Exploring the interaction of microcystin-LR with proteins and DNA L. Vela a,1 , E. Sevilla a,1 , C. Gonzalez b , M.T. Bes a , M.F. Fillat a , M.L. Peleato a, * a Departamento de Bioquimica y Biologia Molecular y Celular, Facultad de Ciencias, and BIFI, Universidad de Zaragoza, Pedro Cerbuna 12, 50009-Zaragoza, Spain b Instituto de Quimica-Fisica Rocasolano (CSIC), Madrid, Spain article info Article history: Received 1 April 2008 Accepted 7 July 2008 Available online 15 July 2008 Keywords: Microcystin-LR Cyanotoxin Hepatotoxin abstract The physiological role of microcystin-LR is still under discussion, and since binding of microcystin-LR to proteins different from their main cellular targets was described, we have performed experiments in order to explore this interaction. A non-specific interaction of microcystin-LR with a variety of soluble proteins in vitro is disrupted when using organic solvents such as methanol. The isoelectric point of pro- teins is not affected by their interaction with microcystin-LR, even though the presence of microcystin-LR alters the pool of peptides obtained by tryptic digestions. Under the conditions tested, microcystin-LR does not exhibit affinity for DNA. Although it is unlikely that the non-specific binding of microcystin- LR to proteins has a physiological meaning, one must be aware of the fact that determinations of the toxin extracted from any biological sample may be affected by the presence of proteins in the extracts. Conse- quently, we strongly recommend use organic solvents and to lyophilise the tissue samples to guarantee the accessibility of these organic solvents to microcystin-LR when performing experiments with tissue or cell extracts. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Microcystins are cyclic nonribosomal heptapeptides produced by some cyanobacterial strains that entail serious ecological problems and a health risk to humans and livestock for their toxicity. Microcystins are the most prevalent of cyanobacterial toxins and specifically inhibit eukaryotic protein phosphatases 1 and 2A (Runnegar et al., 1993). As consequence of phosphory- lation/dephosphorylation disfunction, one of the predominant ef- fects of microcystins on eukaryotic organisms is the alteration of the cytoskeleton, which is involved in key functions such as cell shape determination, cell movement, cell-to-cell recognition and cell division (Zurawell et al., 2005). Despite the large amount of published literature concerning such toxins, very little is known about the role of microcystins. These toxins are considered as intracellular secondary metabolites and must exhibit some sort of function for their structural complexity and the energetic cost involved in their biosynthesis. Microcystins reach high concen- trations inside the cyanobacteria, up to mM (Wiedner et al., 2003), and Juttner and Luthi (2008) proposed that such high concentration of microcystin is only possible when bound to proteins. Immunogold localization of microcystins in the producing cells showed that microcystin-LR was mostly associated with thylakoids, and to a lesser extend located in the nucleoplasmic region (Young et al., 2005). In contrast, other authors found that microcystin-LR was hardly detected within the thylakoid membranes of Microcystis aeruginosa, where the photosystems I and II are located and the pho- tosynthetic activity takes place, while polyphosphate bodies and carboxysomes were found to be microcystin-rich regions (Young et al., 2005; Gerbersdorf, 2006). Since polyphosphate bodies have been reported to trap metals such as zinc (Andrade et al., 2004), Young et al. (2005) proposed that the presence of microcystin-LR may well be involved in a metal detoxification mechanism inside the cell. In fact, Utkilen and Gjolme (1995) suggested a putative role for microcystin-LR as an intracellular chelating molecule. Also, microcystins have been shown to induce DNA damage in vitro and in vivo, and its genotoxicity seems to be mediated by reactive oxygen species (Zegura et al., 2004; Gaudin et al., 2008). Sev- eral host defense peptides, such as indolicidin, forms covalent links with DNA (Marchand et al., 2006), but no information is available concerning microcystins and a possible direct interaction with the nucleic acid. Binding of microcystin-LR to proteins different from their main cellular target was described (Dittmann et al., 2006; Juttner and Luthi, 2008), opening a new perspective in the study of the physi- ological status of the toxin. In this paper, we present a set of exper- iments designed to clarify the nature and significance of the interaction of microcystin-LR with proteins. 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.07.008 * Corresponding author. Tel.: +34 976 762479; fax: +34 976 762123. E-mail address: mpeleato@unizar.es (M.L. Peleato). 1 These authors contributed equally to this work. Toxicology in Vitro 22 (2008) 1714–1718 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit