Oxidative stress and dietary micronutrient deficiencies contribute to overexpression of epigenetically regulated genes by lupus T cells.

Patients with active lupus have altered T cells characterized by low DNA methyltransferase levels. We hypothesized that low DNA methyltransferase levels synergize with low methionine levels to cause greater overexpression of genes normally suppressed by DNA methylation. CD4+ T cells from lupus patients and controls were stimulated with PHA then cultured in custom media with normal or low methionine levels. Oxidative stress was induced by treating the normal CD4+ T cells with peroxynitrite prior to culture. Methylation sensitive gene expression was measured by flow cytometry. Results showed low methionine levels caused greater overexpression of methylation sensitive genes in peroxynitrite treated T cells relative to untreated T cells, and in T cells from lupus patients relative to T cells from healthy controls. In conclusion, low dietary transmethylation micronutrient levels and low DNA methyltransferase levels caused either by oxidative stress or lupus, have additive effects on methylation sensitive T cell gene expression.

Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity.

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/µ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.

Protective Effect of Val129-PrP against Bovine Spongiform Encephalopathy but not Variant Creutzfeldt-Jakob Disease.

Bovine spongiform encephalopathy (BSE) is the only known zoonotic prion that causes variant Creutzfeldt-Jakob disease (vCJD) in humans. The major risk determinant for this disease is the polymorphic codon 129 of the human prion protein (Hu-PrP), where either methionine (Met129) or valine (Val129) can be encoded. To date, all clinical and neuropathologically confirmed vCJD cases have been Met129 homozygous, with the exception of 1 recently reported Met/Val heterozygous case. Here, we found that transgenic mice homozygous for Val129 Hu-PrP show severely restricted propagation of the BSE prion strain, but this constraint can be partially overcome by adaptation of the BSE agent to the Met129 Hu-PrP. In addition, the transmission of vCJD to transgenic mice homozygous for Val129 Hu-PrP resulted in a prion with distinct strain features. These observations may indicate increased risk for vCJD secondary transmission in Val129 Hu-PrP-positive humans with the emergence of new strain features.

Effects of graded dietary levels of soy protein concentrate supplemented with methionine and phosphate on the immune and antioxidant responses of gilthead sea bream (Sparus aurata L.).

The effects of a dietary soy protein concentrate (SPC) as a fish meal (FM) substitute, on selected innate immune responses, the oxidative status, hepatic and intestinal morphology of gilthead sea bream, Sparus aurata, were evaluated after a three-month feeding trial. Isonitrogenous (45% crude protein) and isoenergetic (23 kJ/g gross energy) diets with 20% (SPC20), 40% (SPC40) and 60% (SPC60) of SPC inclusion, supplemented with methionine and phosphate, were evaluated against a diet containing FM as the sole protein source. Diets were allocated in triplicate groups of 26-g fish (8 kg m-3/tank) and administered for three months. Immune responses were evaluated by performing immunological assays in blood (respiratory burst activity) and serum (myeloperoxidase content, bacteriolytic and lysozyme activity), as well as by gene expression analysis of immune-associated genes (MHCIIα, ß2m, CSF-1R, NCCRP-1, TGF-ß1, HSP70) in the head kidney and distal intestine. In addition, oxidative stress was evaluated by measuring the activity of liver enzymes associated with the antioxidant system. The respiratory burst activity of blood was significantly decreased in the SPC40 group, while serum myeloperoxidase content and bacteriolytic and lysozyme activities were affected. Significantly higher expression levels of NCCRP-1 and HSP70 were found in SPC60 head kidneys, while increased intestinal MHCIIα and NCCRP-1 transcripts were observed in SPC40. Hepatic antioxidant enzyme activity of glutathione reductase and glutathione-S-transferase was significantly enhanced in the SPC40 and SPC60 groups, while superoxide dismutase activity was increased only in the SPC40 group. Moreover, increased lipid accumulation in the enterocytes of the distal intestine was observed in the SPC60 group. Overall, a three-month feeding period with diets over 40% of dietary SPC inclusion as a FM substitute, indicated increases on immune and antioxidant enzyme responses, suggesting the dietary SPC levels that gilthead sea bream can tolerate.

Oxidation of M252 but not M428 in hu-IgG1 is responsible for decreased binding to and activation of hu-FcγRIIa (His131).

Oxidation of monoclonal therapeutic antibodies (mAbs) can affect binding to Fc-receptors and potentially influence pharmacokinetics or effector functions like e.g. antibody dependent cellular phagocytosis (ADCP). Recently, it has been demonstrated that binding to FcγRIIa (H131) is affected by methionine oxidation of the Fc-portion but it is currently unknown which methionine is responsible for decreased binding. We separated an oxidized IgG1 monoclonal antibody based on the oxidation state of methionine 252 and analyzed fractionated material in receptor binding experiments as well as in functional (cell-based) assays. Although the unfractionated mixture demonstrated weaker interaction/activation of the receptor, differently oxidized isolated subspecies can lead both to stronger as well as weaker binding and activation of the histidine variant of FcγRIIa.

Lys63/Met1-hybrid ubiquitin chains are commonly formed during the activation of innate immune signalling.

We have reported previously that activation of the MyD88-signaling network rapidly induces the formation of hybrid ubiquitin chains containing both Lys63-linked and Met1-linked ubiquitin (Ub) oligomers, some of which are attached covalently to Interleukin Receptor Associated kinase 1. Here we show that Lys63/Met1-Ub hybrids are also formed rapidly when the TNFR1/TRADD, TLR3/TRIF- and NOD1/RIP2-signaling networks are activated, some of which are attached covalently to Receptor-Interacting Protein 1 (TNFR1 pathway) or Receptor-Interacting Protein 2 (NOD1 pathway). These observations suggest that the formation of Lys63/Met1-Ub hybrids are of general significance for the regulation of innate immune signaling systems, and their potential roles in vivo are discussed. We also report that TNFα induces the attachment of Met1-linked Ub chains directly to TNF receptor 1, which do not seem to be attached covalently to Lys63-linked or other types of ubiquitin chain.

Dietary tryptophan and methionine as modulators of European seabass (Dicentrarchus labrax) immune status and inflammatory response.

Amino acids regulate key metabolic pathways important to immune responses and their nutritional supply may increase synthesis of immune-related proteins. The present study aimed to evaluate the effects of dietary supplementation of tryptophan and methionine on European seabass (Dicentrarchus labrax) cellular and humoral status. The immunomodulatory effects of tryptophan and methionine during an inflammatory insult was also evaluated after intraperitoneal injection with inactivated Photobacterium damselae subsp. piscicida (Phdp). A practical isonitrogenous (45% crude protein) and isolipidic (16% crude fat) diets was formulated to include fish meal and a blend of plant feedstuffs as protein sources and fish oil as the main lipid source (CRL diet). Two other diets were formulated similar to the control but including L-tryptophan or L-methionine at ×2 the requirement level (diets TRP and MET, respectively). European seabass weighing 275 g were fed the experimental diets for a period of 15 days before being sampled (trial 1). Then, fish were subjected to a peritoneal inflammation by intraperitoneally injecting UV killed Phdp (10(6) colony forming units ml(-1)) and sampled following 4 and 24 h post-injection (trial 2). Fish injected with a saline solution served as control. The haematological profile, peripheral cell dynamics and several plasma immune parameters were determined in trials 1 and 2, whereas cell migration to the inflammatory focus was also determined in trial 2. MET positively affected European seabass immune status by improving the peripheral leucocyte response, complement activity and bactericidal capacity, a stronger cellular recruitment to the inflammatory focus, and higher plasma peroxidase and bactericidal activities. TRP also seemed to improve immunostimulation, as there was a trend to augment both cell-mediated immunity and humoral capacity. However, TRP failed to improve an inflammatory response, verified by a decrease in blood phagocyte numbers and lack of immune cells recruitment. In summary, it is confirmed that MET has a pronounced influence on the innate immune response to inflammation, which is more evident than TRP, and raises its potential to incorporate in functional feeds to be used in prophylactic strategies against predictable unfavourable events.

Identify the key amino acid of BAFF binding with TACI.

B-cell activating factor (BAFF) has been used as a therapeutic target. To develop BAFF-specific small molecular inhibitors, it is necessary to know the key amino acid in the BAFF binding with its receptor. The key binding amino acid of BAFF interacting with its receptor TACI (trans-membrane activator and calcium modulator and cyclophilin ligand interactor) was analyzed based on the computer-guided molecular modeling method. According to theoretical prediction, a series of key amino acid mutants of BAFF, including M204 (Lys(204) to Ala), M208 (Met(208) to Ala), M209 (Gly(209) to Ala), M210 (His(210) to Ala), M234 (Gln(234) to Ala), M236 (Met(236) to Ala), and M237 (Pro(237) to Ala) were designed and evaluated with biological experiments. The results show that M208, M209, M236, and M237 of BAFF were the key amino acids and in accord with the theoretical results. The results highlight clues for the further development of BAFF-specific small molecular inhibitors.

Induction of methionine-sulfoxide reductases protects neurons from amyloid ß-protein insults in vitro and in vivo.

Self-assembly of amyloid ß-protein (Aß) into toxic oligomers and fibrillar polymers is believed to cause Alzheimer's disease (AD). In the AD brain, a high percentage of Aß contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aß assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aß. We hypothesized that the lower toxicity of Aß-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aß-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aß and Aß-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aß toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.

Detection of oxidized methionine in selected proteins, cellular extracts and blood serums by novel anti-methionine sulfoxide antibodies.

Methionine sulfoxide (MetO) is a common posttranslational modification to proteins occurring in vivo.These modifications are prevalent when reactive oxygen species levels are increased. To enable the detection of MetO in pure and extracted proteins from various sources, we have developed novel antibodies that can recognize MetO-proteins. These antibodies are polyclonal antibodies raised against an oxidized methionine-rich zein protein (MetO-DZS18) that are shown to recognize methionine oxidation in pure proteins and mouse and yeast extracts. Furthermore, mouse serum albumin and immunoglobulin (IgG)were shown to accumulate MetO as function of age especially in serums of methionine sulfoxide reductase A knockout mice. Interestingly, high levels of methionine-oxidized IgG in serums of subjects diagnosed with Alzheimer's disease were detected by western blot analysis using these antibodies. It is suggested that anti-MetO-DZS18 antibodies can be applied in the identification of proteins that undergo methionine oxidation under oxidative stress, aging, or disease state conditions.

Using bispecific antibodies in forced degradation studies to analyze the structure-function relationships of symmetrically and asymmetrically modified antibodies.

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.

Neutrophil Extracellular Traps protein composition is specific for patients with Lupus nephritis and includes methyl-oxidized αenolase (methionine sulfoxide 93).

NETs constitute a network of DNA and proteins released by neutrophils in response to infectious and immunologic triggers. NET proteins are recognized as autoantigens in ANCA vasculitis; limited knowledge is available in other autoimmune pathologies. The composition of NETs produced ex vivo by resting and Phorbol-myristate acetate (PMA) stimulated neutrophils was analyzed by high-throughput Fusion Orbitrap technology in 16 patients with Systemic Lupus Erythematosus/Lupus nephritis (9 SLE/7 LN) and in 11 controls. Seven-hundred proteins were characterized and specific fingerprints discriminated LN from SLE. We focused on methyl-oxidized αenolase (methionine sulfoxide 93) that was markedly increased in NETs from LN and was localized in NET filaments in tight connection and outlying DNA. The isotype of anti-αenolase antibodies was IgG2 in LN and IgG4 in other autoimmune glomerulonephritis (Membranous Nephropathy, MN); serum anti-αenolase IgG2 were higher in LN than in SLE and absent in MN. The same IgG2 antibodies recognized 5 epitopes of the protein one containing methionine sulphoxide 93. In conclusion, specific NET protein fingerprints characterize different subsets of SLE; methyl-oxidized αenolase is over-expressed in LN. Circulating anti-αenolase IgG2 recognize the oxidized epitope and are high in serum of LN patients. Post-translational modified NET proteins contribute to autoimmunity in patients with LN.

Identification of a novel DPA1 allele, DPA1*010602, in an East African population.

We report here a novel DPA1 allele, DPA1*010602, which was identified from an East African population during sequence-based human leukocyte antigen DPA1 typing. Through cloning and sequencing of multiple clones we confirmed that the new allele is identical to DPA1*010301 at exon 2 with the exception of two nucleotide substitutions (ATG to CAG) at codon 31. The substitutions changed the amino acid at codon 31 from methionine to glutamine. The World Health Organization nomenclature committee named the new allele DPA1*010602.

Parasites shape the optimal investment in immunity.

The evolution of optimal functioning and maintenance of the immune system is thought to be driven by the costs arising from the allocation of resources to immune functions rather than to growth and reproduction and by the benefits arising from higher defence if an infection occurs. In young animals there is a high premium for fast growth and competitiveness and a parasite-mediated trade-off is thus predicted between the allocation of resources to growth versus immune function. In a field study on nestling great tits (Parus major), we manipulated simultaneously the level of immune defence by a dietary supplementation of the immunostimulant methionine and ectoparasite (Ceratophyllus gallinae) abundance in the nest and thereby assessed both the costs and benefits of investing in immune defence. Nestlings supplemented with methionine grew slower during the experimental boost of their immune system compared to controls. Thereafter, however, nestlings with a boosted immune system grew at faster rates under parasite pressure compared to unstimulated birds. It experimentally shows the costs and benefits of investment in immunity and suggests that the evolution of optimum host defence is governed by a parasite-mediated allocation trade-off between growth and immune function.

Lipotropes, immunocompetence, and cancer.

Lipotropes (choline, methionine, folic acid, and vitamin B12) are required for normal metabolic function at the cellular and subcellular levels. Deficiencies of any or all of them to a point of influence on methyl group metabolism has a profound effect on synthesis of cellular macromolecules and on cell proliferation. Lipotrope deficiency results in a diminished immunocompetence and an increased susceptibility to a number of types of cancer in experimental animals. A challenge now being addressed is to identify the linkage between lipotropes, the immune system, and cancer and to determine mechanisms which can be useful in cancer prevention.

Immunochemical studies on alpha-lactalbumin.

The antigenic structural features of alpha-lactalbumin have been investigated using a radioimmunoassay, peptide inhibition of the quantitative precipitin reaction, and by immunodiffusion analysis after chemical modification of the molecule. Antigenic activity (in rabbits) was localized to several peptic fragments and the single arginine residue of bovine alpha-lactalbumin. Antigenic activity was also found to be associated with the single methionine residue. A peptic fragment containing a disulfide loop was found to possess antigenic activity in both bovine and goat alpha-lactalbumin. Radioimmunoassay cross-reactivity between the alpha-lactalbumins is correlated with amino acid sequence similarities; bovine alpha-lactalbumin antiserum cross-reacts with goat alpha-lactalbumin more extensively than with human alpha-lactalbumin, while the more distantly homologous protein, chicken lysozyme, does not cross-react at all. Nevertheless our data indicate that the alpha-lactalbumins and lysozyme share a similar distribution of antigenic determinants on their surfaces.

Strategies to generate antibodies capable of distinguishing between proteins with >90% amino acid identity.

Protein engineering is a common strategy for the generation of protein variants with new properties. The engineered variants often have a high degree of similarity with the wild-type progenitor protein, necessitating a tool (e.g., antibody) to distinguish the wild-type and variant protein forms. As part of an overall effort to understand the process of incorporation of amino acids into storage proteins during seed fill in soybean, we have engineered a variant of soybean vegetative storage protein beta (VSPbeta) that is 91.8% identical in amino acid sequence to the wild-type protein, but contains 10% methionine (VSPbeta-Met, unpublished results). Thus, it would be desirable to have antibodies that specifically recognize VSPbeta-Met over the endogenously expressed wild-type protein in transgenic plants. To this end, we compared three strategies for the isolation of VSPbeta-Met-specific antibodies: (1) hybridoma production using VSPbeta-Met protein as the antigen, (2) polyclonal antibody production in rabbits using a peptide antigen corresponding to a methionine-rich region of VSPbeta-Met, and (3) subtractive immunization in mice using VSPbeta-WT as the tolerogen, cyclophosphamide for immunosuppression and VSPbeta-Met as the immunogen. While the first strategy generated antibodies cross-reactive to both antigens, the second strategy generated polyclonal antibodies that preferentially recognized the variant protein in immunoblots. However, using subtractive immunization, we were able to generate mouse polyclonal antibodies that exhibited 10-fold greater reactivity with VSPbeta-Met than VSPbeta-WT in an ELISA.

Immunocompetence and its costs during development: an experimental study in blue tit nestlings.

The allocation into T-cell-mediated immunocompetence was experimentally increased in 68 out of 139 nestlings by supplementary feeding methionine to half of the nestlings in 15 blue tit nests. Methionine-supplemented nestlings had an increased T-cell-mediated immunocompetence, but a reduced growth compared with control siblings. Nestlings that had low initial weights and nestlings that were supplemented with methionine had an increased mortality risk. The investment that nestling blue tits make in immunocompetence appears to be tightly controlled by survival costs paid through a trade-off between immunocompetence and growth.

Molecular detection of methionine in rat brain using specific antibodies.

In order to study the localization of methionine in rat brain, an immunological approach was developed by raising antibodies directed against this amino acid. Methionine was conjugated to bovine serum albumin (BSA) or human serum albumin (HSA) via glutaraldehyde. The conjugates were then reduced by sodium borohydride and injected alternately into rabbits. Antibody affinity and specificity were evaluated using an adapted ELISA method, by competition experiments between conjugated methionine and related conjugated compounds, pre-incubated with anti-methionine antibodies diluted at 1/20,000. The resulting cross-reactivity ratios, calculated at half-displacement, showed that glutaraldehyde-methionine conjugate (methionine-G-BSA) was the best recognized compound. Non-reduced methionine conjugate (methionine=G=BSA) and the related-conjugated molecules such as homocysteine, homocysteic acid, cysteine, cystathionine and glutamate were not recognized at all. Antibodies to methionine were directed against a glutaraldehyde-methionine epitope and their very high affinity and specificity made them reliable tools for molecular detection of methionine in rat brain. Using purified antibodies diluted at 1/20,000, motoneurons were found to be the most methionine-immunoreactive cell bodies in glutaraldehyde-fixed rat brain sections.

Pneumolysin potentiates oxidative inactivation of alpha-1-proteinase inhibitor by activated human neutrophils.

This study was designed to investigate the effects of the Streptococcus pneumoniae-derived, pro-inflammatory toxin, pneumolysin (8.37 and 41.75 ng/ml), on the oxidative inactivation of alpha-1-protease inhibitor (API) by chemoattractant-activated human neutrophils in vitro. The elastase inhibitory capacity (EIC) of API in supernatants from unstimulated neutrophils, neutrophils treated with pneumolysin only, or with the chemoattractant FMLP (1 microM) only, or the combination of the toxin with FMLP was measured by a colorimetric procedure based on the activity of added porcine elastase. The EIC of API was unaffected by exposure to pneumolysin only, unstimulated neutrophils, or neutrophils treated with pneumolysin only. However, exposure to FMLP-activated neutrophils resulted in a reduction of the EIC of API, which was significantly (P<0.05) augmented by pneumolysin (mean reductions of 16%, 43% and 83% for FMLP only and in combination with 8.37 and 41.75 ng/ml pneumolysin, respectively), and was attenuated by wortmannin (1 microM), an inhibitor of NADPH oxidase, the oxidant-scavenger methionine (100 microM), and depletion of Ca2+ from the cell-suspending medium. These pro-proteolytic interactions of pneumolysin with chemoattractant-activated neutrophils may contribute to the invasiveness of the pneumococcus.