Abstract

To elucidate Ca2+-mediated regulation of aflatoxin production, the status of Ca2+/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [γ-32P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca2+/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.

Introduction

The potent carcinogenic, teratogenic, mutagenic and immunosuppressive effects caused by aflatoxins, produced by toxigenic strains of Aspergillus flavus and A. parasiticus, are well-recognized [1]. Despite several attempts made to combat aflatoxin production, basic knowledge of the regulatory mechanisms leading to their production is still limited. Recent studies from our laboratory have indicated the requirement of Ca2+[2] and the importance of Ca2+/calmodulin-dependent phosphorylation/dephosphorylation of proteins for aflatoxin production by A. parasiticus [3]. In order to further elucidate the nature of calmodulin-mediated regulatory events during aflatoxin production, we conducted a comparative study between the toxigenic and non-toxigenic strains of A. parasiticus involving (i) quantification of calmodulin (CaM) and calcineurin (CaN; calmodulin-dependent protein phosphatase) and (ii) assay of calmodulin-dependent protein kinase (CaM kinase II) and CaN activities during aflatoxin production. The obtained results indicate that calmodulin-dependent protein phosphorylation/dephosphorylation of target proteins may be important for the initiation of aflatoxin production in A. parasiticus.

Materials and methods

Organism and culture conditions

The toxigenic strain of A. parasiticus (NRRL 2999; obtained from the US Department of Agriculture, Northern Regional Research Center, Peoria, IL, USA) as well as the non-toxigenic strain of A. parasiticus (SRRC 255; obtained by repeated culturing of NRRL 2999; from Dr. Maren A. Klich, US Department of Agriculture, Southern Regional Research Center, New Orleans, LA, USA) were maintained on potato dextrose agar slants and cultured on chemically defined media as described earlier [3].

Identification of calcineurin and determination of calmodulin and calcineurin contents

Cytoplasmic extracts were prepared by homogenizing the mycelia in a buffer containing 50 mM Tris-HCl, pH 7.8, 3 mM MgSO4, 1 mM EGTA, 0.5 mM dithiothreitol, 0.02% NaN3 and 0.1 mM phenylmethylsulfonyl fluoride. The homogenates were clarified by centrifugation at 15 000×g for 30 min at 4°C and the obtained supernatants were boiled at 100°C for 5 min prior to determining the calmodulin contents in them. Protein content in the extracts was determined by Bradford's method [4].

Calcineurin was identified in the total calmodulin binding proteins of the cytoplasmic extracts, which were obtained by affinity purification on a CaM-agarose column [5]. The proteins were resolved by polyacrylamide gel electrophoresis under non-denaturing conditions on 10% gels and transferred to PVDF membranes at 200 mA for 1 h using Transblot apparatus (Hoeffer, USA). The immunoreactive CaN was detected as per Goto et al. [6] employing monoclonal anti-calcineurin α-antibodies (Sigma, USA; diluted 1:10 000) at a concentration of 580 ng ml−1.

The cytoplasmic contents of CaM or CaN were determined by a competitive ELISA method described earlier [7] with minor modifications. Essentially, samples (20–50 μg protein) were mixed with the respective monoclonal antibodies (specific to either calmodulin or calcineurin; obtained from Sigma, USA) and transferred to 96-well polystyrene microtiter plates (Microlon-600, Greiner Labortechnix, Germany) that were previously coated either with bovine brain CaM (Sigma, USA; 10 ng) or with bovine brain CaN (Sigma, USA; 25 ng) in 50 mM carbonate-bicarbonate buffer (pH 9.6). Non-specific protein binding sites were blocked on the wells with 200 μl of phosphate-buffered saline (PBS) containing 2% bovine albumin for 1 h prior to the addition of samples. After incubation (2 h at 37°C for CaM; overnight at 4°C for CaN), the wells were washed with PBS containing 0.05% Tween 20 and incubated for a further period of 90 min with 50 μl of goat anti-mouse IgG labeled with horseradish peroxidase (1:2000 dilution). The wells were finally washed with PBS containing Tween 20 and incubated for 1 h at 37°C with 100 μl of substrate (4 mg of o-phenylenediamine mixed with 10 μl of H2O2 in 10 ml of 1.5 M citrate-phosphate buffer, pH 5.0). Reactions were terminated by adding 8 N H2SO4 (50 μl) and the color developed was read at 492 nm using an ELISA reader (Spectra II, SLT Instruments, Austria). Suitable calibration curves were obtained by employing bovine brain CaM (1–25 ng) or CaN (1–500 ng) as reference standards.

Assay of calmodulin-dependent protein kinase and calcineurin

The activity of multifunctional CaM-dependent protein kinase (CaM kinase II) was assayed in the cytoplasmic extracts as described earlier [8] measuring the incorporation of [γ-32P]ATP into a synthetic substrate, autocamtide II (KKALRRQETVDAL). The effect of total aflatoxins (0–36 μM) on CaM kinase activity was determined by including aflatoxins (B1, G1, B2 and G2 in the ratio of 66:27:5:2) in the reaction mixture.

Calcineurin activity was assayed according to Wang and Pallen [9] with minor modifications, determining the CaM-dependent protein phosphatase activity in the presence or absence of 150 μM trifluoperazine [7].

Protein phosphorylation and autoradiography

Cytoplasmic proteins that undergo phosphorylation were detected essentially as described earlier [10]. The samples (50 μg protein) were incubated in a reaction mixture containing PIPES (50 mM), MgCl2 (10 mM), CaCl2 (1.2 mM) and [γ-32P]ATP (5 μM; 5 Ci mmol−1; BRIT, India) for a period of 5 min at 28°C in a total volume of 50 μl. After termination of the reaction by addition of SDS sample buffer and boiling, the proteins were resolved by SDS-PAGE on 10% gels and transferred on to PVDF membranes. The membranes were exposed to Kodak X-OMAT AR films overnight and subjected to autoradiography.

Results and discussion

Consequent to its identification in filamentous fungi [11], CaM has been shown to promote important events involved in growth and protease production [12], conidial germination [10], cell wall synthesis [8] as well as dimorphism [13] in fungi. However, there is no conclusive information on its role in fungal secondary metabolism even though earlier studies from our laboratory, employing aflatoxin production by A. parasiticus as the model system, indicated that Ca2+ and CaM might regulate events involved in the initiation of fungal secondary metabolism [2,3]. In continuation of these observations, the present study was conducted to elucidate the role of CaM-dependent protein phosphorylation and dephosphorylation in aflatoxin biosynthesis employing toxigenic and non-toxigenic strains of A. parasiticus. Evidence was obtained to suggest that CaM-dependent phosphorylation of CaN by CaM kinase regulates aflatoxin production and dephosphorylation of target proteins by CaN leads to sustained aflatoxin production by the toxigenic strain. In addition, inhibition of CaM kinase by aflatoxins seems to ensure continued activation of CaN, which is now known to be active in its dephosphorylated form [14]. It was possible to obtain these findings since the experimental system comprised a toxigenic strain of A. parasiticus and a non-toxigenic strain directly obtained from it.

Importance of protein phosphorylation/dephosphorylation in aflatoxin production

It has been realized over the past two decades that phosphorylation and dephosphorylation of specifically targeted proteins may be a final common pathway for mediating Ca2+-dependent regulatory processes involved in cell growth and proliferation [15]. Since the release of aflatoxins into the medium has earlier been noted to start at 36 h and reach a maximum by 72 h [3], preformed mycelia of toxigenic and non-toxigenic strains of similar age were employed in the present study. Several differences could be noted between toxigenic and non-toxigenic strains with regard to protein phosphorylation (Fig. 1). It was noted that phosphorylation was limited to that of a 36-kDa protein at 48 h in the toxigenic strain (Fig. 1, lane A). In comparison, five other proteins (MW∼95, 86, 70, 57, 45 kDa) were phosphorylated, in addition to the 36-kDa protein, in the non-toxigenic strain at this time period (Fig. 1, lane B). However, complete dephosphorylation of proteins was evident at 72 h (associated with maximal production of aflatoxins) in the toxigenic strain (Fig. 1, lane C) but not in the non-toxigenic strain (Fig. 1, lane D).

1

Protein phosphorylation in toxigenic and non-toxigenic strains of A. parasiticus. Cytoplasmic proteins were subjected to in vitro phosphorylation as described in Section 2 and detected by SDS-PAGE on 10% gels followed by autoradiography. Lane A: 48 h toxigenic; B: 48 h non-toxigenic; C: 72 h toxigenic; D: 72 h non-toxigenic.

1

Protein phosphorylation in toxigenic and non-toxigenic strains of A. parasiticus. Cytoplasmic proteins were subjected to in vitro phosphorylation as described in Section 2 and detected by SDS-PAGE on 10% gels followed by autoradiography. Lane A: 48 h toxigenic; B: 48 h non-toxigenic; C: 72 h toxigenic; D: 72 h non-toxigenic.

Calmodulin and calcineurin contents during aflatoxin production

The relevance of Ca2+- and CaM-dependent protein phosphorylation with regard to aflatoxin production could be established earlier in view of the demonstrated ability of trifluoperazine (an anticalmodulin agent) to inhibit such production [3]. During the course of the present study, the cytoplasmic contents of CaM as well as CaN were quantitated in the non-toxigenic and toxigenic strains, both at 48 h and at 72 h. Data presented in Table 1 show that the toxigenic strain was endowed with greater amounts of CaM in comparison to the non-toxigenic strain. Mycelia of the toxigenic strain, harvested at 48 h and 72 h of growth, possessed 2.8- and 3.7-fold greater amounts of CaM respectively. In addition, the presence of CaN was detected in both the strains by immunological cross-reactivity with monoclonal antibodies specific to the α-subunit of calcineurin (Fig. 2). While CaN has been reported in Saccharomyces cerevisiae [16], Neurospora crassa [17], Aspergillus niger and Aspergillus nidulans [18], this is the first report in A. parasiticus. In addition, the cytoplasmic contents of CaN (similar to those of CaM) were higher in the toxigenic strain when compared to the non-toxigenic strain. Significant increase (9-fold) in CaN levels could be observed at 72 h in the toxigenic strain when compared to the non-toxigenic strain (Table 1). The enhanced levels of both CaM and CaN at phases corresponding to maximal aflatoxin production signify their requirement for processes leading to aflatoxin biosynthesis.

1

Calmodulin and calcineurin contents and activity of calmodulin-dependent protein kinase and calcineurin during aflatoxin production by A. parasiticus

Strain Content (μg mg−1 protein) Specific activity (units) 
 CaM CaN CaM kinasea Calcineurinb 
Toxigenic strain 
48 h 1.17±0.02 17.62±1.4 0.40±0.07 0.27±0.03 
72 h 1.50±0.31 24.00±3.8 0.32±0.06 1.81±0.10 
Non-toxigenic strain 
48 h 0.43±0.03 6.15±0.45 1.18±0.27 0.05±0.005 
72 h 0.39±0.0014 2.70±0.65 2.0±0.2 0.07±0.02 
The cytoplasmic extracts from cultures grown for 48 h and 72 h were assayed for calmodulin kinase and calcineurin activity and contents of calmodulin and calcineurin were determined as described in Section 2
Strain Content (μg mg−1 protein) Specific activity (units) 
 CaM CaN CaM kinasea Calcineurinb 
Toxigenic strain 
48 h 1.17±0.02 17.62±1.4 0.40±0.07 0.27±0.03 
72 h 1.50±0.31 24.00±3.8 0.32±0.06 1.81±0.10 
Non-toxigenic strain 
48 h 0.43±0.03 6.15±0.45 1.18±0.27 0.05±0.005 
72 h 0.39±0.0014 2.70±0.65 2.0±0.2 0.07±0.02 
The cytoplasmic extracts from cultures grown for 48 h and 72 h were assayed for calmodulin kinase and calcineurin activity and contents of calmodulin and calcineurin were determined as described in Section 2

aOne enzyme unit denotes the incorporation of 1×106 dpm of [γ-32P]ATP into the synthetic substrate per mg protein per minute.

bOne enzyme unit denotes μmol of p-nitrophenol released from p-nitrophenyl phosphate per mg protein per minute.

2

Immunological cross-reactivity of calcineurin from A. parasiticus. Calmodulin binding proteins isolated from cytoplasmic extracts of the organism were obtained by affinity purification on calmodulin-agarose columns, subjected to electrophoresis on 10% polyacrylamide gels, transferred onto PVDF membranes and probed with monoclonal antibodies to the α-subunit of calcineurin. Lane A: standard calcineurin; B: sample.

2

Immunological cross-reactivity of calcineurin from A. parasiticus. Calmodulin binding proteins isolated from cytoplasmic extracts of the organism were obtained by affinity purification on calmodulin-agarose columns, subjected to electrophoresis on 10% polyacrylamide gels, transferred onto PVDF membranes and probed with monoclonal antibodies to the α-subunit of calcineurin. Lane A: standard calcineurin; B: sample.

Activities of calmodulin-dependent protein kinase and calcineurin in relation to aflatoxin production

The activities of CaM kinase and CaN were monitored in toxigenic and non-toxigenic strains of A. parasiticus at different growth periods. Results depicted in Table 1 show that the activity of CaM kinase was higher (3-fold at 48 h and 6-fold at 72 h) in the non-toxigenic strain in comparison to the toxigenic strain. In contrast, the activity of CaN was greater in the toxigenic strain in relation to the non-toxigenic strain. In addition, the toxigenic strain (as compared to the non-toxigenic strain) was endowed with 5-fold enhanced activity of CaN at 48 h of growth and this increase was further enhanced (26-fold) at 72 h corresponding to maximal production of aflatoxin.

Inhibition of calmodulin-dependent protein kinase by aflatoxin with possible implications in aflatoxin production

Even though the above results suggested that aflatoxin production is concomitant with continuous dephosphorylation of proteins, it was of interest to examine the possible inhibitory effect of aflatoxin on Ca2+/CaM-dependent protein kinase in view of recent reports on the inhibitory effects of aflatoxin B1 on cyclic nucleotide phosphodiesterase activity [19]. Thus, the effect of a mixture of aflatoxins (obtained from culture medium) on the activity of Ca2+/CaM-dependent protein kinase was examined under in vitro conditions. Results obtained in this regard showed that aflatoxins could inhibit the enzyme activity in a dose-dependent manner (Fig. 3). It would, however, be important to establish the susceptibility of CaM kinase to individual aflatoxins and to establish whether the inhibition is directly on the active centers of the enzyme or due to their ability to limit the availability of calmodulin, which is required for the enzyme activity. Interestingly, recent reports indicate that aflatoxin B1 interacts with activated Ca2+-ATPase by binding specifically to the tryptophan 1107 residue in the calmodulin domain of the enzyme resulting in inhibition of the enzyme [20]. Even though the nature of inhibition brought about by aflatoxins on Ca2+/CaM-dependent protein kinase was not completely elucidated in the present study, the combined results permit a hypothesis on the regulation of aflatoxin production by Ca2+/CaM-dependent protein phosphorylation and dephosphorylation. Since phosphorylation of CaN by CaM kinase is known to inactivate its phosphatase activity [14], inhibition of the protein kinase by aflatoxin would ensure that CaN remains in its active state to bring about dephosphorylation of target proteins which regulate aflatoxin production. We are pursuing the above hypothesis to identify the putative targets, which are susceptible to CaM-dependent phosphorylation and dephosphorylation.

3

Dose-dependent inhibition of CaM kinase by aflatoxin. Calmodulin-dependent protein kinase activity was assayed in the absence or presence of varying concentrations (0–36 μM) of aflatoxin as described in Section 2. *One enzyme unit denotes the incorporation of 1×106 dpm of [γ-32P]ATP into the synthetic substrate per mg protein per minute.

3

Dose-dependent inhibition of CaM kinase by aflatoxin. Calmodulin-dependent protein kinase activity was assayed in the absence or presence of varying concentrations (0–36 μM) of aflatoxin as described in Section 2. *One enzyme unit denotes the incorporation of 1×106 dpm of [γ-32P]ATP into the synthetic substrate per mg protein per minute.

Acknowledgements

The authors thank Dr. B. Sashidhar Rao for technical discussions. T.J. and J.P.R. acknowledge Senior Research Fellowships awarded by the University Grants Commission to them.

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