Institute of Microbiology, Bulgarian Academy of Sciences, 26 Academician G. Bonchev, 1113 Sofia, Bulgaria1
Institute of Organic Chemistry, Bulgarian Academy of Sciences, 9 Academician G. Bonchev, 1113 Sofia, Bulgaria2
Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, 23 Academician G. Bonchev, 1113 Sofia, Bulgaria3
Anorganische Biochemie, Physiologisch-chemisches Institut der Universität, Tübingen, Hoppe-Seyler-Straße 4, D-72076, Tübingen, Germany4
Abteilung für Physikalische Biochemie des Physiologisch-chemischen Instituts der Universität, Tübingen, Hoppe-Seyler-Straße 4, D-72076, Tübingen, Germany5
Author for correspondence: Wolfgang Voelter. Tel: +49 707 12973041. Fax: +49 707 1293361. e-mail: wolfgang.voelter{at}uni-tuebingen.de
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ABSTRACT |
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Keywords: protein sequencing, myeloid Graffi tumour, influenza infection, protective effect
Abbreviations: DO, dissolved oxygen; MST, mean survival time; SOD, superoxide dismutase; TBH, tumour-bearing hamsters
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INTRODUCTION |
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The large scientific and practical interest in SODs has resulted in intensive developments of new technologies for enzyme production from various sources, including animal and human erythrocytes, plants and micro-organisms. Filamentous fungi are especially suitable for SOD production because of their potential advantages, i.e. rich abundant mycelium, intensive respiration and high level of cyanide-resistant respiration (Sakajo et al., 1993 ). Microbial technology would also be more effective and inexpensive compared to the production of this enzyme from bovine erythrocytes. Moreover, despite great efforts, efficient production of recombinant human SOD in prokaryotic systems or simple eukaryotes has failed. This lack of success has greatly complicated large-scale production and genetic engineering of the protein (Stenlund & Tibell, 1999
).
To improve the in vivo pharmacological activity of SOD, the enzyme has been conjugated with mono- and polysaccharides (Fujita et al., 1992 ), haemoglobin (DAgnillo & Chang, 1998
) and lecithin (Nakauchi et al., 1996
), etc. A major advantage of such modified SODs is their longer half life in plasma and blood (Maksimenko et al., 1993
). Our preliminary investigations have shown that the fungal strain Humicola lutea 103 mainly produces the Cu/Zn enzyme. Moreover, H. lutea SOD is a naturally glycosylated enzyme (unpublished data) which could be isolated in very few other cases. The secretory tetrameric extracellular mammalian SOD is the only glycosylated SOD, besides the H. lutea enzyme, described so far (Edlund et al., 1992
). Glycoenzymes of this kind do not need additional processing for conjugation and modification as do non-glycosylated enzymes.
Here, we present the conditions for the production of a novel, naturally glycosylated Cu/Zn SOD from the fungal strain H. lutea 103. We report also on the characterization of the purified enzyme, as well as on its protective effect in experimental influenza virus infection and on myeloid Graffi tumour.
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METHODS |
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Cultivation, equipment and conditions.
Cultivation was performed in a 3 l bioreactor, ABR-09, developed and constructed by the former Central Laboratory for Bioinstrumentation and Automatisation (CLBA) of the Bulgarian Academy of Sciences. The bioreactor was equipped with pH and automatic dissolved oxygen (DO) monitoring equipment and a control system.
The composition of the culture medium has been described previously (Angelova et al., 1996 ). For the inoculum, 80 ml seed medium was inoculated with 5 ml spore suspension at a concentration of 2x108 spores ml-1 in 500 ml Erlenmeyer flasks. The cultivation was performed on a shaker (220 r.p.m.) at 30 °C for 24 h. For bioreactor cultures, 160 ml of the seed culture was brought into the 3 l bioreactor, containing 1850 ml of the production medium. The cultures were grown at 30 °C for 120 h. The fermentation parameters under DO-uncontrolled conditions were: impeller speed, 600 r.p.m.; air flow, 1 v.v.m. [1 vol. air (1 vol. liquid)-1 (min)-1]. In this case, only the changes in DO level during fermentation were measured. For the DO-controlled culture system, aeration and impeller speed were regulated in such a way as to produce 20% oxygen saturation in the liquid. The results obtained in this investigation were evaluated from experiments repeated using three or five parallel runs.
Analytical methods.
The cell-free extract was prepared as described previously (Angelova et al., 1995 ). SOD activity was measured by the nitro blue tetrazolium (NBT) reduction method of Beauchamp & Fridovich (1971)
. One unit of SOD activity was defined as the amount of SOD required for inhibition of the reduction of NBT by 50% (A560) and was expressed as U (mg protein)-1. Cyanide (2 mM) was used to distinguish between the cyanide-sensitive isoenzyme Cu/Zn SOD and the cyanide-resistant Mn SOD. Cu/Zn SOD activity was obtained as total activity minus the activity in the presence of 2 mM cyanide. Protein was estimated by the Lowry procedure, using crystalline bovine albumin as standard. Soluble reducing sugars were determined by the SomogyiNelson method (Somogyi, 1952
). Dry weight determination was performed on samples of mycelia harvested throughout the culture period. The culture fluid was filtered through a Whatman (Clifton, USA) no. 4 filter. The separated mycelia were washed twice with distilled water and dried to a constant weight at 105 °C.
Purification of H. lutea Cu/Zn SOD.
The frozen mycelium was partially thawed and then suspended in 4 vols 25 mM potassium phosphate buffer, pH 7·8, containing 1 mM PMSF. The suspended material was disrupted by an Ultra-Turrax-IKA-Werk homogenizer. Cell debris was removed by filtration through a Buchner funnel and the mixture was clarified by centrifugation at 13000 g for 20 min at 4 °C. All subsequent steps were performed at 4 °C. The supernatant was concentrated approximately three times by membrane ultrafiltration (Amicon PM-10). The concentrated crude extract was further fractionated using acetone, chilled to -20 °C. Cooled acetone was slowly added to the culture filtrates to a concentration of 0·4 vols with vigorous stirring for 30 min. The mixture was incubated for 2 h at -20 °C and the precipitate was collected, centrifuged at 13000 g for 20 min and discarded, while the supernatant was treated with 1·1 vols of chilled acetone. The mixture was held for 12 h at -20 °C; then the precipitate was collected by centrifugation (13000 g for 20 min) and suspended in distilled H2O. The clear viscous supernatant was loaded on a Sephadex G-75 column (2x50 cm; Pharmacia), previously equilibrated with 50 mM potassium phosphate buffer, pH 7·8, and the column was eluted with the same buffer.
Further purification was achieved by ion exchange chromatography on a DEAE cellulose-52 column (3·5x10 cm; Serva), equilibrated in 50 mM KH2PO4 buffer, pH 7·8. The Cu/Zn SOD was eluted with an NaCl gradient (00·1 M). The active fractions were loaded for additional purification onto an FPLC system, equipped with a 10/10 Mono Q anion exchange column. The column was previously equilibrated with 50 mM potassium phosphate buffer, pH 7·8, and the elution was effected employing stepwise increases in NaCl (00·1 M). Fractions with 3100 U (mg protein)-1 were further purified and desalted on a Nucleosil RP C18 column (250x10 mm; Macherey-Nagel) to allow determination of the N-terminal sequence and amino acid composition. The conditions used for HPLC separation were as follows: eluant A, 0·058% trifluoroacetic acid (TFA); eluant B, 80% acetonitrile in A. The gradient was run from 5 to 100% B within 60 min at a flow rate of 1 ml min-1.
Characterization of H. lutea Cu/Zn SOD.
Flameless atomic absorption spectroscopy of Cu, Zn and Mn was performed on a Perkin-Elmer 400 S spectrometer, equipped with a HGA 76B graphite furnace.
Purity control of the enzyme and measurement of molecular mass were performed on 10% polyacrylamide gels, as described by Laemmli (1970) , and the gels, stained for protein detection, were compared with duplicate gels stained for SOD activity as described by Misra & Fridovich, (1977)
. The following molecular mass standards were used: trypsinogen (24 kDa), egg albumin (45 kDa), bovine albumin (66 kDa) and Limulus polyphemus haemocyanin (monomer, 70 kDa).
N-terminal amino acid sequence analysis was performed using an Applied Biosystems sequencer, model 473A.
Mass spectra were obtained by matrix-assisted laser desorption ionization MS (MALDI 1-MS) (Kratos, MALDI III equipment; Shimadzu). The sample (10 pmol) was dissolved in 0·1% (v/v) TFA and applied onto the target. Analysis was carried out in -cyano-4-hydroxycinnamic acid. Solutions of chicken egg ovalbumin (44400 kDa) and bovine serum albumin (66430 Da) were used to calibrate the molecular mass scale.
The protein was analysed for carbohydrates with the orcinol/H2SO4 test (Francois et al., 1962 ).
Experiments concerning the effect of H. lutea SOD on myeloid Graffi tumour.
The protective effect was examined by the following parameters: mean latent time (d) for tumour appearance; mean survival time (MST, d), estimated at day 45 after tumour transplantation; and inhibition (%) of tumour growth, estimated according to Toshkova (1995)
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Experimental animals.
Forty, 2-month-old Golden Siberian hamsters of both sexes, weighing 80 to 100 g, were used for the experiments. For the examination of the SOD-protective effect, the animals were separated into four experimental groups.
Group 1. Animals were treated intraperitoneally (i.p.) with a 65 U H. lutea SOD single dose per animal, twice a week, 1 week before and 2 weeks after tumour transplantation.
Group 2. Animals were treated with a 65 U H. lutea SOD single dose per animal, twice a week, 2 weeks after tumour transplantation, starting on the day of transplantation.
Group 3. Animals were treated with a 125 U H. lutea SOD single dose per animal, twice a week, 2 weeks after tumour transplantation, starting on the day of transplantation.
Group 4. Control animals with tumours without treatment.
Tumour.
The myeloid tumour was induced by the mouse Graffi virus, adapted for hamsters by Yakimov et al. (1979) . The tumour was maintained in vivo by subcutaneous (s.c.) inoculation of 2x104 viable cells in the interscapular region. The tumour was 100% transplantable and 100% lethal for hamsters (Yakimov et al., 1979
; Toshkova, 1995
).
Experiments concerning the effect of H. lutea SOD in experimental influenza infection.
Male and female inbred ICR mice (1618 g) were obtained from the Experimental Animal Station of the Bulgarian Academy of Sciences in Slivnitza, Sofia, Bulgaria. They were quarantined 24 h prior to use and maintained under standard laboratory conditions with tap water ad libitum for the duration of the studies. The experimental groups were of 10 animals each. At the end of the experiments, surviving mice were sacrificed by cervical dislocation.
Virus infection.
In mice, the infection was induced under ether anaesthesia by intranasal inoculation of A/Aichi/2/68 (H3N2), adapted to the mouse lung. This virus causes haemorrhagic pneumonia in mice. The strain was from the collection of the Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria. To cause lethal infection, mice were infected by passages in the lungs with 510 LD50 of the virus in a 0·05 ml volume of physiological saline per mouse.
Experimental design.
Mice were treated with H. lutea Cu/Zn SOD (500 U per mouse d-1) in 0·1 ml saline administered intravenously (i.v.) 47 d after virus exposure and were observed for death daily for 21 d. All experiments were done in parallel with the selective antiviral drug ribavirin (Sigma-Aldrich) and SOD from bovine erytrocytes (Sigma-Aldrich). Ribavirin was inoculated i.p. in a dose of 125 mg per mouse d-1; bovine SOD was inoculated i.v. in a dose of 500 U per mouse d-1.
The protective effect was estimated by the increase in the rate of survival (%), protective index (PI,%) and prolongation of MST (d) as described by Serkedjieva & Ivanova (1997) . PI was determined from the equation (PR-1)/PRx100, where PR (protective ratio) is Mcontrol/Mexperiment (M is mortality).
The results are the mean values from three or four independent experiments. Reduction of mortality rates compared to placebo controls were evaluated using 2 analysis. Students t-test was employed to analyse differences in survival times. Standard deviations were determined within 95% confidence intervals.
Ethical aspects.
Experiments with animals are inevitable and indispensable in investigations concerning the treatment of influenza infection and tumour growth. Only in this way could a matter-of-fact characterization of the protective effect of H. lutea SOD be obtained. That is why the experiments with animals cannot be replaced by experiments with tissue culture cell lines. The number of experimental animals was reduced as much as possible, depending on statistical significance. The animals were sacrificed using ether narcosis and cervical dislocation to minimize the amount of suffering. Refinement of the tests with animals was achieved by careful planning of multifactor experiments.
The animals were bred under standard conditions, accepted by the Bulgarian Veterinary Health Service. Specialized personnel took care of the welfare of the animals. In implementing this study we have adhered most strictly to all national and international ethical provisions applicable to Bulgaria where the investigations with experimental animals were carried out.
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RESULTS AND DISCUSSION |
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Irrespective of accelerated glucose uptake (Fig. 1b), 20% DO-controlled conditions led to a reduction in duration of the stationary phase as well as to a decrease in the maximum level of biomass (Fig. 1a
) and intracellular protein content (data not shown). Petruccioli et al. (1995)
and Guidot et al. (1993)
demonstrated similar data for Penicillium variable and Saccharomyces cerevisiae, respectively, due to increasing DO level.
The effects of two different levels of DO (uncontrolled DO and DO at 20%) on SOD production were evaluated (Fig. 1b). Increasing enzyme activity in H. lutea cells, cultivated under both conditions, was detected in the early exponential phase and an active synthesis was observed during a period of intensive oxygen consumption. Moreover, the time courses of SOD production show two maxima. A similar phenomenon, a secondary increase in SOD activity during the late stationary phase, has been observed for SOD production by filamentous fungi and yeasts (Shilova et al., 1989
; Angelova et al., 1996
). It can be explained by an intensification of the process of ·
generation when the cells utilize endogenous sources of carbon and nitrogen (organic or amino acids).
The results demonstrated that the oxygen level had a significant influence on SOD activity. Under high DO conditions, higher levels of SOD were produced at 1st and 2nd maxima, reaching activities of about 112 and 123 U (mg protein)-1, respectively. Considering enzyme formation in both experiments, DO-controlled cultures produce approximately 1·7-fold higher SOD activity than DO-uncontrolled cultures. Exposure to high DO levels has been reported to cause both enhancement (Vercellone et al., 1990 ) and decrease (Shilova et al., 1989
) in SOD activity for microbial cells.
The isoenzyme profiles of SOD, produced by H. lutea upon cultivation at different DO levels, are illustrated in Fig. 2. The fungal strain produces two isoforms, namely Mn- and Cu/Zn-containing SOD. Cu/Zn SOD activity was obtained as total activity minus the activity in the presence of 2 mM cyanide, and the conclusion from these results is that this isoenzyme was responsible for approximately 90% of total SOD activity in DO-uncontrolled cultures. The numbers of isoforms did not change under hyperoxic conditions when H. lutea was exposed to an atmosphere of 20% O2. There was no significant change in Cu/Zn SOD activity, whereas Mn SOD expression increased 1012-fold by elevated DO. So, it appears that Mn SOD is the primary defence in H. lutea cells against oxygen toxicity. Similar findings about the lack of response of Cu/Zn SOD to oxidative stress have been reported for a variety of cells (Stralin & Marklund, 1994
). The dependence of Cu/Zn SOD on oxygen was found to be related to the availability of copper to H. lutea (M. Angelova, unpublished data), yeasts (Howlett & Avery, 1999
) and mammalian cells (Roughead et al., 1999
).
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The N-terminal sequence of Cu/Zn SOD from H. lutea 103 was compared with corresponding regions of Cu/Zn SODs isolated from other organisms (Bordo et al., 1994 ; Forest et al., 2000
; Fig. 5
). The first 35 residues showed more or less the same percentage (4271%) of identity with the enzymes from eukaryotes. The highest homology was established with the Cu/Zn SODs from fungi and yeasts (71·4 and 62·9% with Neurospora crassa and Saccharomyces cerevisiae, respectively). Comparison with enzymes from higher eukaryotes reveals a level of homology of about 48·5% with human and 42·4% with bovine Cu/Zn SOD. As already mentioned, the enzyme from H. lutea 103 shows a low level of homology (about 20%) with SODs from prokaryotic organisms such as Actinobacillus pleuropneumoniae, Haemophilus ducreyi, Haemophilus influenzae and Haemophilus parainfluenzae.
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The results of the latent time of tumour appearance and the MST are shown in Table 3. The latent time of tumour appearance was elongated in hamsters of all experimental groups treated with H. lutea SOD simultaneously or before tumour transplantation compared to the control group. Furthermore, the application of H. lutea SOD enhanced the MST of Graffi-tumour-bearing hamsters. The effect was better expressed in the 3rd group treated with 125 U H. lutea SOD (39·6 d), compared to the untreated control animals (32·1 d). Though groups 1 and 2 were treated with a lower dose of SOD, a comparable value of MST was established (36·5 and 36 d for groups 1 and 2, respectively).
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On the other hand, it is worthwhile mentioning that groups 2 and 3 show a different response in both determinations (Table 3). Whereas the higher dose (125 U; 3rd group) caused the highest value of MTS, the lower dose (65 U; 2nd group) was more suitable for the prevention of tumour appearance. These data suggest that the effect of exogenous added H. lutea SOD is stage-dependent. In the first stage of carcinogenesis, the levels of antioxidant enzymes (SOD and catalase) in tumour-bearing hamsters (TBH) were almost equal with those in the control animals (E. Ivanova, unpublished data) and a single dose of 65 U SOD was sufficient to restore the oxidant/antioxidant balance in the host organism. In contrast, because of the interference of the endogenous SOD with the higher dose of injected H. lutea SOD (125 U; 3rd group), a significant increase in H2O2 accumulation was established (E. Ivanova, unpublished data). Moreover, the treatment of TBH with H. lutea SOD caused an insignificant increase in catalase activity in liver and tumour cells (E. Ivanova, unpublished data) and consequently enhances the cells ability to generate the highly toxic hydroxyl radical (Fenton reaction).
Regarding the MST, a possible interpretation of the data includes our previous results obtained during the tumour progression stage: (i) the superoxide production by phagocytes was four- to fivefold higher in TBH compared to the control animals (Dimitrova et al., 2000 ); (ii) the levels of SOD and catalase decreased in TBH compared to the healthy animals (E. Ivanova, unpublished data). Probably, this modification of the antioxidant defence during tumour growth resulted in a serious disturbance of the oxidant/antioxidant balance in the cells. It is possible that the higher dose of injected H. lutea SOD (125 U) restores the redox balance to a higher degree compared to the dose of 65 U.
The H. lutea SOD treatment furthermore inhibited tumour growth in the early stage of tumour progression (7375% at day 10 and 4850% at day 15 for the experimental animals in groups 13; Fig. 6). During the following days the most expressed inhibition of tumour growth was established for group 1. It has been reported that reduction of the radicals and oxidants with antioxidants, including SOD, antagonizes tumour promotion activity (Kong & Lillehei, 1998
). Our results supported this concept that SOD has a protective effect during the promotional phases of cancer development (Matès & Sánchez-Jimenez, 2000
).
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The preliminary results from toxicity studies showed that the fungal SOD did not cause either acute or chronic toxicity in experimental animals, treated fourfold with doses of 500 U per mouse d-1. It is significant that no cytotoxicity was observed in cell culture studies (results not shown).
The design of the treatment was evaluated after a series of preliminary experiments. We found that the optimal schedule for treatment included four consecutive i.v. inoculations of 500 U per mouse d-1 on days 47 after viral exposure that caused 80% mortality in the control group. The protective effect of SOD treatment during the critical period of influenza-induced pneumonitis on the survival of mice is presented in Fig. 7. Our results indicate that treatment with H. lutea SOD exhibits a strong protective effect on experimental influenza virus infection in mice and that this effect occurs, if the virus challenge is moderate and when the therapy is initiated late during infection. The lateness of therapy initiation particularly relates well to the time when one would anticipate starting therapy in the human patient. The survival rate increases markedly with respect to placebo control: the time of survival rises within 5·2 d and the protective index reaches 86·1%. The effect of the fungal SOD is comparable to that of the selective antiviral drug ribavirin. Bovine SOD at a dose of 500 U per mouse d-1 also protects mice from mortality significantly, though less efficiently. The higher efficiency of H. lutea SOD could be explained by the presence of a carbohydrate chain attached to the glycosylation site of the fungal enzyme. Human, bovine and recombinant Cu/Zn SOD are metalloproteins lacking oligosaccharides. For all of these a protective effect against influenza infection has been reported which is, however, less than that with the fungal H. lutea SOD glycoprotein. Human SOD, inoculated at a dose of 1000 U per mouse at a late stage of infection caused 6080% survival (Dolganova & Sharonov, 1997
; Sharonov et al., 1991
), bovine Cu/Zn SOD at a dose of 1000 U per mouse resulted in 60% survival of the infected mice (Oda et al., 1989
) and treatment with recombinant Cu/Zn SOD (1 mg per mouse d-1) exhibited low protective activity only (40% survival) (Nimrod et al., 1994
).
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The results from the present investigations on the protective effect of the novel fungal Cu/Zn SOD on experimental influenza infection can be considered as encouraging. They support the findings that the appropriate use of reactive oxygen species scavengers, applied alone or in combination with selective viral inhibitors, is a promising approach for the treatment of influenza virus infection.
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ACKNOWLEDGEMENTS |
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Received 18 September 2000;
revised 19 February 2001;
accepted 5 March 2001.
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