Yeast strains, Plasmids and growth conditions

4. Induction of apoptosis in yeast by ophidian L-amino acid oxidase

4.2.2. Yeast strains, Plasmids and growth conditions

4.2. Materials and methods

4.2.1. General chemicals, reagents and enzymes

DMSO, o-dianisidine, catalase and neuraminidase were from Sigma, propidium iodide and horseradish peroxidase from Roche Diagnostics, Mannheim, Germany. L-Amino acid oxidase (LAAO) from the venom of the Malayan pit viper (Calloselasma rhodostoma) was obtained as described (Macheroux et al., 2001).

4.2.2. Yeast strains, Plasmids and growth conditions

Yeast strains used in this study were isogenic strains of S. cerevisiae, which include BY4741 (MAT a, his 3Δ1, leu2 Δ0, met15 Δ0 and ura3 Δ0) and BY4742 (MATα, his 3Δ1, leu2 Δ0, lys 2Δ0 and ura3 Δ0). Both strains are auxotrophic for leucine due to a deletion of the LEU2 gene. The BY4742 strain was transformed using the lithium acetate method with the plasmid pRS 315 encoding the leucine biosynthesis gene as a marker, to make it prototrophic for leucine (BY4742-pRS315). Transformants were selected on YNB plates lacking leucine.

Cultures were grown in phosphate buffered (100 mM, pH 6.5) minimal medium (0.17%

YNB, 0.5% ammonium sulphate and 2% glucose) supplemented with amino acids (0.25 mM leucine, 0.25 mM methionine, 0.25 mM histidine for BY4741 and lysine, histidine for BY4742 containing pRS315) and 0.25 mM uracil.

Cells at OD600 ≈ 0.3 were incubated with LAAO (1.2 µM) in the presence or absence of catalase (1.5 µM) with the indicated concentrations of leucine at 30 °C in a shaker for 4 hrs. After incubation, cells were counted using a CASY cell counter (Schärfe System GMBH, Reutlingen, Germany) and about 500 cells were plated on YPD plates (2 % tryptone, 1 % yeast extract and 2 % glucose) and incubated at 30 °C incubator for two days. After two days, colony-forming units were assessed by counting, the values of the control were taken as 100%.

43 4.2.3. Dihydrorhodamine staining

Dihydrorhodamine 123 is a non fluorescent compound, which upon reacting with reactive oxygen species is converted into a fluorescent derivative (Klassen and Meinhardt, 2005).

For the detection of reactive oxygen species, cells after incubation with LAAO were washed once with PBS and then incubated with dihydrorhodamine 123 (1: 500 dilution, molecular probes) at 30 °C for 2 hrs. After incubation cells were washed twice with PBS and then analyzed by fluorescence microscopy (Axioskop, Zeiss).

4.2.4. TUNEL staining

After incubation of cells with LAAO, cells were washed once with buffer B (0.5 mM MgCl2, 35 mM potassium phosphate buffer, pH 6.8) and then fixed in formaldehyde (3.7

%) for 1 hour. After that, cells were washed with sorbitol buffer (0.5 mM MgCl2, 35 mM potassium phosphate buffer, pH 6.8, 1.2 M sorbitol) and with glucuronidase / arylsulfatase (20 units, Roche) and lyticase (5 units, Sigma) and applied onto a polylysine coated slide. DNA ends were labelled with the TdT- mediated dUTP nick end labelling (TUNEL) method using the In situ cell death detection kit, POD (Roche) as described previously (Madeo et al., 1997).

4.2.5. Annexin V staining

Exposure of phosphatidylserine to the outer membrane is one of the earliest detectable markers of apoptosis. This phosphatidylserine exposure was detected by FITC labelled Annexin V. After incubation with LAAO for 4 hrs, cells were washed with sorbitol buffer (1.2 M sorbitol, 0.5 mM MgCl2, 35 mM potassium phosphate buffer, pH 6.8) and digested with glucuronidase / arylsulfatase (20 U) and lyticase (5 U) for 1 hr at 30 °C.

After digestion cells were washed in annexin binding buffer provided by the manufacturer (Annexin -V FLUOS staining kit, Roche) and incubated with annexin V (1:250) and propidium iodide (1:250) for 20 min at room temperature. Cells were then

washed, suspended in binding buffer, applied to a microscopic slide and analysed by fluorescence microscopy (Axioskop, Zeiss).

4.2.6. Amino acid analysis

Yeast cells were incubated with LAAO in the presence or in the absence of catalase for 4 h. After incubation cells were centrifuged at 13,000 rpm for 5 minutes. Supernatant was collected and filtered through UFC5 BCC filter (Millipore). The flow through was used for amino acid analysis. The liquid chromatography was carried out on a HP 1100 (Agilent, Waldbronn, Germany). The column was a reversed phase Lichrocart Purosphere Star 100 (55 × 2 mm, 3 µm). The elution was done isocratically with a mixture of acetonitril (5 %), water (93 %) and trifluoroacetic acid (2 %) using a flow rate of 0.3 mL/min and an injection volume of 1 µL. Leucine was detected by electrospray-MS using the mass signal at m/z = 132 in the positive mode (MS parameters: drying gas temperature: 350 °C, vaporizer temperature: 325 °C, drying gas flow: 10 l/min, nebulizer pressure: 40 psig, capillary voltage: 3500 V, fragmentor voltage: 40 V). Using these conditions only one single peak occurred in the chromatogram having the same retention time as the standard.

4.2.7. Protease protection assay

Yeast cells were incubated with LAAO and ds-LAAO in the presence of catalase for 30 min. Cells were then, washed thrice with PBS and then they were incubated in the presence of proteinase K (0.2 mg/ml) for 2 hrs at 37 °C. After the incubation cells were washed thrice with PBS and then lysed with lysis buffer (NaOH 1.85 M, 7.5% of ß- mercaptoethanol). Cells incubated with LAAO and ds- LAAO but without treating with proteinase K were used as controls. Proteins were then precipitated using 50% TCA.

Protein precipitate was washed twice with sterile water and then dissolved in SDS sample buffer. Samples were then separated on 12% SDS PAGE, blotted to a nitrocellulose membrane and blocked in 3% milk powder in TST (0.15 M NaCl, 0.1% tween 20, 50mM

Tris-Hcl pH 7.5) over night. The membrane was then incubated with rabbit polyclonal antibody against LAAO (1:1000) for 2 hrs followed by peroxidase conjugated IgG antibody (1:10000, Amersham Biosciences) for 1 h and visualized by a chemiluminescence kit (Amersham Biosciences).

4.2.8. Western blotting

Yeast cells were incubated with LAAO and catalase at indicated time points, washed twice with PBS and lysed with lysis buffer (NaOH 1.85 M, 7.5% of ß- mercaptoethanol).

Proteins were then precipitated using 50% TCA. Protein precipitate was washed twice with sterile water and then dissolved in SDS sample buffer. Samples were then separated on 12% SDS PAGE, blotted to a nitrocellulose membrane and blocked in 3% milk powder in TST (0.15 M NaCl, 0.1% tween 20, 50mM Tris-Hcl pH 7.5) over night. The membrane was then incubated with rabbit polyclonal antibody against LAAO (1:1000) for 2 hrs followed by peroxidase conjugated IgG antibody (1: 10000, Amersham Biosciences) for 1 h and visualized by a chemiluminescence kit (Amersham Biosciences).

4.2.9. Immunofluorescence

After incubation of cells with LAAO for indicated periods, cells were washed once with buffer B (35 mM potassium phosphate buffer pH 6.8, 0.5 mM MgCl2), resuspended in 1 ml of buffer B and then fixed in formaldehyde (3.7%). After that, cells were washed with sorbitol buffer (0.5 mM MgCl2, 35 mM potassium phosphate buffer, pH 6.8, 1.2 M sorbitol), digested with glucuronidase/arylsulfatase (20 units, Roche) and lyticase (5 units, Sigma) and applied onto a polylysine coated slide. Cells were then blocked in blocking buffer (PBS / 0.1% BSA) for 30 min and incubated with anti- LAAO antibody (1:700) for 2 hrs. Cells were washed three times with PBS / 0.1% BSA. After washing, cells were incubated with anti rabbit IgG antibody conjugated with rhodamine fluorophore (1:100, Molecular probes) for 1 hr. Eventually cells were washed with three

times with PBS / 0.1% BSA and mounted on a coverslide with a drop of Kaiser’s glycerol gelatin (Merck) and observed under a fluorescence microscope (Axioskop, Zeiss).

4.3. Results

4.3.1. L-amino acid oxidase induces cell death in yeast

Both yeast strains used in this study are auxotrophic for leucine as they carry a disruption of a leucine biosynthesis gene, plasmid pRS315 restores leucine auxotrophy. Incubation with LAAO (1.2 µM) of both auxotrophic and prototrophic strains at the indicated concentrations of leucine resulted in cell death as shown in the Fig 9 (left panel). Cell death induced by LAAO in both strains was increasing in parallel with the increase in the concentration of leucine.

4.3.2. The effect of catalase on cell death caused by LAAO

Since previous experiments have indicated a central role for hydrogen peroxide in induction of cell death, we investigated the effect of catalase, an active scavenger of hydrogen peroxide on cell death. Incubation of cells with catalase (1.5 µM) at various concentrations of leucine, did not completely inhibit cell death caused by LAAO (1.2 µM) in leucine auxotrophic strain. As shown in the Fig 9 (right panel), at higher concentrations of leucine ≈ 90% cells are viable but especially at lower concentrations of leucine about ≈ 40% of the cells are not viable. This indicates that cell death caused by LAAO at lower concentrations of leucine involve other factors in addition to the production of hydrogen peroxide.

Fig 9. Effect of LAAO on yeast cells.

Yeast cells were cultured up to OD₆₀₀ ≈ 0.3 in minimal medium followed by incubation with the indicated concentrations of leucine and LAAO (1.2 µM) in the absence (left panel) or in the presence of catalase (1.5 µM) (right panel) for 4 hrs. After respective incubations yeast cells were counted, about 500 cells were plated on YPD plates and incubated at 30 °C for 2 days. After the incubation, number of colony forming units was counted.

4.3.3. Characterization of cell death caused by LAAO

Reactive oxygen species have the potential to induce cell death in yeast (Eisler et al., 2004; Klassen and Meinhardt, 2005; Laun et al., 2001; Ludovico et al., 2001; Madeo et al., 1999). Dihydrorhodamine 123 is a reliable indicator for reactive oxygen species as it is rapidly converted into a highly fluorescent derivative upon interacting with reactive oxygen species. 80% of cells pretreated with LAAO were positive for the fluorescent product of dihydrorhodamine 123 oxidation as shown in the Fig 10. This is a clear indication that LAAO mediated cell death is caused by the generation of reactive oxygen species.

Fig 10. Accumulation of ROS in LAAO treated Yeast cells.

Cells were incubated in the presence of LAAO (1.2 µM) for 4 hrs with indicated concentrations of leucine, then incubated with dihydrorhodamine 123 for 2 hrs and observed under a fluorescence microscope. About 200 cells were counted for each concentration.

Further characterization of cell death was carried out by TUNEL staining, which specifically detects DNA fragmentation. As shown in the Fig 11, control cells were not stained but cells treated with LAAO in the presence of leucine, were positive for the TUNEL test.

Fig 11. DNA fragmentation analyzed by TUNEL staining.

Cells after incubation with LAAO for 4 hrs were stained with TUNEL assay for DNA strand breaks. (1) untreated cells, (2) cells treated with LAAO in the presence of 10 mM leucine and (3) cells treated with LAAO in the presence of 20 mM leucine.

Cells incubated with LAAO were further analyzed for the exposure of phosphatidylserine on the cell surface. This was done by incubating the cells with FITC conjugated annexin V and propidium iodide. As shown in the Fig 12, most of the cells are annexin V positive.

Taken together, all the evidence indicates that LAAO causes cell death by induction of apoptosis.

Fig 12. Annexin V staining.

Cells were incubated with LAAO (1.2 µM) for 4 hrs, then digested with glucuronidase / arylsulfatase (20 U) and lyticase (5 U) followed by incubation with FITC labelled annexin V and propidium iodide for 20 min. Cells were then observed under the fluorescence microscope (1: fluorescence image / FITC channel;

2: fluorescence image / PI channel; 3: transmission image)

4.3.4. Cell death as a function of hydrogen peroxide and amino acid deprivation

Treatment of yeast cells with hydrogen peroxide induces apoptosis in a concentration dependent manner (Madeo et al., 1999). We found that the death rate is increased in leucine auxotrophs when incubated in a medium devoid of leucine (Fig 13). Incubation of cells with 0.2 mM of hydrogen peroxide caused about ≈ 20% of cell death, with the same concentration of hydrogen peroxide causing ≈ 50% cell death in the absence of leucine. In the absence of hydrogen peroxide, leucine deprivation had no effect on cell survival. This clearly indicates that lower concentrations of hydrogen peroxide accompanied by leucine deficiency enhance the cell death in the leucine auxotrophic yeast strain.

Fig 13. Effect of hydrogen peroxide in the medium with and without leucine.

Leucine auxotrophic BY4742 were cultured up to the OD₆₀₀ ≈ 0.3 in minimal medium followed by incubation with the indicated concentrations of hydrogen peroxide in the absence or in the presence of leucine for 4 hrs. After the incubation yeast cells were counted, about 500 cells were plated on YPD plates and incubated at 30 °C for 2 days. After the incubation, colony forming units were counted.

4.3.5. Interaction of LAAO with the yeast cell

Observations in cultured Jurkat cells indicated that hydrogen peroxide is not solely responsible for LAAO induced apoptosis, as catalase can not fully rescue (Ande et al., 2006). In order to further evaluate these possible contributions, we studied the interaction of LAAO with yeast cells. In a first attempt, cells were incubated with LAAO for various time points and the cells were isolated and probed with LAAO antibody. As can be seen in Fig 4, there is a protein fragment recognized by LAAO antibodies in the cells treated with LAAO. The intensity of this protein fragment is time dependent. Interestingly, incubation with desialylated LAAO also results in a single protein fragment (Fig 14, right). To gain further support for the interaction of LAAO with yeast cells, we incubated cells with LAAO and probed the cells by fluorescence microscopy after incubating with LAAO antibody and then with secondary antibody labeled with fluorophore. In the case of native LAAO, the cells are homogeneously labeled indicating that the enzyme is interacting with the cells. In addition to the native LAAO, desialylated enzyme (ds LAAO) does interact with the cells, as it is evident from Fig 15 (lower panel).

Further support of LAAO interaction with the cell surface has come from the protease protection assay. Cells were incubated with LAAO and subsequently treated in the presence or in the absence of proteinase K and then probed with LAAO antibody. As shown in the Fig 6, antibodies could not recognize any protein fragment in the cells treated with LAAO but in the cells which are not treated with proteinase K, they could recognize the protein fragment. This indicates that LAAO interacts with the cell surface and hence it is amenable to proteinase K digestion. Hence no signal was observed in the western analysis. But in the absence of proteinase K there is a signal (Fig 16). This is the same with the cells incubated with ds-LAAO in the presence or in the absence of proteinase K.

Fig 14. Interaction of LAAO with yeast cells.

Cells were incubated with LAAO or ds LAAO (1.2 µM) in the presence of catalase (1.5 µM) for the time indicated. Cells were then washed thrice with PBS, lysed and proteins were analysed on SDS PAGE.

Western blot was performed using LAAO polyclonal antibodies. Untreated cells were used as negative control.

Fig 15. Immunofluorescence.

Cells were incubated with LAAO or ds-LAAO (1.2 µM) in the presence of catalase (1.5 µM) washed twice with PBS and fixed with formaldehyde (3.7%) then digested with glucuronidase / arylsulfatase (20 U) and lyticase (5 U). Then cells were placed on polylysine coated slides, and incubated with LAAO antibody followed by rhodamine conjugated anti-rabbit IgG antibody and then observed under the fluorescence microscope.

Fig 16. Protease protection assay with yeast cells.

Cells were incubated with LAAO or ds LAAO (1.2 µM) in the presence of catalase (1.5 µM) for 30 min.

Cells were then washed thrice with PBS and digested with proteinase K (0.2 mg/ml). After digestion, cells were washed thrice with PBS, lysed and proteins were analysed on SDS PAGE. Western blot was performed using LAAO polyclonal antibodies. Untreated cells were used as negative control

4.4. Discussion

L-amino acid oxidase from Calloselasma rhodostoma is a homodimeric and glycosylated flavoprotein. Apoptosis caused by LAAO is mainly attributed to the hydrogen peroxide generated upon oxidation of leucine. Incubation of LAAO with the isogenic strains of S.

cerevisiae which are auxotrophic (BY4741) and prototrophic (BY4742-pRS315) to leucine induces apoptosis in parallel with the increase in the concentration of leucine (Fig 9A). Speaking in terms of extent of insult, number of colonies that die apoptotically in case of the leucine auxotrophic strain is higher, when compared with the leucine prototrophic strain (Fig 9A). This may be attributed to the different insults acting upon the cells at the same time and that could trigger more cells to undergo apoptotic cell death. We have described that depletion of essential amino acid can trigger apoptotic cell death in yeast (Eisler et al., 2004). Other members of LAAO family, such as achacin from the body surface mucus of the giant African snail (Achatina fulica Ferussac) and the apoptosis inducing protein (AIP) from parasite-infected fish respectively, deplete arginine, lysine, tryptophane, and tyrosine, or only lysine from mammalian cell culture medium (Kanzawa et al., 2004; Murakawa et al., 2001). Depletion of these essential amino acids has been shown to contribute to the cell demise. In our earlier report we could also show that LAAO from C. rhodostoma is very effective in the degradation of L-amino acids. It has a preference for aromatic and hydrophobic amino acids such as Tyr, Phe, Val and Leu, which are rapidly metabolized and depleted within 4 h from the medium (Ande et al., 2006). In the present study we show that LAAO depletes leucine from yeast minimal medium. This leads to a higher extent of apoptotic cell demise in the case of leucine auxotrophic strain, because of a combined effect of H2O2 production and depletion of an essential nutrient. In the case of the leucine prototrophic strain, however, fewer cells are undergoing apoptotic cell death.

Incubation of cells with catalase did not prevent apoptosis in case of the leucine auxotrophic strain (Fig 9B). There are two different cases here, at the higher

concentrations of leucine, there is no cell death indicating that hydrogen peroxide is the main toxic insult. Interestingly, the cell death is prevalent only in the lower concentrations of leucine. We assume that in the case of lower concentrations of leucine, the enzyme degrades all of the leucine. This deprivation of leucine alone may not be enough for the cells to undergo cell death in a short time, because there is a report in the literature that deprivation of essential amino acid can trigger reactive oxygen species mediated apoptosis in yeast only after 24 h. But in our case it is very fast and within 4 h about 40% of the cells die. There are reports that LAAO binds to the cell, so that some of the hydrogen peroxide generated diffuses directly into the cell, unavailable for the catalase (Suhr and Kim, 1996). Intriguingly LAAO has a funnel in the active center and the glycan moieties are in the vicinity of the funnel (Pawelek et al., 2000). It is assumed that LAAO with the help of glycan moieties binds to the cell and generates small amounts of hydrogen peroxide locally. So, local concentrations of hydrogen peroxide generated by the enzyme and depletion of leucine acting together and making the cells to die faster.

Either local concentrations of hydrogen peroxide generated by the enzyme alone or deprivation of leucine alone are not sufficient to cause cell death with in 4 h (Fig 9B).

This assumption is supported by the observation that the leucine prototrophic strain when incubated with LAAO in the presence of catalase did not show increased apoptosis (Fig 9 B), though the enzyme does bind to the cell (data not shown).

Interaction of LAAO with the yeast cells was shown by western analysis and by immunofluorescence. Antibodies of LAAO recognize protein fragment in the western blot, indicating that LAAO is interacting with the cell surface. Cells treated with desialylated LAAO also showed that particular fragment indicating that ds-LAAO also interacts with the cell surface (Fig 14). Protease protection assay indicates that LAAO and ds-LAAO are interacting to the cell surface but not getting internalized. This is different from our earlier report (Ande et al., 2006). So, it will be interesting to study how LAAO’s interact with the surface of the different cells and possible factors that help them to interact.

5. Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

Sudharsana Rao Ande*, Phaneeswara Rao Kommoju*, Sigrid Draxl, Michael Murkovic, Peter Macheroux, Sandro Ghisla and Elisa Ferrando-May.

(Manuscript accepted for publication in Apoptosis)

The work presented in this chapter was as a part of collaboration between Prof. Dr.

Sandro Ghisla and Dr. Elisa Ferrando-May. * PRK and SRA have contributed equally to this work. All the experiments with mammalian cells was done by myself. Sigrid Draxl, Dr. Michael Murkovic and Prof. Peter Macheroux of Technical University Graz, Austria are responsible for the amino acid analysis of LAAO media. PRK has provided the antibodies of LAAO, he has carried out H2O2 measurements with LAAO and DAAO. He has provided the western analysis of cells incubated with LAAO, and has estimated the sialic acid content of native LAAO and of desialylated LAAO. PRK has assessed the activity of pH and of freeze inactivated LAAO.

5.1. Introduction

Apoptosis is a controlled and regulated form of cell death that plays an important role in the development and maintenance of higher organisms. It is defined by several morphological and biochemical hallmarks, like the exposure of phosphatidylserine to the

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