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     

Studies on the reducing systems for plant and animal thioredoxin-independent methionine sulfoxide reductases B

Two distinct stereospecific methionine sulfoxide reductases (Msr), MsrA and MsrB reduce the oxidized methionine (Met), methionine sulfoxide [Met(O)], back to Met. In this report, we examined the reducing systems required for the activities of two chloroplastic MsrB enzymes (NtMsrB1 and NtMsrB2) from tobacco (Nicotiana tabacum). We found that NtMrsB1, but not NtMsrB2, could use dithiothreitol as an efficient hydrogen donor. In contrast Escherichia coli thioredoxin (Trx) could serve as a reducing agent for NtMsrB2, but not for NtMsrB1. Similar to previously reported human Trx-independent hMsrB2 and hMsrB3, NtMsrB1 could also use bovine liver thionein and selenocysteamine as reducing agents. Furthermore, the unique plant Trx-like protein CDSP32 was shown to reduce NtMsrB1, hMsrB2 and hMsrB3. All these tested Trx-independent MsrB enzymes lack an additional cysteine (resolving cysteine) that is capable of forming a disulfide bond on the enzyme during the catalytic reaction. Our results indicate that plant and animal MsrB enzymes lacking a resolving cysteine likely share a similar reaction mechanism.     

Functional characterization of the Aspergillus nidulans methionine sulfoxide reductases (msrA and msrB)

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here, we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage.     

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.     

Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases

Oxidative damage to proteins is considered to be one of the major causes of aging and age-related diseases, and thus mechanisms have evolved to prevent or reverse these modifications. Methionine is one of the major targets of reactive oxygen species (ROS), where it is oxidized to methionine sulfoxide (MetO). Recently, evidence has accumulated suggesting that methionine (Met) oxidation may play an important role in the development and progression of neurodegenerative diseases like Alzheimer's and Parkinson's diseases. Oxidative alteration of Met to Met(O) is reversed by the methionine sulfoxide reductases (consisting of MsrA enzymes that reduce S-MetO and MsrB enzymes that reduce R-MetO, respectively). A major biological role of the Msr system is suggested by the fact that the MsrA null mouse (MT) exhibits a neurological disorder in the form of ataxia ("tip toe walking"), is more sensitive to oxidative stress, and has a shorter life span (by approximately 40%) than wild-type (WT) mice. By their action, the Msr enzymes can regulate protein function, be involved in signal-transduction pathways, and prevent cellular accumulation of faulty proteins. Malfunction of the Msr system can lead to cellular changes resulting in compromised antioxidant defense, enhanced age-associated diseases involving neurodegeneration, and shorter life span. In this review, the function and possible roles of the Msr system in prokaryotes and eukaryotes, in general, and in neurodegenerative diseases, in particular, will be discussed.     

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.     

Calorimetry and mass spectrometry study of oxidized calmodulin interaction with target and differential repair by methionine sulfoxide reductases

Calmodulin is known to be a target for oxidation, which leads to conversion of methionine residues to methionine sulfoxides. Previously, we reported that both methionine sulfoxide reductases MsrA and MsrB were able to reduce methionine sulfoxide residues in oxidized calmodulin. In the present study, we have made use of the interaction between calmodulin and RS20, a peptide model for calmodulin targets, to probe the structural consequences of oxidation and mode of repair both by MsrA and MsrB. Isothermal titration calorimetry and differential scanning calorimetry showed that oxidized calmodulin interacts with RS20 via its C-terminal domain only, resulting in a non-productive complex. As shown by spectrofluorometry, oxidized calmodulin treated with MsrA exhibited native binding affinity for RS20. In contrast, MsrB-treatment of oxidized calmodulin resulted in 10-fold reduced affinity. Mass spectrometry revealed that the sulfoxide derivative of methionine residue 124 was differentially repaired by MsrA and MsrB. This provided a basis for rationalizing the difference in binding affinities of oxidized calmodulin reported above, since Met124 residue had been shown to be critical for interaction with some targets. This study provides the first evidence that in an oxidized polypeptide chain MetSO residues might be differentially repaired by the two Msr enzymes.     

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.     

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.     

High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 +/- 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 +/- 4 microM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.     

The methionine sulfoxide reductases: Catalysis and substrate specificities

Oxidation of Met residues in proteins leads to the formation of methionine sulfoxides (MetSO). Methionine sulfoxide reductases (Msr) are ubiquitous enzymes, which catalyze the reduction of the sulfoxide function of the oxidized methionine residues. In vivo, the role of Msrs is described as essential in protecting cells against oxidative damages and to play a role in infection of cells by pathogenic bacteria. There exist two structurally-unrelated classes of Msrs, called MsrA and MsrB, with opposite stereoselectivity towards the S and R isomers of the sulfoxide function, respectively. Both Msrs present a similar three-step catalytic mechanism. The first step, called the reductase step, leads to the formation of a sulfenic acid on the catalytic Cys with the concomitant release of Met. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the catalysis, in particular of the reductase step, and in structural specificities.     

New membrane-associated and soluble peptide methionine sulfoxide reductases in Escherichia coli

It is known that reactive oxygen species can oxidize methionine residues in proteins in a non-stereospecific manner, and cells have mechanisms to reverse this damage. MsrA and MsrB are members of the methionine sulfoxide family of enzymes that specifically reduce the S and R forms, respectively, of methionine sulfoxide in proteins. However, in Escherichia coli the level of MsrB activity is very low which suggested that there may be other enzymes capable of reducing the R epimer of methionine sulfoxide in proteins. Employing a msrA/B double mutant, a new peptide methionine sulfoxide reductase activity has been found associated with membrane vesicles from E. coli. Both the R and S forms of N-acetylmethionine sulfoxide, D-ala-met(o)-enkephalin and methionine sulfoxide, are reduced by this membrane associated activity. The reaction requires NADPH and may explain, in part, how the R form of methionine sulfoxide in proteins is reduced in E. coli. In addition, a new soluble Msr activity was also detected in the soluble extracts of the double mutant that specifically reduces the S epimer of met(o) in proteins.     

The enzymology and biochemistry of methionine sulfoxide reductases

The methionine sulfoxide reductase (Msr) family is composed of two structurally unrelated classes of monomeric enzymes named MsrA and MsrB, which display opposite stereo-selectivities towards the sulfoxide function. MsrAs and MsrBs, characterized so far, share the same chemical mechanism implying sulfenic acid chemistry. The mechanism includes three steps with (1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mol of enzyme; (2) formation of an intramonomeric disulfide Msr bond followed by; (3) reduction of the oxidized Msr by thioredoxin (Trx). This scheme is in accordance with the kinetic mechanism of both Msrs which is of ping-pong type. For both Msrs, the reductase step is rate-determining in the process leading to the formation of the disulfide bond. The overall rate-limiting step takes place within the thioredoxin-recycling process, likely being associated with oxidized thioredoxin release. The kinetic data support structural recognition between oxidized Msr and reduced thioredoxin. The active sites of both Msrs are adapted for binding protein-bound methionine sulfoxide (MetSO) more efficiently than free MetSO. About 50% of the MsrBs binds a zinc atom, the location of which is in an opposite direction from the active site. Introducing or removing the zinc binding site modulates the catalytic efficiency of MsrB.     

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.     

Specific activity of methionine sulfoxide reductase in CD-1 mice is significantly affected by dietary selenium but not zinc

Reactive oxygen species-mediated oxidation of methionine residues in protein results in a racemic mixture of R and S forms of methionine sulfoxide (MetO). MetO is reduced back to methionine by the methionine sulfoxide reductases MsrA and MsrB. MsrA is specific toward the S form and MsrB is specific toward the R form of MetO. MsrB is a selenoprotein reported to contain zinc (Zn). To determine the effects of dietary selenium (Se) and Zn on Msr activity, CD-1 mice (N=16/group) were fed, in a 2×2 design, diets containing 0 or 0.2 μg Se/g and 3 or 15 ∥ Zn/g. As an oxidative stress, half of the mice received L-buthionine sulfoximine (BSO; ip; 2 mmol/kg, three times per week for the last 3 wk); the others received saline. After 9.5 wk, Msr (the combined specific activities of MsrA and MsrB) was measured in the brain, kidney, and liver. Se deficiency decreased (p<0.0001) Msr in all three tissues, but Zn had no direct effect. BSO treatment was expected to result in increased Msr activity; this was not seen. Additionally, we found that the ratio of MetO to methionine in liver protein was increased (indicative of oxidative damage) by Se deficiency. The results show that Se deficiency increases oxidation of methionyl residues in protein, that Se status affects Msr (most likely through effects on the selenoprotein MsrB), and that marginal Zn deficiency has little effect on Msr in liver and kidney. Finally, the results show that the oxidative effects of limited BSO treatment did not upregulate Msr activity.     

The metal-catalyzed oxidation of methionine in peptides by Fenton systems involves two consecutive one-electron oxidation processes.

The one-electron oxidation of methionine (Met) plays an important role in the redox reactions of Met in peptides and proteins under conditions of oxidative stress, e.g., during the metal-catalyzed oxidation of beta-amyloid peptide (beta A). However, little information is available with regard to mechanisms and product formation during the metal-catalyzed oxidation of Met. Here, we demonstrate that two-electron oxidation of Met in Fenton reactions, carried out aerobically by [Fe(II)(EDTA)](2-) and H(2)O(2) (EDTA = ethylenediaminetetra acetate) is the consequence of two consecutive one-electron transfer reactions carried out by either free or complexed hydroxyl radicals, followed by the reaction of an intermediary sulfur-nitrogen bonded radical cation (sulfuranyl radical) with O(2). The model peptide Met-Met represents an ideal substrate for these investigations as its one-electron oxidation, followed by reaction with molecular oxygen, produces unique intermediates, azasulfonium diastereomers, which can be chemically isolated before hydrolysis to sulfoxide occurs.     

Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A

Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29>Met30>Met13, with Met79 being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in vivo.     

Modulating protein activity and cellular function by methionine residue oxidation

The sulfur-containing amino acid residue methionine (Met) in a peptide/protein is readily oxidized to methionine sulfoxide [Met(O)] by reactive oxygen species both in vitro and in vivo. Methionine residue oxidation by oxidants is found in an accumulating number of important proteins. Met sulfoxidation activates calcium/calmodulin-dependent protein kinase II and the large conductance calcium-activated potassium channels, delays inactivation of the Shaker potassium channel ShC/B and L-type voltage-dependent calcium channels. Sulfoxidation at critical Met residues inhibits fibrillation of atherosclerosis-related apolipoproteins and multiple neurodegenerative disease-related proteins, such as amyloid beta, α-synuclein, prion, and others. Methionine residue oxidation is also correlated with marked changes in cellular activities. Controlled key methionine residue oxidation may be used as an oxi-genetics tool to dissect specific protein function in situ.     

Specificity of thioredoxins and glutaredoxins as electron donors to two distinct classes of Arabidopsis plastidial methionine sulfoxide reductases B

Methionine sulfoxide reductases (MSRs) A and B reduce methionine sulfoxide (MetSO) S- and R-diastereomers, respectively, back to Met using electrons generally supplied by thioredoxin. The physiological reductants for MSRBs remain unknown in plants, which display a remarkable variety of thioredoxins (Trxs) and glutaredoxins (Grxs). Using recombinant proteins, we show that Arabidopsis plastidial MSRB1 and MSRB2, which differ regarding the number of presumed redox-active cysteines, possess specific reductants. Most simple-module Trxs, especially Trx m1 and Trx y2, are preferential and efficient electron donors towards MSRB2, while the double-module CDSP32 Trx and Grxs can reduce only MSRB1. This study identifies novel types of reductants, related to Grxs and peculiar Trxs, for MSRB proteins displaying only one redox-active cysteine.