Immunogenicity of mitochondrial DNA modified by hydroxyl radical

Cellular Immunology 247 (2007) 12–17 www.elsevier.com/locate/ycimm

Immunogenicity of mitochondrial DNA modified by hydroxyl radical Khurshid Alam *, Moinuddin, Suraya Jabeen Department of Biochemistry, Faculty of Medicine, J.N. Medical College, A.M.U., Aligarh 202 002, India Received 9 April 2007; accepted 28 June 2007 Available online 22 August 2007

Abstract Mitochondria consume about 90 percent of oxygen used by the body, and are a particularly rich source of reactive oxygen species (ROS). In this research communication mitochondrial DNA (mtDNA) was isolated from fresh goat liver and modified in vitro by hydroxyl radical generated from UV irradiation (254 nm) of hydrogen peroxide. As a consequence of hydroxyl radical modification, mtDNA showed hyperchromicity and sensitivity to nuclease S1 digestion as compared to control mtDNA. Animals immunized with mtDNA and ROS-modified mtDNA induced antibodies as detected by direct binding and competition ELISA. The data suggest that immunogenicity of mtDNA got augmented after treatment with hydroxyl radical. IgG isolated from immune sera showed specificity for respective immunogen and cross-reaction with other nucleic acids. Binding of induced antibodies with array of antigens clearly indicates their polyspecific nature. Moreover, the polyspecificity exhibited by induced antibodies is unique in view of similar multiple antigen binding properties of naturally occurring anti-DNA antibodies derived from SLE patients.  2007 Elsevier Inc. All rights reserved. Keywords: mtDNA; Hydroxyl radical; Immunogenicity; Antibodies; SLE

1. Introduction Human mitochondrial DNA is a double-stranded circular molecule of 16,568 base pairs that codes for thirteen essential genes of oxidative phoshorylation [1–3]. Each human cell has hundreds of mitochondria and multiple copies of mtDNA and is the only extra chromosomal DNA in human cells [4]. Lack of a chromatin structure, histones protection, and inefficient repair system makes mtDNA susceptible to oxidative damage. ROS-induced damage to mtDNA has resulted in accumulation of 8oxodGuo (damage marker) in UV-irradiated hepatoma cell line [5]. Approximately 1–5% of the oxygen consumed by mitochondria is converted to ROS under physiological conditions [6] and thus ROS production appears to be essentially a function of oxygen consumption. Furthermore, increased mitochondrial activity per se can be an oxi-

*

Corresponding author. Fax: +91 571 2702758. E-mail address: kalam786@rediffmail.com (K. Alam).

0008-8749/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2007.06.007

dative stress to cell [7]. Electron transport chain of the organelle is a major source of ROS and mtDNA is therefore exposed to high levels of damaging oxygen species. Indeed, oxidative DNA-base damage measured as 8hydroxydeoxyguanosine (oxodGuo) has been detected in mtDNA at steady-state levels several fold higher than in nuclear DNA [8]. This apparent difference in damage could be due to proximity of mtDNA to ROS generated during electron transport. Mitochondrial dysfunction is important cause of certain human diseases [9,10] and cumulative mtDNA damage is implicated in the aging process and in the progression of such common diseases as diabetes, cancer, and heart failure [1,7]. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis [11,12]. Activation of apoptotic pathway, in turn, inhibits the rate of mitochondrial translation. The damaged mtDNA could accumulate in senescent cells. The cells loaded with perturbed DNA undergo apoptosis and the contents are eliminated.

K. Alam et al. / Cellular Immunology 247 (2007) 12–17

However, some molecules of in vivo ROS-modified mtDNA might escape elimination and present itself to the immunoregulatory network as neoantigen or migrate to nucleus and integrate into the nuclear genome [13]. In certain pathology increased apoptosis and delayed removal of apoptotic cell debris may also favor persistence of ROSmodified mtDNA in circulation inter alia exposure to immunoregulatory network and thus making the molecule immunogenic. Double stranded DNA (B-conformation) against which most of the antibodies are detected in human lupus is no longer regarded as an antigen initiating the disease because immunization with B-form of DNA does not induce clinical features typical of SLE [14]. Involvements of major organs like heart, lungs, kidneys and central nervous system have been shown to be the cause of mortality resulting from SLE [15]. The anti-dsDNA autoantibodies, marker antibody for the confirmed diagnosis of SLE, show diverse antigen binding [16,17] which include components of DNA, its different conformations and chemically modified structures [16,18]. In this communication immunology of mtDNA and its hydroxyl radical modified counterpart has been studied. The binding properties of experimentally induced antibodies were compared with SLE derived pathogenic anti-DNA antibodies. 2. Materials and methods 2.1. Isolation and purification of mitochondrial DNA Mitochondrial DNA was isolated as per the procedures described elsewhere [19]. Briefly, fresh goat liver was homogenized in ice-cold buffer of following composition: 0.3 M sucrose, 25 mM Tris, 10 mM EDTA (pH 7.0). The homogenate was centrifuged at 4000 rpm (0 C) for 10 min and supernatant was preserved. The pellet was suspended in one-tenth volume of above-mentioned buffer and re-centrifuged. The mitochondrial pellet, thus obtained, was suspended in 20 ml of 10 mM Tris, 0.1 M NaCl, 0.1 M EDTA (pH 8.0). SDS was added to a final concentration of 2% and the mixture was incubated for 60 s at 37 C followed by 9 min incubation at room temperature to complete the lysis. The contaminant high molecular weight nuclear DNA (if any) was precipitated by one-third volume of 4 M NaCl (final concentration, 1 M). The contents were gently mixed by inverting the tube and mixture was placed at 0 C for 4–16 h. The material was centrifuged at 27,000g for 15 min at 0 C and supernatant was deproteinized thrice using chloroform/isoamylalcohol (24:1) mixture. The aqueous layer rich in mitochondrial nucleic acids was concentrated by precipitation with two volumes of ethanol at 20 C. The alcohol precipitate was collected by centrifugation and dissolved in minimum volume of Tris–EDTA containing 0.1 M NaCl. The material was passed through Sepharose CL 4B column to obtain pure mtDNA.

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2.2. Modification of mitochondrial DNA Mitochondrial DNA was modified by hydroxyl radical as described earlier [20]. Aqueous solution of mitochondrial DNA (0.15 mM base pair) in PBS was irradiated under 254 nm UV light for 30 min at room temperature in presence of hydrogen peroxide (15.1 mM). Excess hydrogen peroxide was removed by extensive dialysis against PBS (pH 7.4). 2.3. SLE patients Informed consent was obtained from patients (all females) before taking blood samples. All patients fulfilled at least four criteria of American College of Rheumatology for the classification of SLE. 2.4. Immunization scheme Mitochondrial DNA and its hydroxyl-modified counterpart (25 lg) were separately complexed with equal amount of methylated BSA and emulsified with complete Freund’s adjuvant. The complex was injected intramuscularly in the hind leg muscles of rabbits (three animals in each group). Subsequent injections were given in incomplete Freund’s adjuvant. Weekly injections of antigen (mtDNA/hydroxyl-modified mtDNA) were given for seven weeks and thus each animal received a total of 175 lg antigen during the course of immunization. One week after the last dose of immunogen, blood was collected; serum separated and decomplemented by heating at 56 C for 30 min. Preimmune blood was collected prior to immunization. The material was stored in small aliquots at 80 C with sodium azide (0.1%) as preservative. 2.5. Isolation of IgG by protein-A-Sepharose Protein-A-Sepharose CL 4B was used to purify serum IgG. Serum (0.5 ml) diluted with equal volume of PBS (pH 7.4), was applied to the column (0.9 · 15 cm) preequilibrated with above buffer. The wash through was recycled 2–3 times. Unbound IgG was removed by extensive washing with PBS (pH 7.4). The bound IgG was eluted with 0.58% acetic acid in 0.85% sodium chloride [21]. Three milliliter fractions were collected in a measuring cylinder already containing 1 ml of 1 M Tris–HCl, pH 8.5, and absorbance was recorded. The IgG concentration was determined considering 1.4 OD280 = 1.0 mg mammalian IgG/ml. The isolated IgG was then dialyzed against PBS (pH 7.4) and stored at 80 C. 2.6. Enzyme linked immunosorbent assay (ELISA) Serum antibodies were detected by ELISA on flat bottom microtiter wells [22]. Test wells were filled with 100 ll DNA antigens (2.5 lg/ml in TBS) and incubated for 2 h at 37 C and overnight at 4 C. The wells were

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K. Alam et al. / Cellular Immunology 247 (2007) 12–17

washed thrice with TBS-T to remove unbound antigen. Unoccupied sites were blocked with 150 ll of 1.5% BSA (in TBS) for 5 h at room temperature. The plates were washed once with TBS-T and preimmune/immune sera (100 ll per well of 1:100 dilution), or IgG isolated from sera, were absorbed for 2 h at room temperature and overnight at 4 C. The unbound antibody was washed off four times using TBS-T. An appropriate anti-immunoglobulin alkaline phosphate conjugate (100 ll of 1:2000 dilution) was added to each well. In case of inhibition ELISA, immune complex was added instead of serum/IgG. The immune complex was formed by mixing varying concentrations of antigen (inhibitor) with fixed amount of antibody. The mixture was incubated for 2 h at room temperature and overnight at 4 C and complex thus formed was coated in place of serum/IgG. After incubation at 37 C for 2 h, the plates were washed four times with TBS-T and three times with distilled water and developed using p-nitrophenyl phosphate. Absorbance was read at 410 nm. 3. Results 3.1. Characterization of mtDNA and its modification by hydroxyl radical Incubation of isolated mtDNA with single strand specific nuclease S1 enzyme did not affect its co-migration with mtDNA alone in agarose gel and the material was considered to be pure (Fig. 1a). The circular mtDNA was modified with hydroxyl radical generated by UV irradiation (254 nm) of hydrogen peroxide. The UV absorption profile of hydroxyl-modified mtDNA showed hyperchromicity compared to control (Fig. 1b). Furthermore, at 260 nm wavelength the hyperchromicity was approximately 35 percent. The observed hyperchromicity in mtDNA suggest structural changes in the molecule as a consequence of hydroxyl radical attack.

3.2. Physico-chemical characterization of ROS-modified mtDNA Heat induced melting of mtDNA and hydroxyl-modified mtDNA was monitored at 260 nm from 30 to 95 C at the rate of 1.5 C/min [19]. While mtDNA did not show heat-induced denaturation under our experimental conditions, the melting temperature (Tm) of hydroxyl-mtDNA was observed to be 78 C. Table 1 summarizes the UV and thermal denaturation properties of mtDNA and its modified form. 3.3. Nuclease S1 treatment of hydroxyl-modified mtDNA Mitochondrial DNA and its modified form were incubated with nuclease S1 (20 IU/lg DNA) for 30 min at 37 C. EDTA terminated the reaction and samples were electrophoresed in agarose gel. The enzyme digested hydroxyl-modified mtDNA (figure not shown), which is indicative of structural changes in mtDNA as a consequence of hydroxyl radical attack. Mitochondrial DNA, used as control, did not show any sensitivity towards nuclease S1. 3.4. Immunogenicity of mtDNA and hydroxyl-modified mtDNA Rabbits were immunized with mtDNA and hydroxylmodified mtDNA. At the end of immunization blood was withdrawn and serum separated. Direct binding ELISA revealed that hydroxyl-modified mtDNA is more immunogenic than mtDNA (Fig. 2). Preimmune sera, used as control, did not show appreciable binding with either immunogen. Specificity of the induced antibodies was evaluated by competitive binding assays [22,23]. Specificity of induced antibodies towards respective immunogen was evident from inhibition in antibody activity using immunogen as inhibitor (Fig. 3). 3.5. Cross-reactivity of induced antibodies IgG was isolated from anti-mtDNA and anti-hydroxylmodified mtDNA sera and subjected to binding studies on solid phase coated with nucleic acid antigens. Although induced antibodies were highly specific for their immunogen, they did cross-react with nucleic acids of varying size

Table 1 UV and thermal denaturation characteristics of mtDNA and hydroxylmodified mtDNA

Fig. 1. (a) Agarose gel electrophoretogram of mtDNA (lane 1) treated with nuclease S1 (lane 2). (b) Ultraviolet absorption spectra of mtDNA (- - -) and hydroxyl-modified mtDNA (—).

Parameter

mtDNA

Hydroxyl-modified mtDNA

Physical shape Absorbance ratio (A260/A280) Percent hyperchromicity at 95 C Melting temperature (Tm), C Onset of melting, C

Circular 1.84 Not observed Not observed Not observed

Linear 1.47 20 78 65

K. Alam et al. / Cellular Immunology 247 (2007) 12–17 Table 2 Competitive inhibition data of anti-ROS-mtDNA IgG

Absorbance at 410 nm

1.5

Inhibitor

Maximum percent inhibition at 20 lg/ml

Concentration required for 50% inhibition (lg/ml)

ROS-mtDNA mtDNA ssDNA DNA ROS-DNA Superoxide-modified DNA Z-DNA RNA ROS-RNA Poly (G) ROS-poly(G)

80.5 63 58 48 65 27 8 40 75 25 29

6 9 17 — 13 — — — 8 — —

1.0

0.5

0.0 2.0

2.3

2.6

2.9

3.2

3.5

3.8

- log serum dilution

The microtiter plates were coated with ROS-mtDNA (2.5 lg/ml).

Fig. 2. Direct binding ELISA of anti-mtDNA antibodies (n) and antihydroxyl-modified mtDNA antibodies (d) with their respective immunogen. Corresponding unfilled symbols (h, s) represent binding of preimmune serum with immunogen.

Table 3 Competitive ELISA of anti-mtDNA IgG Inhibitor

Maximum percent inhibition at 20 lg/ml

Concentration required for 50% inhibition (lg/ml)

mtDNA ROS-mtDNA ssDNA DNA ROS-DNA Superoxide-modified DNA Z-DNA RNA ROS-RNA Poly (G) ROS-poly(G)

72 41 31 59 47 32 No inhibition 35 28 22 33

8 —

100

75

Percent inhibition

15

50

25

11 — — — — — —

The microtiter plates were coated with mtDNA (2.5 lg/ml).

0 0.1

1.0

10.0

100.0

Inhibitor concentration, μg/ml Fig. 3. Inhibition ELISA of anti-mtDNA antibodies (s) and antihydroxyl-modified mtDNA antibodies (d) with their respective immunogen.

and structure (Tables 2 and 3). The cross-reactive properties of induced antibodies appear to be due to their polyspecific nature, a characteristic feature of most of the SLE derived pathogenic anti-DNA autoantibodies. 3.6. Binding of human SLE autoantibodies with DNA, mtDNA and hydroxyl-modified mtDNA Anti-dsDNA positive SLE samples were subjected to competitive binding assay with DNA, mtDNA and hydroxyl-modified mtDNA. The human autoantibodies were well recognized by both mtDNA and hydroxy-modified mtDNA (Table 4). Serum autoantibodies of normal healthy subjects (NHS) showed little binding with inhibitors.

Table 4 Inhibition of SLE anti-DNA antibodies by native DNA, mtDNA, and OH-mtDNA SLE serum

Maximum percent inhibition at 20 lg/ml nDNA

mtDNA

OH-mtDNA

1 2 3 4 5 6 7 8

49 66 22 62 68 69 37 59

45 51 35 46 48 52 45 54

51 62 41 56 59 64 56 65

Means ± SEM a NHS (n = 5)

54 ± 5.96 15 ± 6.17

47 ± 2.08 12 ± 8.06

56.75 ± 2.78 18 ± 4.2

The microtiter wells were filled with nDNA (2.5 lg/ml). a Means ± SEM values of serum autoantibodies of normal healthy subjects binding with respective inhibitors.

4. Discussion The mtDNA is not protected by histones, as is the nuclear DNA, and it lies in close proximity to the free

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radical producing process of oxidative phosphorylation. Approximately 1–5% of the oxygen consumed by mitochondria is converted to reactive oxygen species under physiological conditions. The production of ROS is essentially a function of O2 consumption. Hence, increased mitochondrial activity per se can be an oxidative stress to cell. Mammalian cells have numerous enzymatic and non-enzymatic antioxidant defenses to guard the level of potentially dangerous reactive oxygen species. Cumulative damage of mtDNA is implicated in the progression of many human diseases. The objective of this study was twofold. The first aim was to evaluate the immunogenicity of mtDNA in experimental animals and secondly the mtDNA was subjected to in vitro modification by hydroxyl radical and experimental animals were challenged with this neo-antigen. Native form of mammalian DNA (B-conformation) is non-immunogenic. In contrast, bacterial DNA is a strong immunogen [24]. The immunogenicity of bacterial DNA has been attributed to the presence of nucleotide hexamers containing unmethylated CpG motifs. Similarly, the role of unmethylated CpG dinucleotide sequences in the immunogenicity of plasmid DNA is well recognized [25–27]. Other modified forms of DNA and polynucleotides [28,29], modified self determinants [30] and self proteins [31] have been reported to be immunogenic and the antibodies thus generated are cross reactive with native DNA. Mammalian DNA complexed with synthetic peptide Fus-1 elicited anti-dsDNA response in mice [32]. Mitochondrial DNA modified with ROS exhibited hyperchromicity at 260 nm and showed single strand breaks. These changes in mtDNA are attributed to semi loss of secondary structure as a consequence of hydroxyl radical insult. Mitochondrial DNA and its ROS form were subjected to nuclease S1 digestion. The enzyme selectively chopped off the single stranded regions in ROS-modified mtDNA. Unmodified mtDNA was not affected by nuclease treatment. Both mtDNA and its ROS counterpart were immunologically active and induced antibodies in experimental animals. Comparative studies revealed that ROS-modified mtDNA is a better immunogen. Antibodies against ROSmodified mtDNA were highly specific for its immunogen and showed cross-reactivity with mtDNA. Cross-reactions of antibodies (experimentally induced or naturally occurring) with closely related antigens are attributed to epitopes sharing between/among antigens. The preferential binding of anti-ROS-mtDNA IgG with ROS forms of DNA, RNA and poly (G) is a quasi evidence of active role being played by activated oxygen species in modifying antigenic structure of macromolecules. Raised respiration of mitochondrial cells will increase ROS production to levels beyond detoxifying defenses, which may damage mtDNA. Subsequent in vitro studies on ROS and mtDNA revealed base modification and strand breaks in mtDNA [33]. Furthermore, based on the finding that mtDNA is fragmented by ROS, Richter [34]

suggested that mtDNA fragments carrying modified bases escape from mitochondria and accumulate in a time-dependent manner in nuclear DNA. In our view there are fair chances that body’s immune cells might consider this fragmented and modified mtDNA as a foreign body and elicit antibody response typical of anti-DNA autoantibodies. Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by high titer polyspecific anti-dsDNA antibodies in patients’ sera. What causes SLE is really not known and all attempts to induce antibodies against nDNA in experimental animals have failed. Anti-DNA positive SLE sera were subjected to competitive binding assay using mtDNA and ROS-modified-mtDNA as inhibitors (Table 4). Analysis of data in Table 4 clearly indicates that mtDNA and its modified form were fairly recognized by anti-nDNA positive SLE samples. In view of the nonimmunogenic nature of nDNA and the immunogenicity exhibited by mtDNA and hydroxyl-modified mtDNA in our experimental conditions, it is possible that mtDNA and its modified from might act as triggering antigen in a subgroup of lupus patients. Acknowledgment This study was supported by a research grant [37(980)/ 98/EMR-II] to K.A. from Council of Scientific and Industrial Research, New Delhi. References [1] S. Ohta, A multifunctional organelle mitochondria is involved in cell death, proliferation and disease, Curr. Med. Chem. 10 (2003) 2485–2494. [2] T. Stevnsner, T. Thorslund, N.C. Souza-Pinto, V.A. Bohr, Mitochondrial repair of 8-oxoguanine and changes with aging, Exp. Gerontol. 37 (2002) 1189–1196. [3] J.M. Shoffner, D.C. Wallace, Oxidative phosphorylation diseases. Disorders of two genomes, Adv. Hum. Genet. 19 (1990) 267–330. [4] T.M. Wardell, E. Ferguson, P.F. Chinnery, G.M. Borthwick, R.W. Taylor, G. Jackson, A. Craft, R.N. Lightowlers, N. Howell, D.M. Turnbull, Changes in the human mitochondrial genome after treatment of malignant disease, Mutat. Res. 525 (2003) 19–27. [5] D. Takai, S.H. Park, Y. Takada, S. Ichinose, M. Kitagawa, M. Akashi, UV-irradiation induces oxidative damage to mitochondrial DNA primarily through hydrogen peroxide: analysis of 8-oxodGuo by HPLC, Free Radic. Res. 40 (2006) 1138–1148. [6] S. Papa, Mitochondrial oxidative phosphorylation changes in the life span. Molecular aspects and physiopathological implications, Biochim. Biophys. Acta 1276 (1996) 87–105. [7] D. Kang, N. Hamasaki, Mitochondrial oxidative stress and mitochondria, Clin. Chem. Lab. Med. 41 (2003) 1281–1288. [8] C. Richter, Reactive oxygen and DNA damage in mitochondria, Mutat. Res. 275 (1992) 249–255. [9] R. Luft, The development of mitochondrial medicine, Proc. Natl. Acad. Sci. USA 91 (1994) 8731–8738. [10] D.C. Wallace, Mitochondrial diseases in man and mouse, Science 283 (1999) 1482–1488. [11] M. Mirabella, S. DiGiovanni, G. Silvestri, P. Tonali, S. Servidei, Apoptosis in mitochondrial encephalomyopathies with mitochondrial DNA mutations: a potential pathogenic mechanism, Brain 123 (2000) 93–104.

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