Fischer A1 and Dietrich DR1
1 Human and Environmental Toxicology, University of Konstanz, Konstanz, Germany
in preparation
3.1 Abstract
The unusual D-amino acid 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, abbreviated Adda, represents part of the toxic moiety of the cyclic cyanobacterial peptides microcystins (MCs) and nodularins (NODs), potent inhibitors of several serine/threonine-specific phophatases (PP). We examined the inhibitory potential of isolated Adda on recombinant PP1 and PP2A in comparison with one of its well-studied parental molecules MCLR.
Adda had no effect on the activity of neither PP1 nor PP2A in concentrations up to 55.7 µM, whereas MCLR concentration-dependently inhibited both phosphatases in the low nanomolecular range. These findings clearly demonstrate the relevance of the remaining structural units of the MC molecule for its biological activity.
Keywords: cyanobacteria, microcystin, Adda, protein phosphatase
3.2 Introduction
Microcystins (MCs) and the closely related nodularins (NODs) are toxic cyanobacterial oligopeptides that share three amino acids: D-methylaspartic acid, D-glutamic acid acid and the unusal D-amino acid 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda). Their general structures are cyclo(-D-Ala1-L-X2-D-erythro-β-methylAsp3-L-Z4-Adda5-D-Glu6
-N-methyl-Chapter III Inhibitory capacity of Adda on PP1 and 2A
dehydro-Ala7) and cyclo(-D-erythro-β-methylAsp1-L-Z2-Adda3-D-Glu4 -N-methyldehydrobutyrine5), respectively. In NODs Z either stands for L-arginine (nodularin-R) or L-valine (nodularin-V or motuporin) contrary to MCs, where X and Y are variable L-amino acid residues that mainly contribute to the high diversity of structural analogs (Botes et al., 1984; Botes et al., 1985; Rinehart et al., 1988; Rinehart et al., 1994) and are therefore used for the nomenclature of MCs (e.g. MCLR is a microcystin congener with L-leucine and L-arginine in position 2 and 7, respectively) (Carmichael et al., 1988a).
The toxicity of MCs and NODs predominantly stems from the very potent inhibition of several serine/threonine-specific protein phosphatases (PPs), especially PP1 and PP2A (MacKintosh, 1993; Honkanen et al., 1994; Runnegar et al., 1995a; Toivola et al., 1997; Hastie et al., 2005). The IC50s (concentration that inhibits 50% of the enzyme’s activity) of both toxins for PP1 and PP2A were found to be in the low nanomolar range. The inhibition relies on the non-covalent binding of the Adda-glutamate domain that represents the toxic moiety of both oligopeptides. In contrast to NODs most MC congeners are capable of forming an additional covalent, thus, irreversible binding via the N-methyldehydroalanine residue that is linked to a cysteine residue of the PP (MacKintosh et al., 1995; Runnegar et al., 1995a; Craig et al., 1996; Bagu et al., 1997; Hastie et al., 2005; Maynes et al., 2006). Structural alterations in Adda (e.g. isomerization of its diene) or D-glutamate (e.g. acetylation or esterification) have been shown to drastically decrease or abolish the inhibitory potential and hence toxicity of MCs and NODs indicating the importance of this moiety for bioactivity (Harada et al., 1990a; Harada et al., 1990b; Nishiwaki-Matsushima et al., 1991; Namikoshi et al., 1992; Stotts et al., 1993; Rinehart et al., 1994;
Harada, 1996b).
The purpose of this study was to examine the inhibitory potential of isolated Adda on recombinant PP1 and PP2A in comparison with MCLR, the most intensively studied MC congener, using a colorimetric PP inhibition assay.
3.3 Material & Methods
Chemicals and reagents
All chemicals were of the highest analytical grade commercially available. Adda was synthesized as previously reported (Fischer et al., 2001). Microcystin-LR (MCLR) was obtained from Alexis (Switzerland) and dissolved in 75% MeOH.
The concentrations of the stock and the working solutions were confirmed photometrically using the molar absorption coefficient of MCLR (39800 mol l-1 cm-1) published by Harada et al. (Harada et al., 1990a). This coefficient refers to MCLR dissolved in 100% MeOH, however, was shown to be applicable for 75%
MeOH as well (Meriluoto et al., 2004; Meriluoto and Spoof, 2005). Additionally, MCLR concentrations were confirmed by HPLC-DAD analysis according to Lawton et al. (Lawton et al., 1994). MCLR and Adda were sterilized by filtration using a 0.22 µm filter (Millex-GV, sterile; Millipore, Ireland).
Colorimetric protein phosphatase inhibition (cPPIA) assay with recombinant PP1 and 2A
Adda and MCLR were diluted serially (1:3) from 55.7 µM to 0.21 nM and 500 nM to 7.63 pM, rspectively, and analyzed at least four times in duplicates. The assay was carried out as described by Herersztyn and Nicholson (Heresztyn and Nicholson, 2001) using PP 1 (rabbit skeletal muscle, recombinant (E. coli), New England Biolabs, USA) and PP 2A (isolated from human red blood cells, Promega, USA) in final concentrations of 3 units/ml and 1.5 units/ml, respectively. P-nitrophenylphosphate from Acros Organics, USA, was used as substrate for the PPs.
Statistics
Colorimetric protein phosphatase inhibition assays were carried out at least four times in duplicates. The mean values of each duplicate yielded the values for calculation of the standard deviation (n>3). For calculation of the respective R2, EC50 values and statistical analysis GraphPad Prism 4.03 software was used.
Briefly, the respective mean values were logarithmized and normalized.
Resulting curves were fitted by nonlinear regression. An F-test (P<0.05) was employed for for the comparison of the EC50 values, hillslopes and curves.
Chapter III Inhibitory capacity of Adda on PP1 and 2A
3.4 Results
The results obtained from the cPPIAs clearly demonstrate that Adda neither reduced phosphatase activity in PP1 (Fig. 3.1 (A)) nor PP2A (Fig. 3.1 (B)) at the concentrations applied. On the contrary, MCLR concentration-dependently inhibited both phosphatases resulting in IC50 values of 1.3 nM (0.73 - 2.3 nM; R2
= 0.78) for PP1 (Fig. 3.1 (A)) and 0.29 nM (0.22 - 0.38 nM; R2 = 0.95) for PP2A (Fig. 3.1 (B)).
Fig 3.1: Inhibition of the catalytic subunit of recombinant PP1 (A) and PP2A (B) by Adda and MCLR. Values represent mean ± standard error of the mean of at least four independent analyses.
10-3 10-2 10-1 100 101 102 103 104 105 0
25 50 75 100 125
MC-LR ADDA B
[nM]
% phosphatase activity
10-3 10-2 10-1 100 101 102 103 104 105 0
25 50 75 100 125
MC-LR ADDA A
[nM]
% phosphatase activity
3.5 Discussion
The capability of MCs and NODs of potently inhibiting serine/threonine-specific PPs, especially PP1 and PP2A has led to numerous, in part lethal, poisonings of animals and humans (Kuiper-Goodman et al., 1999).
The i.p. LD50s (dose of toxin that kills 50% of the exposed animals) of both MCLR, the most prominent MC congener, and NOD were found to be 50 µg/kg mouse (Krishnamurthy et al., 1986; Eriksson et al., 1988). However, some MC and NOD congeners revealed to be less or even non-toxic as determined by mouse bioassays (i.p. administration) (Rinehart et al., 1994; Sivonen and Jones, 1999). Substitutions in the variable L-amino acids of MCs appeared to have only little effects on the toxicity except for MCRR whose LD50 was found to be about one order of magnitude higher than the corresponding MCLR congener (Krishnamurthy et al., 1986; Watanabe et al., 1988). On the other hand, modifications in the Adda residue such as isomerization of the diene from 6(E) to 6(Z) rendered MCs and NODs non-toxic (Harada et al., 1990a; Harada et al., 1990b; Namikoshi et al., 1994). Nishiwaki-Matsushima et al. (Nishiwaki-Matsushima et al., 1991) reported the inhibitory activity of 6(Z)-Adda-MCLR and -MCRR on PP2A to be 100-times weaker than of the corresponding maternal MCLR and MCRR. The authors concluded that the Adda moiety is not only crucial for toxicity, but for phosphatase inhibition as well.
We therefore examined the inhibitory activity of isolated Adda on PP1 and PP2A in comparison with MCLR. Our results clearly demonstrate that Adda neither reduced activity of PP1 nor PP2A even at 27.85 µM. By contrast, MCLR concentration-dependently inhibited both phosphatases in the low nanomolecular range as expected. This is supported by the findings of Harada et al. (Harada et al., 2004): isolated Adda showed no inhibitory effects on PP1 (at 10 µM) and revealed to be non-toxic in a mouse bioassay at concentrations up to 10 mg/kg body weight (i.p.) in contrast to the native toxin MCLR.
In conclusion, the inhibitory activity and hence toxicity of MCs and NODs relies not on Adda itself, but requires the remaining structural units of the maternal toxins. However, even though Adda is ineffective in PP inhibition, it remains to be elucidated wether it may induce any other effects in exposed organisms.
Chapter III Inhibitory capacity of Adda on PP1 and 2A
Acknowledgements
We would like to acknowledge the Arthur & Aenne Feindt Foundation (Hamburg, Germany) for kindly funding parts of this study.