Abstract
purpose. Methionine-sulfoxide reductases are unique, in that their ability to repair oxidized proteins and MsrA, which reduces S-methionine sulfoxide, can protect lens cells against oxidative stress damage. To date, the roles of MsrB1, -B2 and -B3 which reduce R-methionine sulfoxide have not been established for any mammalian system. The present study was undertaken to identify those MsrBs expressed by the lens and to evaluate the enzyme activities, expression patterns, and abilities of the identified genes to defend lens cells against oxidative stress damage.
methods. Enzyme activities were determined with bovine lens extracts. The identities and spatial expression patterns of MsrB1, -B2, and -B3 transcripts were examined by RT-PCR in human lens and 21 other tissues. Oxidative stress resistance was measured using short interfering (si)RNA–mediated gene-silencing in conjunction with exposure to tert-butyl hydroperoxide (tBHP) and MTS viability measurements in SRA04/01 human lens epithelial cells.
results. Forty percent of the Msr enzyme activity present in the lens was MsrB, whereas the remaining enzyme activity was MsrA. MsrB1 (selenoprotein R, localized in the cytosol and nucleus), MsrB2 (CBS-1, localized in the mitochondria), and MsrB3 (localized in the endoplasmic reticulum and mitochondria) were all expressed by the lens. These genes exhibit asymmetric expression patterns between different human tissues and different lens sublocations, including lens fibers. All three genes are required for lens cell viability, and their silencing in lens cells results in increased oxidative-stress–induced cell death.
conclusions. The present data suggest important roles for both MsrA and -Bs in lens cell viability and oxidative stress protection. The differential tissue distribution and lens expression patterns of these genes, coupled with increased oxidative-stress–induced cell death on their deletion provides evidence that they are important for lens cell function, resistance to oxidative stress, and, potentially, cataractogenesis.
The eye lens consists of a single layer of epithelial cells that overlie concentric layers of differentiated and elongated fiber cells. Damage to lens cells and their components results in protein aggregation associated with age-related cataract, an opacity of the eye lens that is the major cause of blindness worldwide.
1 Oxidative stress is believed to play a major role in cataract formation, since oxidation of proteins results in loss of protein function.
2 3 4 5 One major modification resulting from oxidative stress is oxidation of methionine, which affects a multitude of biological functions through the direct inactivation of proteins.
6 The content of oxidized methionine residues increases in the lens with age, and in cataracts as much as 60% of the total membrane-bound protein methionine is found as methionine sulfoxide (Met (O)), suggesting a possible link between methionine oxidation and age-related cataract.
7 8 9
Reaction of methionine with reactive oxygen species (ROS) results in the formation of two epimers of methionine sulfoxide, referred to as Met-
R-(O) and Met-
S-(O). Unlike most protein modifications, these oxidations are reversible through the action of the methionine sulfoxide reductase (Msr) family of enzymes that exhibit two specific activities: MsrA and -B. MsrA is stereospecific for Met-
S-(O), and MsrB is specific for Met-
R-(O).
10 11 To date, the only naturally occurring system for Met (O) reduction in cells is reduced thioredoxin.
6 In mammals, there is only one gene encoding MsrA, but there are at least three MsrB genes called MsrB1 (selenoprotein R), MsrB2 (CBS-1) and MsrB3.
12 13 Previous studies have indicated that MsrB1 is localized to the cell nucleus and cytoplasm, MsrB2 is localized to the mitochondria, and MsrB3 resides in the endoplasmic reticulum (ER).
12 MsrA has been found in both mitochondria and cytoplasm.
The Msr family of genes is conserved throughout evolution and influences longevity in species, including
Drosophila,
14 mice,
15 and yeast.
16 For instance, overexpression of MsrA in transgenic flies renders them more resistant to oxidative stress and dramatically increases their lifespan.
14 Overexpression of MsrA in yeast can extend lifespan and protect against oxidative stress in yeast,
16 17 human T-lymphocytes
17 and PC12 cells.
18 By contrast, deletion of the MsrA gene in mice results in increased sensitivity to oxidative stress, a shortened lifespan, and neurologic impairment.
15
In previous studies, we have demonstrated that overexpression of MsrA protects lens cells against oxidative stress, whereas deletion of MsrA renders them more resistant to oxidative stress and decreases cell viability in the absence of oxidative stress.
19 In contrast to MsrA, no functional studies have been reported on any of the MsrB family of genes in lens or other mammalian systems.
In the present study, we examined the levels and spatial expression patterns of the MsrB gene family in the human lens and tested the viability of lens cells in which these genes were silenced when exposed to oxidative stress. The data demonstrate that the lens contains both MsrA and -B enzyme activity, show that all three MsrB genes are expressed by the human lens, and show that silencing of each of the three MsrB genes results in loss of lens cell viability and decreased resistance of lens cells to oxidative stress exposure. The varied expression of these genes in different tissues, lens sublocations, and cellular sublocations, coupled with their requirement for oxidative stress defense suggests that they play important roles in the repair of oxidized lens proteins and, potentially, in cataract formation.
Analysis of Msr Transcript Levels in Microdissected Components of Whole Human Lenses
A cell proliferation assay kit (Cell Titer 96 Aqueous One Solution; Promega, Madison, WI) containing the tetrazolium compound [3-(4,5-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) was used to monitor cell viability according to the manufacturer’s protocol. The final concentration of MTS added to the cells was 317 μg/μL, and the MTS color change at 490 nm was monitored with a universal plate reader (model ELX-800; Bio-Tek Instruments, Winooski, VT).
Effect of Silencing of Individual MsrB Genes in Lens Cells on Cell Viability and Resistance to Oxidative Stress
The results in the present study provide evidence that the lens contains both MsrA and -B enzyme activity and that all three MsrB genes are important for lens cell viability and oxidative stress resistance. We also found asymmetric distribution of MsrB transcripts between the lens and other human tissues, and we showed that MsrA is the predominant Msr expressed by the lens epithelium, whereas MsrB2 is the predominant Msr expressed by the lens fibers.
Previous studies have detected the presence of Msr activity in human and cow lenses
22 ; however, in these studies, a substrate that does not distinguish between
R-and
S-methionine sulfoxide was used. In the present report, we used a stereo specific enzyme inhibitor to demonstrate that approximately 40% of lens Msr activity is due to MsrB activity. Although our enzyme assays cannot distinguish the individual activities of the separate MsrB enzymes, because they act on the same substrates, our semiquantitative RT-PCR data demonstrate that all three Msr genes are expressed by the lens, although they exhibit different abundances in different lens sublocations. In contrast to MsrA, which is evenly distributed in the different tissues examined, individual MsrB genes were differentially expressed between different tissues. This could indicate different roles for these genes in these tissues.
Antibodies for MsrBs are not available, and therefore corresponding protein levels could not be determined. Although we are confident that Msr protein levels parallel the mRNA levels, we cannot rule out the possibility that differences between mRNA and protein levels exist in the gene expression or gene-silencing studies.
Because only MsrA acts on the
S-form of methionine sulfoxide, whereas all three MsrB genes act on the
R-form, it is not surprising that MsrA would be ubiquitously expressed and the MsrB genes asymmetrically expressed. Previous studies have demonstrated that MsrA is expressed in multiple tissues, including the kidney, retinal pigmented epithelium, brain, blood, and alveolar macrophages.
23 In the lens, MsrA transcripts were detected in the epithelium and nuclear fibers.
19
MsrB1 and -B2 transcripts have been detected in liver kidney, heart skeletal muscle, and brain.
24 Subcellular localization in mammalian cells indicated that MsrB1 was localized to the cytoplasm and nucleus. MsrB2 was localized to the mitochondria and MsrB3 to the ER, suggesting specialized subcellular roles for these proteins.
12 Like MsrA,
19 all three MsrB proteins were found in lens fibers, suggesting that both MsrA and -Bs participate in protein repair of both
S- and
R-methionine sulfoxides formed in lens fibers. MsrA has been shown to play a role in defense against oxidative stress in multiple cell systems, including human lens cells.
19 In the present study, we provide evidence that all three MsrB genes are also important for cell viability and oxidative stress protection of lens cells.
MsrA and -B2 are localized to the mitochondria, and it is likely that these Msrs are important in the maintenance of mitochondrial function through direct scavenging of reactive oxygen species produced during mitochondrial respiration. Approximately 0.1% to 1% of respiratory oxygen is estimated to form reactive oxygen species during normal respiration
25 and mitochondria are a major target for reactive oxygen species. It is interesting to note that both MsrA and -B2 were the predominant transcripts expressed in lens epithelia and lens fibers, respectively, suggesting that maintenance of mitochondrial function is important for these lens components.
MsrB1 and MsrB3 are localized to the cytosol and the ER,
12 respectively, suggesting that they play specialized roles in these cellular compartments that could include repairing damage to cytosolic proteins and proteins in the ER, including newly synthesized proteins. These transcripts were detected at high levels in the lens and in bone marrow, intestine, heart, kidney, colon, and skeletal muscle, all of which are tissues with high levels of protein synthesis that could require coordinated repair from oxidative stress.
Although the targets for Msr action in the lens have yet to be defined, it has been shown that methionine oxidation of α-crystallin results in loss of chaperone activity,
2 26 which could play a role in cataract formation.
27 Other likely targets for Msr repair are the γ-crystallins which are rich in methionine residues and are one of the first lens proteins to aggregate on cataract formation.
28 MsrA function depends on the reducing system, and NADPH levels have been shown to decrease rapidly on cataract formation,
29 which could reduce the ability of Msrs to repair proteins damaged through oxidation.
Regardless of the exact roles of the individual MsrB genes in lens cells, the present study provides evidence that these genes are important for the maintenance of lens cell viability and resistance to oxidative stress damage. These properties of Msrs, coupled with their presence in lens fibers, suggests that they play important roles in the repair of oxidized lens proteins and that loss of their normal activities is likely to contribute to cataract and other age-related diseases.
Supported by National Eye Institute Grant EY13022 (MK) and Grant P200415 from the State of Florida Center of Excellence in Biomedical and Marine Biotechnology.
Submitted for publication January 6, 2005; revised February 9, 2005; accepted February 16, 2005.
Disclosure:
M.A. Marchetti, None;
G.O. Pizarro, None;
D. Sagher, None;
C. DeAmicis, None;
N. Brot, None;
J.F. Hejtmancik, None;
H. Weissbach, None;
M. Kantorow, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Marc Kantorow, Biomedical Science, Florida Atlantic University, 777 Glades Road, PO Box 3091, Boca Raton, FL 33431-0991;
[email protected].
RT-PCR Primer | Product Size (bp) | Primer Sequence |
MsrA forward | 329 | 5′-AGTACCTGAGCAAGAACCCCA-3′ |
MsrA reverse | | 5′-TCACTCAGACCCCAGAAGACA-3′ |
MsrB1 forward | 328 | 5′-GACGTTACACCCTCACCTT-3′ |
MsrB1 reverse | | 5′-AGCTACTTCCGCACAGATT-3′ |
MsrB2 forward | 308 | 5′-CAAGGAAGCAGGAATGTATCA-3′ |
MsrB2 reverse | | 5′-ATGGTCAGTGTTTCCTTGGTTT-3′ |
MsrB3 forward | 328 | 5′-CCGGGTCGTGTAGGGATAAA-3′ |
MsrB3 reverse | | 5′-TGAGCACCACACTGAGAGCA-3′ |
GAPDH forward | 600 | 5′-CCACCCATGGCAAATTCCATGGCA-3′ |
GAPDH reverse | | 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ |
siRNA | siRNA Sequence |
MsrA: siRNA A-8 | r(CAAAGUACAAAGGAAUUUAUU) and (UAAAUUCCUUUGUACUUUGUG) |
MsrB1/SelX: siRNA B1-1 | r(GCGUCCGGAGCACAAUAGA)d(TT) and r(UCUAUUGUGCUCCGGACGC)d(TT) |
MsrB2/CBS1: siRNA B2-1 | r(GUUCUACGUCACAAGAGAA)d(TT) and r(UUCUCUUGUGACGUAGAAC)d(TG) |
MsrB3: siRNA B3-1 | r(GUGCCUUUGAAGGAGAAUA)d(TT) and r(UAUUCUCCUUCAAAGGCAC)d(TT) |
Table 2. MsrA and MsrB Enzyme Activity in Lens Fractions
Table 2. MsrA and MsrB Enzyme Activity in Lens Fractions
Enzyme | % Activity/Fiber Fraction | % Activity/Epithelial Fraction |
MsrA | 63 | 60 |
MsrB | 37 | 40 |
The authors thank Venkat Reddy for providing the SRA04/01 lens epithelia cells and the West Virginia Eye Bank and the Lions Eye Bank of Oregon for providing the human lenses used in the study.
KupferC, UnderwoodB, GillenT. Leading causes of visual impairment world wide. Principles and Practice of Ophthalmology Basic Science. 1994;1249–1255.WB Saunders Philadelphia.
SmithJB, JiangX, AbrahamEC. Identification of hydrogen peroxide oxidation sites of alpha A- and alpha B-crystallins. Free Radic Res. 1997;26:103–111.
[CrossRef] [PubMed]McNamaraM, AugusteynRC. The effects of hydrogen peroxide on lens proteins: a possible model for nuclear cataract. Exp Eye Res. 1984;38:45–56.
[CrossRef] [PubMed]BodanessRS, LeclairM, ZiglerJS, Jr. An analysis of the H2O2-mediated crosslinking of lens crystallins catalyzed by the heme-undecapeptide from cytochrome c. Arch Biochem Biophys. 1984;231:461–469.
[CrossRef] [PubMed]ZiglerJS, Jr, HuangQL, DuXY. Oxidative modification of lens crystallins by H2O2 and chelated iron. Free Radic Biol Med. 1989;7:499–505.
[CrossRef] [PubMed]WeissbachH, EtienneF, HoshiT, et al. Peptide methionine sulfoxide reductase: structure, mechanism of action, and biological function. Arch Biochem Biophys. 2002;397:172–178.
[CrossRef] [PubMed]SpectorA. Oxidative stress-induced cataract: mechanism of action. FASEB J. 1995;9:1173–1182.
[PubMed]TruscottRJ, AugusteynRC. Oxidative changes in human lens proteins during senile nuclear cataract formation. Biochim Biophys Acta. 1977;492:43–52.
[CrossRef] [PubMed]GarnerMH, SpectorA. Selective oxidation of cysteine and methionine in normal and senile cataractous lenses. Proc Natl Acad Sci USA. 1980;77:1274–1277.
[CrossRef] [PubMed]MoskovitzJ, SinghVK, RequenaJ, WilkinsonBJ, JayaswalRK, StadtmanER. Purification and characterization of methionine sulfoxide reductase from mouse and Staphylococcus aureus and their substrate stereospecificity. Biochem Biophys Res Commun. 2002;290:62–65.
[CrossRef] [PubMed]GrimaudR, EzratyB, MitchellJ.K, et al. Repair of oxidized proteins: identification of a new methionine sulfoxide reductase. J Biol Chem. 2001;276:48915–48920.
[CrossRef] [PubMed]KimHY, GladyshevVN. Methionine sulfoxide reduction in mammals: characterization of methionine-R-sulfoxide reductases. Mol Biol Cell. 2004;15:1055–1064.
[PubMed]HuangW, EscribanoJ, SarfaraziM, Coca-PradosM. Identification, expression and chromosome localization of a human gene encoding a novel protein with similarity to the pilB family of transcriptional factors (pilin) and to bacterial peptide methionine sulfoxide reductase. Gene. 1999;233:233–240.
[CrossRef] [PubMed]RuanH, TangXD, ChenML, et al. High-quality life extension by the enzyme peptide methionine sulfoxide reductase. Proc Natl Acad Sci USA. 2002;99:2748–2753.
[CrossRef] [PubMed]MoskovitzJ, Bar-NoyS, WilliamsWM, RequenaJ, BerlettBS, StadtmanER. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc Natl Acad Sci USA. 2001;98:12920–12925.
[CrossRef] [PubMed]KocA, GaschAP, RutherfordJC, KimHY, GladyshevVN. Methionine sulfoxide reductase regulation of yeast lifespan reveals reactive oxygen species-dependent and-independent components of aging. Proc Natl Acad Sci USA. 2004;101:7999–8004.
[CrossRef] [PubMed]MoskovitzJ, FlescherE, BerlettBS, AzareJ, PostonJM, StadtmanER. Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress. Proc Natl Acad Sci USA. 1998;95:14071–14075.
[CrossRef] [PubMed]YermolaievaO, XuR, SchinstockC, et al. Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation. Proc Natl Acad Sci USA. 2004;101:1159–1164.
[CrossRef] [PubMed]KantorowM, HawseJR, CowellTL, et al. Methionine sulfoxide reductase A is important for lens cell viability and resistance to oxidative stress. Proc Natl Acad Sci USA. 2004;101:9654–9659.
[CrossRef] [PubMed]MatsuiH, LinLR, HoYS, ReddyVN. The effects of up- and downregulation of MnSOD enzyme on oxidative stress in human lens epithelial cells. Invest Ophthalmol Vis Sci. 2003;44:3467–3475.
[CrossRef] [PubMed]BrotN, WerthJ, KosterD, WeissbachH. Reduction of N-acetyl methionine sulfoxide: a simple assay for peptide methionine sulfoxide reductase. Anal Biochem. 1982;122:291–294.
[CrossRef] [PubMed]SpectorA, ScottoR, WeissbachH, BrotN. Lens methionine sulfoxide reductase. Biochem Biophys Res Commun. 1982;108:429–434.
[CrossRef] [PubMed]MoskovitzJ, JenkinsNA, GilbertDJ, et al. Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues. Proc Natl Acad Sci USA. 1996;93:3205–3208.
[CrossRef] [PubMed]HanselA, JungS, HoshiT, HeinemannSH. A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1. Redox Rep. 2003;8:384–388.
[CrossRef] [PubMed]NichollsDG. Mitochondrial membrane potential and aging. Aging Cell. 2004;3:35–40.
[CrossRef] [PubMed]CherianM, AbrahamEC. Diabetes affects alpha-crystallin chaperone function. Biochem Biophys Res Commun. 1995;208:675–679.
[CrossRef] [PubMed]BradyJP, GarlandD, Duglas-TaborY, RobisonWG, Jr, GroomeA, WawrousekEF. Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin E. Proc Natl Acad Sci USA. 1997;94:884–889.
[CrossRef] [PubMed]BrownNP, BronAJ. Lens Disorders: A Clinical Manual of Cataract Diagnosis. 1996;Butterworth-Heineman Oxford.
LeeSM, SchadeSZ, DoughtyCC. Aldose reductase, NADPH and NADP+ in normal, galactose-fed and diabetic rat lens. Biochim Biophys Acta. 1985;841:247–253.
[CrossRef] [PubMed]