Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met[O]) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.Key words: immunoglobulin gamma antibody, methionine sulfoxide, oxidation, photo-oxidation, methionine sulfoxide reductase     

Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met(O)) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.     

Lens methionine sulfoxide reductase

Investigation of human and bovine lenses has demonstrated the presence of a methionine sulfoxide (Met(O)) peptide reductase activity. The reductase can use either dithiothreitol or thioredoxin but not glutathione as a reducing agent. The enzyme is present primarily in the water soluble fraction. The highest specific activity is in the outer epithelial layer with decreasing activity in the inner layers of the tissue. The known high level of methionine sulfoxide residues in cataractous lens protein is not due to a decreased level of Met (O)-peptide reductase itself since a comparison of normal and cataractous human lenses showed no statistically significant decrease in reductase activity in the cataract population. However, it is not known whether the reducing system for Met (O)-peptide reductase (probably the thioredoxin system) is deficient in cataractous lenses.     

Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase.

Methionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D-sulfoxide diastereomer of CaM-bound L-MetSO (L-Met-D-SO). After exhaustive reduction by pMSR, the ratio of L-Met-D-SO to L-Met-L-SO decreased to about 1:25 for hydrogen peroxide-oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR.     

A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide

It is known that Escherichia coli methionine mutants can grow on both enantiomers of methionine sulfoxide (met(o)), i.e., met-R-(o) or met-S-(o), indicating the presence of enzymes in E. coli that can reduce each of these enantiomers to methionine (met). Previous studies have identified two members of the methionine sulfoxide reductase (Msr) family of enzymes, MsrA and fSMsr, that could reduce free met-S-(o), but the reduction of free met-R-(o) to met has not been elucidated. One possible candidate is MsrB which is known to reduce met-R-(o) in proteins to met. However, free met-R-(o) is a very poor substrate for MsrB and the level of MsrB activity in E. coli extracts is very low. A new member of the Msr family (fRMsr) has been identified in E. coli extracts that reduces free met-R-(o) to met. Partial purification of FRMsr has been obtained using extracts from an MsrA/MsrB double mutant of E. coli.     

Structure of Mycobacterium tuberculosis methionine sulfoxide reductase A in complex with protein-bound methionine

Peptide methionine sulfoxide reductase (MsrA) repairs oxidative damage to methionine residues arising from reactive oxygen species and reactive nitrogen intermediates. MsrA activity is found in a wide variety of organisms, and it is implicated as one of the primary defenses against oxidative stress. Disruption of the gene encoding MsrA in several pathogenic bacteria responsible for infections in humans results in the loss of their ability to colonize host cells. Here, we present the X-ray crystal structure of MsrA from the pathogenic bacterium Mycobacterium tuberculosis refined to 1.5 A resolution. In contrast to the three catalytic cysteine residues found in previously characterized MsrA structures, M. tuberculosis MsrA represents a class containing only two functional cysteine residues. The structure reveals a methionine residue of one MsrA molecule bound at the active site of a neighboring molecule in the crystal lattice and thus serves as an excellent model for protein-bound methionine sulfoxide recognition and repair.     

Methionine sulfoxide and the methionine sulfoxide reductase system as modulators of signal transduction pathways: a review

Methionine oxidation and reduction is a common phenomenon occurring in biological systems under both physiological and oxidative-stress conditions. The levels of methionine sulfoxide (MetO) are dependent on the redox status in the cell or organ, and they are usually elevated under oxidative-stress conditions, aging, inflammation, and oxidative-stress related diseases. MetO modification of proteins may alter their function or cause the accumulation of toxic proteins in the cell/organ. Accordingly, the regulation of the level of MetO is mediated through the ubiquitous and evolutionary conserved methionine sulfoxide reductase (Msr) system and its associated redox molecules. Recent published research has provided new evidence for the involvement of free MetO or protein-bound MetO of specific proteins in several signal transduction pathways that are important for cellular function. In the current review, we will focus on the role of MetO in specific signal transduction pathways of various organisms, with relation to their physiological contexts, and discuss the contribution of the Msr system to the regulation of the observed MetO effect.

     

Salmonella Typhimurium methionine sulfoxide reductase A (MsrA) prefers TrxA in repairing methionine sulfoxide

Intraphagocytic survival of Salmonella Typhimurium (ST) depends (at least in part) upon its ability to repair oxidant-damaged macromolecules. Met residues either free or in protein bound form are highly susceptible to phagocyte-generated oxidants. Oxidation of Mets leads to Met-SO formation, consequently loss of protein functions that results in cell death. Methionine sulfoxide reductase (Msr) reductively repairs Met-SO to Met in the presence of thioredoxin (trx) and thioredoxin reductase (trxR). Earlier we reported that methionine sulfoxide reductase A (msrA) gene deletion strain of ST suffered oxidative stress.^[1 Trivedi, R.N.; Agarwal, P.; Kumawat, M.; Pesingi, P.K.; Gupta, V.K.; Goswami, T.K.; Mahawar, M. Methionine Sulfoxide Reductase A (MsrA) Contributes to Salmonella Typhimurium Survival Against Oxidative Attack of Neutrophils. Immunobiology 2015, 220(12), 1322–1327.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]^] Thioredoxin system of ST comprises of two thioredoxins (trxA and trxC) and one thioredoxin reductase (trxB). Preferred trx utilized in MsrA-mediated repair of Met-SO is not known. In current study, we cloned, expressed, and purified ST TrxA, TrxB, TrxC, and MsrA in recombinant forms. The migration of TrxA, TrxB, TrxC, and MsrA proteins was approximately 10, 36, 16, and 26?kDa on SDS-gels. The nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-linked reductase assays interpreted that MsrA utilized two times more NADPH for the reduction of S-methyl p-tolyl sulfoxide when TrxA was included in the assays as compared to TrxC.     

Increased catalytic efficiency following gene fusion of bifunctional methionine sulfoxide reductase enzymes from Shewanella oneidensis

Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and together function as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes are fused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate how gene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressed the MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. For comparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a genetically engineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatory protein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymes together at 25 degrees C. A restoration of the normal protein fold is observed co-incident with the repair of MetSO in oxidized CaM (CaMox by MsrBA, as monitored by time-dependent increases in the anisotropy associated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). Underlying the efficient repair of MetSO in CaMox is the coordinate activity of the two catalytic domains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement in comparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of both domains of MsrBA permits the full repair of all MetSO in CaMox. The common expression of Msr fusion proteins in bacterial pathogens is consistent with an important role for this enzyme activity in the maintenance of protein function necessary for bacterial survival under highly oxidizing conditions associated with pathogenesis or bioremediation.     

Reduction of N-acetyl methionine sulfoxide: A simple assay for peptide methionine sulfoxide reductase

A simple and rapid assay has been developed to measure the enzymatic activity of peptide methionine sulfoxide reductase. The assay is based on the reduction of labeled N-acetylmethionine sulfoxide to N-acetylmethionine. The N-acetylmethionine can be separated from the substrate by extraction into ethyl acetate.     

Analyses of methionine sulfoxide reductase activities towards free and peptidyl methionine sulfoxides

There have been insufficient kinetic data that enable a direct comparison between free and peptide methionine sulfoxide reductase activities of either MsrB or MsrA. In this study, we determined the kinetic parameters of mammalian and yeast MsrBs and MsrAs for the reduction of both free methionine sulfoxide (Met-O) and peptidyl Met-O under the same assay conditions. Catalytic efficiency of mammalian and yeast MsrBs towards free Met-O was >2000-fold lower than that of yeast fRMsr, which is specific for free Met-R-O. The ratio of free to peptide Msr activity in MsrBs was 1:20-40. In contrast, mammalian and yeast MsrAs reduced free Met-O much more efficiently than MsrBs. Their k(cat) values were 40-500-fold greater than those of the corresponding MsrBs. The ratio of free to peptide Msr activity was 1:0.8 in yeast MsrA, indicating that this enzyme can reduce free Met-O as efficiently as peptidyl Met-O. In addition, we analyzed the in vivo free Msr activities of MsrBs and MsrAs in yeast cells using a growth complementation assay. Mammalian and yeast MsrBs, as well as the corresponding MsrAs, had apparent in vivo free Msr activities. The in vivo free Msr activities of MsrBs and MsrAs agreed with their in vitro activities.     

Methionine sulfoxide reductase contributes to meeting dietary methionine requirements

Methionine sulfoxide reductases are present in all aerobic organisms. They contribute to antioxidant defenses by reducing methionine sulfoxide in proteins back to methionine. However, the actual in vivo roles of these reductases are not well defined. Since methionine is an essential amino acid in mammals, we hypothesized that methionine sulfoxide reductases may provide a portion of the dietary methionine requirement by recycling methionine sulfoxide. We used a classical bioassay, the growth of weanling mice fed diets varying in methionine, and applied it to mice genetically engineered to alter the levels of methionine sulfoxide reductase A or B1. Mice of all genotypes were growth retarded when raised on chow containing 0.10% methionine instead of the standard 0.45% methionine. Retardation was significantly greater in knockout mice lacking both reductases. We conclude that the methionine sulfoxide reductases can provide methionine for growth in mice with limited intake of methionine, such as may occur in the wild.     

Plant methionine sulfoxide reductase A and B multigenic families

Methionine oxidation to methionine sulfoxide (MetSo), which results in modification of activity and conformation for many proteins, is reversed by an enzyme present in most organisms and termed as methionine sulfoxide reductase (MSR). On the basis of substrate stereospecificity, two types of MSR, A and B, that do not share any sequence similarity, have been identified. In the present review, we first compare the multigenic MSR families in the three plant species for which the genome is fully sequenced: Arabidopsis thaliana, Oryza sativa, and Populus trichocarpa. The MSR gene content is larger in A. thaliana (five MSRAs and nine MSRBs) compared to P. trichocarpa (five MSRAs and four MSRBs) and O. sativa (four MSRAs and three MSRBs). A complete classification based on gene structure, sequence identity, position of conserved reactive cysteines and predicted subcellular localization is proposed. On the basis of in silico and experimental data originating mainly from Arabidopsis, we report that some MSR genes display organ-specific expression patterns and that those encoding plastidic MSRs are highly expressed in photosynthetic organs. We also show that the expression of numerous MSR genes is enhanced by environmental conditions known to generate oxidative stress. Thioredoxins (TRXs) constitute very likely physiological electron donors to plant MSR proteins for the catalysis of MetSO reduction, but the specificity between the numerous TRXs and methionine sulfoxide reductases (MSRs) present in plants remains to be investigated. The essential role of plant MSRs in protection against oxidative damage has been recently demonstrated on transgenic Arabidopsis plants modified in the content of cytosolic or plastidic MSRA.     

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.     

Peptide methionine sulfoxide reductase: biochemistry and physiological role

The oxidation of methionine to methionine sulfoxide both in vivo and in vitro can lead to the loss of biological activity in a variety of proteins. This loss of activity can be reversed by an enzyme called methionine sulfoxide reductase. The gene for this enzyme has been cloned and sequenced from a variety of prokaryotic and eukaryotic cells, and the deduced amino acid sequence is very highly conserved. The mechanism of action of the bovine enzyme has been shown to involve a critical cysteine residue located at position 72 of the protein. In addition to its role as a "repair" enzyme, other evidence suggests that the enzyme may be involved in bacterial adherence and regulation of protein activity.     

Myristoylated methionine sulfoxide reductase A protects the heart from ischemia-reperfusion injury

Methionine sulfoxide reductase A (MsrA) catalytically scavenges reactive oxygen species and also repairs oxidized methionines in proteins. Increasing MsrA protects cells and organs from a variety of oxidative stresses while decreasing MsrA enhances damage, but the mechanisms of action have not been elucidated. A single gene encodes MsrA of which ～25% is targeted to the mitochondria, a major site of reactive oxygen species production. The other ～75% is targeted to the cytosol and is posttranslationally modified by myristoylation. To determine the relative importance of MsrA in each compartment in protecting against ischemia-reperfusion damage, we created a series of transgenic mice overexpressing MsrA targeted to the mitochondria or the cytosol. We used a Langendorff model of ischemia-reperfusion and assayed both the rate pressure product and infarct size following ischemia and reperfusion as measures of injury. While the mitochondrially targeted MsrA was expected to be protective, it was not. Notably, the cytosolic form was protective but only if myristoylated. The nonmyristoylated, cytosolic form offered no protection against injury. We conclude that cytosolic MsrA protects the heart from ischemia-reperfusion damage. The requirement for myristoylation suggests that MsrA must interact with a hydrophobic domain to provide protection.     

Functional characterization of methionine sulfoxide reductase A from Trypanosoma spp

Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.     

Deletion of both methionine sulfoxide reductase A and methionine sulfoxide reductase C genes renders Salmonella Typhimurium highly susceptible to hypochlorite stress and poultry macrophages

Molecular Biology Reports - Salmonella Typhimurium survives and replicates inside the oxidative environment of phagocytic cells. Proteins, because of their composition and location, are the...     

The Arabidopsis plastidic methionine sulfoxide reductase B proteins. Sequence and activity characteristics, comparison of the expression with plastidic methionine sulfoxide reductase A, and induction by photooxidative stress

Two types of methionine (Met) sulfoxide reductases (Msr) catalyze the reduction of Met sulfoxide (MetSO) back to Met. MsrA, well characterized in plants, exhibits an activity restricted to the Met-S-SO-enantiomer. Recently, a new type of Msr enzyme, called MsrB, has been identified in various organisms and shown to catalytically reduce the R-enantiomer of MetSO. In plants, very little information is available about MsrB and we focused our attention on Arabidopsis (Arabidopsis thaliana) MsrB proteins. Searching Arabidopsis genome databases, we have identified nine open reading frames encoding proteins closely related to MsrB proteins from bacteria and animal cells. We then analyzed the activity and abundance of the two chloroplastic MsrB proteins, MsrB1 and MsrB2. Both enzymes exhibit an absolute R-stereospecificity for MetSO and a higher catalytic efficiency when using protein-bound MetSO as a substrate than when using free MetSO. Interestingly, we observed that MsrB2 is reduced by thioredoxin, whereas MsrB1 is not. This feature of MsrB1 could result from the lack of the catalytical cysteine (Cys) corresponding to Cys-63 in Escherichia coli MsrB that is involved in the regeneration of Cys-117 through the formation of an intramolecular disulfide bridge followed by thioredoxin reduction. We investigated the abundance of plastidial MsrA and B in response to abiotic (water stress, photooxidative treatment) and biotic (rust fungus) stresses and we observed that MsrA and B protein levels increase in response to the photooxidative treatment. The possible role of plastidic MsrB in the tolerance to oxidative damage is discussed.