Archives of Biochemistry and Biophysics Vol. 383, No. 2, November 15, pp. 303–308, 2000 doi:10.1006/abbi.2000.2093, available online at http://www.idealibrary.com on
Decreased Mitochondrial Hydrogen Peroxide Release in Transgenic Drosophila melanogaster Expressing Intramitochondrial Catalase Linda K. Kwong, 1 Robin J. Mockett, 2 Anne-Ce´cile V. Bayne, 2 William C. Orr, and Rajindar S. Sohal 2,3 Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275
Received August 8, 2000, and in revised form August 25, 2000
The objective of this study was to develop strategies for manipulating oxidative stress transgenically in a multicellular organism. Ectopic catalase was introduced into the mitochondrial matrix, which is the main intracellular site of H 2O 2 formation and where catalase is normally absent. Transgenic Drosophila melanogaster were generated by microinjection of a P element construct, containing the genomic catalase sequence of Drosophila, with the mitochondrial leader sequence of ornithine aminotransferase inserted upstream of the coding region. Total catalase activities in whole-body homogenates of 10-day-old flies from four transgenic lines were ⬃30 –160% higher than those from the parental and four vector-only control lines. Expression of catalase in the mitochondrial matrix was confirmed by immunoblotting and catalase activity assays. Mitochondrial release of H 2O 2 was decreased by ⬃90% in the transgenic lines when compared to levels in vector-only controls. This in vivo system provides a novel model for examining the functional significance of decreased mitochondrial H 2O 2 release. © 2000 Academic Press Key Words: catalase; mitochondria; transgenic; chimeric proteins; ectopic expression; signal peptides; oxidative stress; H 2O 2.
1
Present address: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107-2609. 2 Present address: Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA 90089-9121. 3 To whom correspondence should be addressed. Fax: (323) 2247473. E-mail:
[email protected]. 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
It is now widely recognized that mitochondria are the major intracellular source of superoxide anion (O 2•⫺) and hydrogen peroxide (H 2O 2) production. Approximately 1–3% of the oxygen consumed by the mitochondria has been estimated to be converted to O 2•⫺, the first in a series of reactive oxygen species (ROS) 4 (1, 2). The O 2•⫺ is converted to H 2O 2 by the action of manganese-containing superoxide dismutase (Mn-SOD), an enzyme located in the mitochondrial matrix (3). The major enzymatic defenses against H 2O 2 are catalase and glutathione peroxidase. In mammals, glutathione peroxidase is present in the cytosol as well as in the mitochondrial matrix, but catalase is found only in the cytosol and peroxisomes, the only known exception being in the rat heart mitochondria (4). In insects, glutathione peroxidase activity is absent (5) and extramitochondrially located catalase is thought to be the sole enzyme that detoxifies H 2O 2. Most nuclear-encoded mitochondrial matrix proteins are synthesized in the cytosol as precursors, containing an N-terminal presequence, which is employed for targeting and translocating the proteins into the matrix (6). After the proteins are transported into the matrix, the presequences are cleaved by mitochondrial proteases and the proteins are folded into the proper conformation. Although the effectiveness of the targeting presequence is dependent on the nature of the passenger protein (7, 8), many chimeric proteins, consisting of ectopic presequences and passenger proteins, have been shown to be importable into the mitochondria of yeast, plant cells, and cultured mammalian cells (7, 9 –11). Originally, the import of chimeric proteins was 4
Abbreviations used: ROS, reactive oxygen species; Mn-SOD, manganese-containing superoxide dismutase; OAT, ornithine aminotransferase; SMP, submitochondrial particle. 303
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used to study the mechanics of mitochondrial targeting and protein translocation (12–14), but, more recently, it has also been used to study the effects of specific passenger proteins in mitochondria (9, 10, 15). To date, there have been no studies in which ectopic proteins were targeted to the mitochondria of muticellular eukaryotic animals in order to test hypotheses about their structure and function. Thus, protein chimeras offer a novel and largely unexplored experimental approach to modify mitochondrial function in intact animals. In an earlier study, we showed that overexpression of catalase in the cytosol of Drosophila melanogaster attenuated H 2O 2 toxicity in transgenic flies (16). Similar improvement in resistance to experimental oxidative stress was reported in cultured cells overexpressing catalase in the cytosol (9, 17, 18). A recent noteworthy finding is that ectopic expression of catalase in the mitochondrial matrix, which is the major site of H 2O 2 production, increased the resistance of HepG2 cells to H 2O 2-induced apoptosis (9). However, a similar in vivo expression of functional cytosolic proteins in the mitochondria of a multicellular animal has not yet been demonstrated. Thus, the objective of the present study was to target cytosolic catalase to the mitochondrial matrix of D. melanogaster by inserting DNA, encoding a mitochondrial matrix presequence, upstream of the coding region of the gene. The resulting transgenic flies were hypothesized to express ectopic catalase in the mitochondria and to have diminished mitochondrial H 2O 2 release. Flies with such alterations in mitochondrial physiology would represent a powerful model system in which to study the importance of mitochondrial oxidative stress in the aging process. MATERIALS AND METHODS Materials. Restriction enzymes were obtained from New England Biolabs (Beverly, MA). DNA isolation kits (QIAquick PCR Purification Kit, QIAprep Spin Miniprep Kit, EndoFree Plasmid Maxi Kit, and QIAquick Gel Extraction Kit) were obtained from Qiagen, Inc. (Valencia, CA). Primers used in PCR amplifications were synthesized by Life Technologies (Grand Island, NY). All other enzymes and reagent grade chemicals were purchased from Sigma Chemical Co. (St Louis, MO). Transgene design and cloning strategy. A genomic catalase sequence isolated from a Drosophila genomic DNA library (19) and cloned into the pBluescript SK⫹ vector was used for the construction of the transgene. The 22 amino-terminal codons of D. melanogaster ornithine aminotransferase (OAT), which include a putative mitochondrial presequence, were inserted at the beginning of the coding region of the genomic catalase gene, replacing the first codon, using a two-step PCR amplification approach. In the first PCR reaction, the primers used were 5⬘-CGTGGTGCTGGGCTATAAAAACA-3⬘ (upper) and 5⬘-GCATGAACGCGTGGCAATGCCTCGAGTGGAAAGCTTGGAGAACATTTTGCTTAAAATTTAGGATATTTGCTCAAC-3⬘ (lower). The upper primer corresponds to positions ⫺175 to ⫺153 in relation to the translation start site. The lower primer corresponds to positions ⫺1 to ⫺30, with a 42-nucleotide 5⬘ overhang containing the first 13 codons of the OAT presequence. The PCR product was
FIG. 1. pCaSpeR4 –OAT-Cat construct. A 7084-bp fragment (broad arc) containing the Drosophila catalase (Cat) gene [promoter and poly(A) consensus signal, open arcs; OAT presequence and exons, solid arcs with arrowheads; introns, stippled arcs] was subcloned between the NotI and the KpnI sites of the pCaSpeR4 vector (narrow arc). A partial restriction map is shown, indicating some of the sites used for transgene construction and for diagnostic digests prior to microinjection of the construct into Drosophila embryos.
cleaved at the EcoRI and MluI sites and inserted into the pBluescript SK⫹ vector between the EcoRI and SalI polylinker sites. The selection of codons for the presequence introduced a XhoI site not normally present at the 5⬘ end of the Drosophila catalase coding region. The primers for the second PCR reaction were 5⬘-ATGGACACGCGTATCGGTTATTTGGCCCAAAAAGCGGCTGCTGGACGCGATGCGGCTTC-3⬘ (upper) and 5⬘-GGACTACTCGAGAATGGAAAGTACAGGCTCATAGCAATCGTTA-3⬘ (lower). The upper primer corresponds to positions ⫹4 to ⫹23, with a 39-nucleotide 5⬘ overhang containing codons 14 –22 of the OAT presequence. The lower primer corresponds to positions ⫹243 to ⫹214, with a 12-nucleotide 5⬘ overhang. The PCR product was incubated with the Klenow large fragment polymerase, followed by digestion with MluI, and ligated into the product from the first cloning reaction between the MluI site and the blunt-ended polylinker ApaI site. The resulting construct was cleaved with EcoRI and NaeI. The fragment containing the OAT presequence was ligated with a pBluescript SK⫹ vector containing the genomic catalase gene, which was partially digested with EcoRI and NaeI. The 7084-base-pair modified genomic catalase fragment, which contained the catalase promoter, ornithine aminotransferase presequence, complete exon and intron sequences, and poly(A) consensus signal, was shuttled into the pCaSpeR4 vector (kindly provided by Carl Thummel, Howard Hughes Medical Institute, University of Utah) between the polylinker NotI and KpnI sites, generating a 15-kilobase-pair construct (Fig. 1). The OAT DNA insert and the ligation sites were sequenced commercially to verify the accuracy of the final construct (Lone Star Lab., Houston, TX). Creation of transgenic lines. Drosophila strains and methods used for P element-mediated transformation, generation of transgenic lines, and chromosomal mapping were as described previously
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FIG. 2. Southern analysis of DNA from OAT-Cat and control flies. DNA was isolated from OAT-Cat (OC#1) and parental control (y w) flies. After digestion with XhoI (Cat probe) or SacI (pCaSpeR probe), the DNA was analyzed as described under Materials and Methods.
(20). Experimental transgenic flies were generated by microinjection of the pCaSper4-OAT-catalase construct into embryos of the strain y w; Sb⌬(2-3)/TM6,Ubx, which possesses endogenous P element transposase activity, followed in some cases by transient remobilization of the transgene. Preexisting transgenic lines with pCaSpeR vectoronly sequence served as controls. The presence of the transgene was verified by Southern analysis with digoxigenin-labeled probes complementary to either catalase or white gene sequences. DNA isolated from possible transgenic lines was digested with XhoI or SacI to confirm the presence of the exogenous modified catalase gene and the number of pCaSpeR white genes, respectively. XhoI digestion of the transgenic DNA produced two constant fragments of ⬃4.0 and ⬃1.6 kb, and a fragment of variable size, distinct from the two ⬎9-kb fragments resulting from the digestion of the native (endogenous) catalase gene (Fig. 2). The catalase transgene contained a SacI site in the C-terminal noncoding region. Therefore, in addition to the ⬃8.5-kb and ⬃3.9-kb fragments from the native (endogenous) white gene, SacI digestion produced a constant fragment of ⬃1.9 kb and a fragment of variable size for each inserted transgene (Fig. 2). After electrophoresis through a 1.0% agarose gel and transfer to a nitrocellulose membrane, the samples were analyzed with the digoxigenin probes. Biochemical assays. All extraction and isolation procedures were performed at 4°C. For measurement of catalase activity in wholebody homogenates, flies were homogenized in 0.1% Triton X-100 (100 l/fly). Cellular debris was removed from the homogenate by centrifugation at 3000g for 3 min. The supernatant was further centrifuged at 12,000g for 15 min. Catalase activity was determined on the final supernatant at 30°C by a modification of the method of Lu¨ck (21), as described previously (22). Activity was calculated using an extinction coefficient of 0.043 mM ⫺1cm ⫺1. One unit of catalase activity was defined as 1 mol H 2O 2 decomposed/min. Protein concentration was determined by the BCA method (Pierce, Rockford, IL), using bovine serum albumin as standard. For catalase activity and immunoblot analysis, mitochondria were isolated from the thoracic flight muscles of 2-day-old control and transgenic flies by a modification of the procedure of Wood and Nordin (23), as described previously (24). Mitochondrial fractionation was performed according to Lass et al. (25). Submitochondrial particles (SMPs) were resuspended in 0.1% Triton X-100. Catalase activity was measured in the Triton X-100-solubilized mitochondria, mitochondrial matrix, and SMPs. To measure H 2O 2 release from the mitochondria, purified mitochondria from the thoracic flight muscles of 3-day-old control and
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transgenic flies were incubated at 30°C in 800 l of reaction buffer (154 mM KCl, 10 mM potassium phosphate, 3 mM MgCl 2, 0.1 mM EGTA, pH 7.4) containing 7 mM ␣-glycerol-3-phosphate and 150 – 250 g mitochondrial protein. After 5, 8, and 12 min of incubation, 200 l of the reaction mixture was removed and the amounts of H 2O 2 released from the mitochondria were determined by the method of Hyslop and Sklar (26), as described previously (27). Immunoblot analysis. Total mitochondrial proteins and mitochondrial matrix proteins were denatured with SDS loading buffer at 95°C for 5 min and then resolved by electrophoresis on a 7.5% SDS–polyacrylamide gel with a 4% stacking gel under reducing conditions (28) and electroblotted onto a PVDF membrane (Millipore Corp., Bedford, MA). After the membrane was blocked with 5% (wt/vol) nonfat milk in TBST buffer (20 mM Tris–HCl, pH 7.5, containing 500 mM NaCl and 0.1% Tween-20), it was incubated with rabbit affinity-purified, polyclonal antibodies to Drosophila catalase followed by incubation with rabbit anti-Drosophila Mn-SOD. The production and isolation of the rabbit anti-catalase and anti-MnSOD antibodies are described elsewhere ((29); Radyuk et al., manuscript submitted). The membrane was then incubated with horseradish-peroxidase-conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO). Immunoreactive protein bands were visualized using the ECL detection system (Amersham Pharmacia Biotech, UK) and BioMax film (Kodak, Rochester, NY). Statistical analysis. All data are expressed as mean ⫾ SD or SEM. The significance of differences in catalase activity was determined by one-way analysis of variance, with post hoc Tukey tests, using SYSTAT 7.0.1 software. Probability values less than or equal to 0.05 were considered to be statistically significant.
RESULTS
Molecular Verification of Transgene Insertion A fusion construct (OAT-Cat) was generated, consisting of the amino-terminal 22 codons of D. melanogaster ornithine aminotransferase, the putative mitochondrial presequence, fused to the beginning of the coding region of the genomic catalase gene fragment. The OAT-Cat fragment was inserted into the pCaSpeR4 vector (Fig. 1), and introduced into the fly genome by P element transformation, as described under Materials and Methods. Southern analysis confirmed the presence of a single OAT-Cat insert in each transgenic line. Figure 2 shows the Southern analysis of a representative trangenic line, OC#1, and the parental line, y w. Total Catalase Activity The effect of the transgene on total catalase activity (i.e., mitochondrial and extramitochondrial) was determined by comparing the activities in four transgenic lines (OC#1, OC#2d5a, OC#6b, and OC#9a5) with those in parental (y w) and four vector-only transgenic control lines (C2, C5, C6, and C7). Total catalase activities in whole-body homogenates of 10-day-old transgenic flies from the four lines were ⬃30 –160% higher than those of y w and vector-only control flies (P ⬍ 0.005; Fig. 3). There were no statistically significant differences between the y w and four vector-only lines.
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FIG. 3. Total catalase activities in OAT-Cat and control flies. Catalase activities were measured in whole-body homogenates from 10day-old flies of parental (solid bar), vector-only control (striped bars), and OAT-Cat transgenic lines (stippled bars), as described under Materials and Methods. Data are mean ⫾ SEM for three to five measurements.
Presence of Catalase in Mitochondria To determine if the insertion of the transgene resulted in the expression of catalase in the mitochondria, immunoblot analysis and enzymic assay of the mitochondria were performed. Mitochondria were isolated from thoraces of 2-day-old OC#1, y w, and C6 flies. Immunoblot analysis showed the presence of the catalase protein in mitochondria from OC#1 flies, but not in those from the y w and C6 lines (Fig. 4). The relative amounts of mitochondria used in SDS–PAGE were assessed using the Mn-SOD antibody. As shown in Fig. 4, the electrophoretic mobility of the mitochondrial catalase (OC#1) was similar to that of cytosolic catalase (Cy), with an apparent molecular weight of ⬃58 kDa. Mitochondria were fractionated to determine the compartmental localization of catalase. Catalase pro-
FIG. 5. Catalase activities in mitochondria and mitochondrial fractions from OAT-Cat and control flies. Catalase activities were measured in mitochondria, matrix, and SMPs, isolated from 2-day-old flies, as described under Materials and Methods. *Statistically significant difference between OC#1 and either y w or C6 flies (P ⬍ 0.0005). Data are mean ⫾ SD of three to six determinations.
tein, with electrophoretic mobility similar to that of cytosolic catalase, was detected in the matrix from OC#1 mitochondria, but not in the matrix from y w and C6 mitochondria (Fig. 4). No catalase protein was detected in the SMP fraction (mitochondrial membrane fraction) isolated from the three lines (data not shown). Enzyme activity assays on the mitochondrial fractions mirrored the immunoblot data (Fig. 5). High levels of catalase enzyme activity were measured in the transgenic line mitochondria and mitochondrial matrix, while negligible levels were detected in mitochondrial fractions from the control lines, y w and C6 (P ⬍ 0.0005). In addition, very low levels of catalase activity were measured in SMPs isolated from all three lines. Overall, the results indicate that catalase was localized almost entirely to the mitochondrial matrix. The presence of mitochondrial catalase in the remaining transgenic lines was also confirmed by enzymic activity assay. Mitochondria were isolated from thoraces of 3-day-old flies from the transgenic and vector-only control lines. While varying levels of mitochondrial catalase activity were measured in the transgenic lines, little or no activity was detected in the vectoronly control lines (Fig. 6). Mitochondrial H 2O 2 Release
FIG. 4. Presence of catalase protein in mitochondria and mitochondrial matrix from different Drosophila lines. Cytosolic (Cy), mitochondrial, and matrix proteins (5 g) were resolved by SDS–PAGE, and analyzed by immunoblotting with rabbit polyclonal (A) antiDrosophila catalase and (B) anti-Drosophila Mn-SOD antibodies, as described under Materials and Methods. Lane 1, cytosolic protein reference (Cy); lanes 2 and 5, OAT-Cat transgenic line (OC#1); lanes 3 and 6, vector-only control line (C6); lanes 4 and 7, parental control line (y w).
Time-dependent release of H 2O 2 from mitochondria was measured in the transgenic and control lines. As shown in Fig. 7, high levels of H 2O 2 were released from mitochondria of control lines, but not from those of transgenic lines. Thus, the presence of catalase in the mitochondria of transgenic flies resulted in a marked decrease in H 2O 2 release from the mitochondria.
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FIG. 6. Mitochondrial catalase activities of OAT-Cat and control lines. Catalase activities were measured in mitochondria isolated from 3-day-old flies of vector-only control lines (striped bars), and transgenic lines (stippled bars), as described under Materials and Methods. Data are mean ⫾ SEM for three to five measurements.
DISCUSSION
This study demonstrates the successful targeting of catalase, a nonmitochondrial protein, to the mitochondrial matrix of a multicellular organism, D. melanogaster, by insertion of an ectopic mitochondrial targeting sequence into the catalase gene. The presence of catalase protein and activity in the mitochondrial matrix of transgenic flies confirmed that the modified transgene was expressed, and that the protein was translocated into the mitochondria and correctly processed, folded and assembled into an active form. The expression of catalase activity in the mitochondria resulted in a dramatic decrease in H 2O 2 release from the mitochondria. Most mitochondrial matrix proteins are synthesized as precursors in the cytosol. After insertion into the mitochondria, the N-terminal presequence is cleaved by protease(s) and the proteins are folded into their functional conformation. Catalase is a heme-containing protein, whose functional form is a homotetramer. Each subunit has four domains, with the amino-terminal domain of each monomer buried inside the neighboring monomer. Therefore, precise folding of the monomers is critical for correct assembly of the functional tetramer. For this reason, and because introns play an important regulatory role in gene expression (30 –33), the design of the OAT-Cat construct in the present study minimized the modification of the genomic catalase gene. In an earlier experiment with the Drosophila catalase cDNA and Mn-SOD promoter and leader peptide DNA, the 5⬘ end of the cDNA was
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substituted with Mn-SOD extending several codons downstream of the putative presequence cleavage site. Additionally, the 3⬘ end of the coding sequence was substituted with an influenza hemagglutinin epitope and Mn-SOD 3⬘ codons and regulatory sequences. No functional product was obtained by this approach, presumably because these extensive modifications prevented correct assembly of the protein, leading to rapid proteolysis (34). An alternative approach employing the maize Cat3, which is associated with mitochondria, was also unsuccessful because there was little or no expression of the cat3 gene in transgenic Drosophila. A recent study reported the successful expression of functional catalase in the mitochondria of HepG2 cells transfected with a modified human catalase cDNA containing a Mn-SOD mitochondrial leader sequence (9). The present study extends this finding to a multicellular organism, showing for the first time that all required processing and assembly of ectopic catalase is performed properly in the Drosophila mitochondrial matrix. Such targeting will permit simultaneous elevation of both Mn-SOD (29) and catalase activities in the mitochondrial matrix. Previous studies showed that simultaneous overexpression of Cu,Zn-SOD and catalase in the cytosol of transgenic Drosophila decreased oxidative damage and increased the life span by up to 34% (24), whereas overexpression of either enzyme alone had only marginal effects (16, 20). By analogy, stronger beneficial effects may be predicted
FIG. 7. Cumulative mitochondrial H 2O 2 release in OAT-Cat and control flies. Mitochondria were isolated from 3-day-old flies of transgenic and vector-only control lines. The total amount of H 2O 2 released was measured after varying periods of incubation, as described under Materials and Methods.
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with enhancement of both Mn-SOD and catalase activities in the mitochondrial matrix than with either enzyme alone, reflecting the importance of balanced, targeted elevation of antioxidant levels. In the present study, a decrease in H 2O 2 release from mitochondria to cytosol was observed in the transgenic lines. This represents a completely novel feature of the animal model reported here, since, from the perspective of the extramitochondrial compartment, it is expected to lower oxidative stress by effectively reducing oxidant production rather than simply elevating antioxidative defense levels. This strategy has particularly important implications because numerous studies have shown a close association between mitochondrial ROS production and both aging and species maximum life-span potential, whereas the relationship with antioxidant levels is more ambiguous (35–38). In conclusion, this study has successfully targeted ectopic catalase to the mitochondrial matrix of Drosophila, thus reducing the amount of H 2O 2 released from the mitochondria. This creates a unique model system in which to study the role of ROS production and oxidative stress in the aging process. ACKNOWLEDGMENTS The authors thank S. N. Radyuk, V. I. Klichko, and J. G. Hubbard for their intellectual contributions to the transgene design and cloning strategy. This research was supported by Grant RO1 AG7657 from the National Institute on Aging–National Institutes of Health.
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