Molecular mechanisms underlying the interaction of motuporin and microcystins with type-1 and type-2A protein phosphatases

Abstract

Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA, and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase-toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and nodularin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.

Developmental Biology of Neoplastic Growth

“…Although motuporin contains valine instead of the arginine in nodularin, these compounds are equipotent inhibitors of PP1 and PP2A (Craig et al 1996). The results are consistent with the report that the arginine residue in microcystin-LR does not significantly contribute to biological activity, based on a comparison of enzyme inhibition and receptor binding activity ).…”

“…Sponges of the order Haplosclerida often contain 3-alkylpyridinium salts (Andersen et al 1996;Sep i 2000), some of which were reported to be toxic to mammals. The first example of this class is halitoxin (29) isolated from Haliclona spp.…”

Quantitatively evaluating detoxification of the hepatotoxic microcystin-LR through the glutathione (GSH) pathway in SD rats

“…Second, part of the MC-LR may covalently bind with protein phosphatases in liver and then released gradually over time. Covalent binding of MCs with protein phosphatases reduces extractability of microcystins (Craig et al 1996), which could result in an underestimation of the amount of toxin really present into the liver (Malbrouck et al 2004). Although the mechanism of sustainedrelease behavior of MC-LR is not well studied so far, the binding form and subsequent release may account for the fluctuation of MC-LR detected in the rat liver during the experimental process.…”

Bioaccumulation of microcystins in two freshwater gastropods from a cyanobacteria-bloom plateau lake, Lake Dianchi

“…Indeed, MCs can covalently bind to various proteins (Craig et al, 1996;Gehringer et al, 2004). However, our investigation of the accumulation of MCs in these two gastropods used ELISA methods, which detects only free forms of MCs (Hastie et al, 2005;Maynes et al, 2006).…”