.

Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization.

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.

Autoreduction of ferryl myoglobin: discrimination among the three tyrosine and two tryptophan residues as electron donors.

Ferric myoglobin undergoes a two-electron oxidation in its reaction with H(2)O(2). One oxidation equivalent is used to oxidize Fe(III) to the Fe(IV) ferryl species, while the second is associated with a protein radical but is rapidly dissipated. The ferryl species is then slowly reduced back to the ferric state by unknown mechanisms. To clarify this process, the formation and stability of the ferryl forms of the Tyr --> Phe and Trp --> Phe mutants of recombinant sperm whale myoglobin (SwMb) were investigated. Kinetic studies showed that all the mutants react normally with H(2)O(2) to give the ferryl species. However, the rapid phase of ferryl autoreduction typical of wild-type SwMb was absent in the triple Tyr --> Phe mutant and considerably reduced in the Y103F and Y151F mutants, strongly implicating these two residues as intramolecular electron donors. Replacement of Tyr146, Trp7, or Trp14 did not significantly alter the autoreduction, indicating that these residues do not contribute to ferryl reduction despite the fact that Tyr146 is closer to the iron than Tyr151 or Tyr103. Furthermore, analysis of the fast phase of autoreduction in the dimer versus recovered monomer of the Tyr --> Phe mutant K102Q/Y103F/Y146F indicates that the Tyr151-Tyr151 cross-link is a particularly effective electron donor. The presence of an additional, slow phase of reduction in the triple Tyr --> Phe mutant indicates that alternative but normally minor electron-transfer pathways exist in SwMb. These results demonstrate that internal electron transfer is governed as much by the tyrosine pK(a) and oxidation potential as by its distance from the electron accepting iron atom.

Intra- and intermolecular transfers of protein radicals in the reactions of sperm whale myoglobin with hydrogen peroxide.

Reaction of sperm whale metmyoglobin (SwMb) with H2O2 produces a ferryl (MbFeIV=O) species and a protein radical and leads to the formation of oligomeric products. The ferryl species is maximally formed with one equivalent of H2O2, and the maximum yields of the dimer (28%) and trimer (17%) with 1 or 2 eq. Co-incubation of the SwMb Y151F mutant with native apoSwMb and H2O2 produced dimeric products, which requires radical transfer from the nondimerizing Y151F mutant to apoSwMb. Autoreduction of ferryl SwMb to the ferric state is biphasic with t = 3.4 and 25.9 min. An intramolecular autoreduction process is implicated at low protein concentrations, but oligomerization decreases the lifetime of the ferryl species at high protein concentrations. A fraction of the protein remained monomeric. This dimerization-resistant protein was in the ferryl state, but after autoreduction it underwent normal dimerization with H2O2. Proteolytic digestion established the presence of both dityrosine and isodityrosine cross-links in the oligomeric proteins, with the isodityrosine links primarily forged by Tyr151-Tyr151 coupling. The tyrosine content decreased by 47% in the dimer and 14% in the recovered monomer, but the yields of isodityrosine and dityrosine in the dimer were only 15.2 and 6.8% of the original tyrosine content. Approximately 23% of the lost tyrosines therefore have an alternative but unknown fate. The results clearly demonstrate the concurrence of intra- and intermolecular electron transfer processes involving Mb protein radicals. Intermolecular electron transfers that generate protein radicals on bystander proteins are likely to propagate the cellular damage initiated by the reaction of metalloproteins with H2O2.

Tryptophan-14 is the preferred site of DBNBS spin trapping in the self-peroxidation reaction of sperm whale metmyoglobin with a single equivalent of hydrogen peroxide.

The 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS)-metmyoglobin adduct formed following the horse metmyoglobin-H(2)O(2) reaction has been assigned to both a tyrosyl and a tryptophanyl residue radical. At low H(2)O(2), hyperfine coupling to a (13)C atom in sperm whale metmyoglobin labeled at the tryptophan residues with (13)C allowed the unequivocal assignment of the primary adduct to a tryptophanyl radical. Trapping at Trp-14 of sperm whale myoglobin was indicated by greatly decreased electron paramagnetic resonance (EPR) spectral intensity of the DBNBS adducts of the Trp-14-Phe recombinant proteins. Complex EPR spectra with partially resolved hyperfine splittings from several atoms were obtained by pronase treatment of the DBNBS/*W14F metmyoglobin adducts. The EPR spectra of authentic DBNBS/*Tyr adducts were incubation time-dependent; the late time spectra resembled the spectra of pronase-treated DBNBS/*W14F sperm whale myoglobin adducts, suggesting formation of an unstable tyrosyl radical adduct in the latter proteins. When the H(2)O(2):metmyoglobin ratio was increased to 5:1, the EPR spectrum after pronase treatment supported trapping of a tyrosyl radical, although similar decreases in tryptophan content were detected at H(2)O(2):metmyoglobin ratios of 1:1 and 5:1.