SEPTEMBER 2006 VOLUME 19, NUMBER 9 © Copyright 2006 by the American Chemical Society
Letters to the Editor Molecular Mimicry as a Mechanism for the Uptake of Cysteine S-Conjugates of Methylmercury and Inorganic Mercury To the Editor: The authors of the article entitled “Molecular Mimicry in Mercury Toxicology” compared the structures of methionine and cystine with cysteine (Cys) S-conjugates of methylmercury and inorganic mercury, respectively, and suggested that the uptake of these mercuric complexes into target cells does not involve molecular mimicry. While the findings of this study are interesting, the authors do not provide sufficient evidence to disprove the theory that molecular mimicry is involved in the transport of these mercuric species by membrane transporters. The aforementioned manuscript focuses primarily on comparing the structural features of methionine and the Cys S-conjugate of methylmercury; yet, surprisingly, the authors have failed to acknowledge the initial work by Clarkson and Aschner (1-4), who were the first to implicate molecular mimicry in the cellular uptake of this conjugate via membrane transporters. In addition, the authors have overlooked the important review by Clarkson entitled “Molecular and Ionic Mimicry of Toxic Metals” (5). This review, which was the first of its kind published after the review by Wetterhahn-Jennette (6), was the first to suggest the Cys S-conjugate of methylmercury mimics the amino acid, methionine, at the site of a membrane transporter as a means to gain access to the intracellular compartment of cells. Our work, on the other hand, shows that Cys S-conjugates of inorganic mercury and the amino acid cystine are both transported by the amino acid transporter, system b0,+, via a mechanism whereby this mercuric conjugate acts as a structural and/or functional homologue of cystine (7, 8). The authors of the above study utilized mass spectrometry and computational chemistry to generate their versions of Cys S-conjugates of methylmercury and inorganic mercury. In the discussion of these models, the authors were quite critical of space-filled models that were published in our recent reviews (6, 9). We agree with the authors’ conclusion that there are
obvious structural differences between the Cys S-conjugates of methylmercury and inorganic mercury and the methionine and cystine, respectively. However, we never intended, nor do we intend, to imply that these molecules exist only in the forms proposed in our reviews. Our models are simplified threedimensional diagrammatic or cartoon illustrations that were meant to show a linearized form of the amino acids bonded to a mercuric ion. These models were provided as a means to visually illustrate the similarities between certain amino acids and the corresponding mercuric conjugates. The three-dimensional space-filled structures were produced by converting twodimensional stick drawings (similar to those drawn by the authors in Figure 1) using Chem-Draw. As our intent was to show simplified versions of these complexes, we did not utilize optimization procedures. It is important to note that the authors carried out their structural analyses of mercuric complexes in a simple aqueous solution containing 50% methanol. This solution in no way represents the complex milieu of molecular species, including proteins, amino acids, and other molecules and metals, found in intracellular and extracellular compartments within humans and other mammals. Because of molecular forces, including van der Walls forces and intermolecular hydrogen bonding, these mercuric complexes are not static molecules in vivo but, rather, are undergoing dynamic changes. Because of these factors, it is difficult to make definitive conclusions regarding the in vivo conformation of a particular molecule. In addition, the authors state that similarities between the LR region of cysteine within the mercuric complex and that of other amino acids may account for the uptake of metal complexes into cells. If this were the mechanism for substrate recognition, transporters such as system b0,+, which recognizes cysteine and cystine as distinct substrates, would likely not be able to distinguish between these two amino acids. Given this, it seems unlikely that the ability of a transport protein to recognize a substrate is based solely on the LR region of a molecule. The precise nature by which Cys S-conjugates of methylmercury and inorganic mercury are recognized by membrane transport proteins has not been defined. Thus, one cannot determine the
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exact structural conformation or mechanism involved in the docking and transport of these mercuric species. Moreover, it appears that the authors have a narrow definition of molecular mimicry that is based on near identical structural matches of metal complexes and endogenous compounds. The authors state that “the structural similarities between metal compounds and natural products are insufficient to support a mechanism based on molecular mimicry.” It should be pointed out that Clarkson has defined molecular mimicry as the process whereby “a toxic metal forms a complex with an endogenous ligand such that the resulting compound mimics the behavior of a normal substrate” (5). In our recent review, which was miscited in the Chemical Research in Toxicology paper, we define molecular mimicry as the “phenomenon whereby the bonding of metal ions to nucleophilic groups on certain biomolecules results in the formation of organo-metal complexes that can behave or serve as a structural and/or functional homologue of other endogenous biomolecules or the molecule to which the metal ion has bonded” (8). These two definitions of molecular mimicry do not state that the mercuric complexes are exact structural matches to the endogenous substrates. Moreover, these definitions state that metal complexes can be functional or behavioral mimics of certain endogenous compounds. There is a great deal of data supporting molecular mimicry. The authors of the Chemical Research in Toxicology paper appear to discount much of these previous data yet do not offer alternative explanations for the mechanisms by which mercuric complexes are taken up by cells. To date, the most logical explanation for the entry of S-conjugates of mercury into cells is that these complexes are similar enough to certain amino acids and organic anions to serve as substrates for amino acid and organic anion transporters, respectively. Indeed, studies in multiple biological systems provide support for the theory of molecular mimicry. Data from Xenopus laeVis oocytes (10), isolated perfused proximal tubules (11, 12), membrane vesicles (13), in vivo studies in rats (14, 15), and in vitro studies in nontransfected (16, 17) and stably transfected (7, 18) cultured cells indicate that Cys S-conjugates of methylmercury and inorganic mercury are taken up by amino acid or organic anion transporters, presumably via molecular mimicry. Until it is proven that the binding sites involved in the recognition of amino acids by carrier proteins are not involved in the binding and transport of mercuric complexes, one cannot conclude that molecular mimicry does not exist as a mechanism for the transport of these complexes.
References (1) Ashner, M. (1989) Brain, kidney and liver 203Hg-methylmercury uptake in the rat: Relationship to the neutral amino acid carrier. Pharmacol. Toxicol. 65, 17-20. (2) Aschner, M., and Clarkson, T. W. (1988) Uptake of methylmercury in the rat brain: Effects of amino acids. Brain Res. 462, 31-39. (3) Aschner, M., and Clarkson, T. W. (1989) Methyl mercury uptake across the bovine brain capillary endothelial cells in Vitro: The role of amino acids. Pharmacol. Toxicol. 64, 293-299. (4) Aschner, M., Eberle, N. B., Goderie, S., and Kimelberg, H. K. (1990) Methylmercury uptake in rat primary astrocyte cultures: The role of the neutral amino acid transport system. Brain Res. 521, 221-228. (5) Clarkson, T. W. (1993) Molecular and ionic mimicry of toxic metals. Ann. ReV. Biochem. 32, 545-571. (6) Wetterhahn-Jennette, K. (1981) The role of metals in carcinogenesis: Biochemistry and Metabolism. EnViron. Health Perspect. 40, 233252. (7) Bridges, C. C., Bauch, C., Verrey, F., and Zalups, R. K. (2004) Mercuric conjugates of cysteine are transported by the amino acid transporter system b0,+: Implications of molecular mimicry. J. Am. Soc. Nephrol. 15, 663-673.
Letters to the Editor (8) Bridges, C. C., and Zalups, R. K. (2005) Molecular and ionic mimicry and the transport of toxic metals. Toxicol. Appl. Pharmacol. 204, 274308. (9) Zalups, R. K. (2000) Molecular interactions with mercury in the kidney. Pharmacol. ReV. 52, 113-143. (10) Simmons-Willis, T. A., Koh, A. S., Clarkson, T. W., and Ballatori, N. (2002) Transport of a neurotoxicant by molecular mimicry: The methylmercury-L-cysteine complex is a substrate for human L-type large neutral amino acid transporter (LAT) 1 and LAT2. Biochem. J. 367, 239-246. (11) Cannon, V. T., Barfuss, D. W., and Zalups, R. K. (2000) Molecular homology and the luminal transport of Hg2+ in the renal proximal tubule. J. Am. Soc. Nephrol. 11, 394-402. (12) Cannon, V. T., Barfuss, D. W., and Zalups, R. K. (2001) Amino acid transporters involved in luminal transport of mercuric conjugates of cysteine in rabbit proximal tubule. J. Pharmacol. Exp. Ther. 298, 780789. (13) Zalups, R. K., and Lash, L. H. (1997) Binding of mercury in renal brush-border and basolateral membrane-vesicles: Implication of a cysteine conjugate of mercury involved in the luminal uptake of inorganic mercury. Biochem. Pharmacol. 53, 1889-1900. (14) Zalups, R. K. (1995) Organic anion transport and action of gammaglutamyl transpeptidase in kidney linked mechanistically to renal tubular uptake of inorganic mercury. Toxicol. Appl. Pharmacol. 132, 289-298. (15) Zalups, R. K, and Barfuss, D. W. (1995) Pretreatment with paminohippurate inhibits the renal uptake and accumulation of injected inorganic mercury in the rat. Toxicology 103, 23-35. (16) Kerper, L. E., Ballatori, N., and Clarkson, T. W. (1992) Methylmercury transport across the blood-brain barrier by an amino acid carrier. Am. J. Physiol. 262, R761-R765. (17) Mokrzan, E. M., Kerper, L. E., Ballatori, N., and Clarkson, T. W. (1995) Methylmercury-thiol uptake into cultured brain capillary endothelial cells on amino acid system L. J. Pharmacol. Exp. Ther. 272, 1277-1284. (18) Zalups, R. K., Aslamkhan, A. G., and Ahmad, S. (2004) Human organic anion transporter 1 mediated cellular uptake of cysteine-Sconjugates of inorganic mercury. Mol. Pharmacol. 54, 353-363.
Christy C. Bridges Rudolfs K. Zalups Mercer University School of Medicine Division of Basic Medical Sciences Macon, Georgia 31207 TX060158I
More on Molecular Mimicry in Mercury Toxicology To the Editor: Bridges and Zalups write concerning our recent paper entitled “Molecular Mimicry in Mercury Toxicity” published in Chemical Research in Toxicology (1). They have several criticisms of our paper, which we have divided into three categories: (i) that we have discounted or ignored previous biological studies by others; (ii) that we have ignored alternative definitions of molecular mimicry, have studied what amounts to “static molecules”, and do not account for the flexibility, which might be induced by interactions in vivo; and (iii) that our experiments were conducted under nonphysiological conditions. Bridges and Zalups complain that we did not cite early work by Clarkson and co-workers and imply that we have ignored important contributions by this group (2). This was absolutely not our intention, and indeed, we did cite more recent studies by these workers (3). We (as Bridges and Zalups suggest) also do not dispute or discount the experimental evidence for uptake of mercury species by cellular transporters. These studies report the phenomenon of uptake by transporters, they provide essential information, and we do not minimize or discount their impor-
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tance in any way. However, they do not directly determine whether the mechanism of uptake is by molecular mimicry, irrespective of how one defines this phenomenon. In fact, these studies provided the motive for our study as reported in our recent Chemical Research in Toxicology paper (1). As Bridges and Zalups point out, we did give an incorrect title for one of their papers (4); this was unintentional, and for this, we apologize. The different definitions of molecular mimicry that Bridges and Zalups quote stress the importance of interactions with other biomolecules. Our study sought to use a combination of spectroscopic methods to understand the solution structures of the mercury species in question and then to apply computational chemistry to examine the possibility of molecular mimicry in a quantitative and objective manner. Our analysis does not require an “exact structural match”, as Bridges and Zalups suggest, but instead, it examines the possible molecular similarities between molecules by searching among allowed conformations (scarcely “static molecules”). These conformations include any that might be induced by binding to biomolecules, and thus, our conclusions clearly apply to both of the alternative definitions of molecular mimicry that Bridges and Zalups cite; irrespective of how one defines molecular mimicry, there must be at least some molecular similarities under some conditions for the term to be applied. In our paper, we addressed the question of how to judge molecular similarity and adopted a method that combines both steric and electrostatic effects. This method was applied to objectively investigate molecular mimicry for two different mercury species, L-cysteinato(methyl)mercury(II) (hereafter called methylmercury cysteine) and mercury(II) bis-Lcysteinate. Methylmercury cysteine was compared with the proposed target of its molecular mimicry methionine and the other LAT1 amino acids, and mercury bis-cysteinate was compared with cystine. The results of our analyses clearly show that there is no a priori chemical basis for the molecular mimicries proposed for these compounds. We do not dispute that they are transported by amino acid transporters, but rather, we dispute that it is specific molecular mimicry that is the underlying mechanism (i.e., we state that methylmercury cysteine is not a probable molecular mimic of methionine). Perhaps underpinning the difficulties that Bridges and Zalups have with our paper is our (mildly stated) criticism of structures that Zalups and co-workers have proposed in various reviews and papers in recent years (4-6). The structures in question clearly show three-dimensional molecular geometries that are incorrect according to well-established chemical understanding. For example, in a recent review (4), not only are a number of mercury compounds shown with incorrect geometries, but the simple tetrahedral anions sulfate, selenate, and molybdate are shown as having square-based pyramidal geometry. The software that Zalups and co-workers employed and any unstated intent are quite immaterial; the structures they show are not the structures of sulfate, selenate, and molybdate. Bridges and Zalups state in their letter that they never intended to “imply that these molecules exist only in the forms proposed”. This statement indicates that Bridges and Zalups believe that these species can at least potentially exist in the forms shown (but not only in these forms). The energetic penalty involved in varying bond lengths and bond angles is far greater than that associated with varying torsion or dihedral angles, and in a conformational analysis, only the latter are typically considered.
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Figure 1. Experimental Hg LIII EXAFS Fourier transforms of methanol-water solutions and buffered aqueous solution (50 mM HEPES, pH 7.5) containing 20% glycerol to protect against ice crystal diffraction artifacts. Data were measured on frozen solutions as previously described (1). Fourier transforms were calculated using a k range of 1.0-14.0 Å-1 and are phase-corrected for Hg-S backscattering.
We calculate that the energy required to convert tetrahedral sulfate to the square-based pyramidal geometry shown by Bridges and Zalups is a staggering 10 eV (an energy sufficient to easily break almost any covalent bond), and the energetic penalty associated with deforming an essentially linear mercury conformation to the bent conformation frequently shown in the papers of Zalups and co-workers is less, but still large, at around 1.7 eV. This certainly is very much more than the energy that can be exerted by hydrogen bonding or van der Waals forces, as Bridges and Zalups suggest. Thus, as we discussed in our Chemical Research in Toxicology paper (1), two-coordinate mercury species adopt an essentially linear geometry, and this will not significantly vary in solution or under physiological conditions. To be blunt, while the papers containing them contain elegant experiments and important results, the structures shown by Zalups and co-workers are wrong. Bridges and Zalups also comment on the fact that our “structural analyses” were conducted in a methanol-water mixture. They correctly point out that this is a poor representation of a cell. These are quite standard conditions for mass spectrometry, and we adopted the same conditions for our mercury LIII X-ray absorption spectroscopy (XAS) measurements in order to be consistent. Figure 1 compares the extended X-ray absorption fine structure (EXAFS) Fourier transform of a frozen buffered aqueous solution of methylmercury cysteine and the frozen methanol/water solution data reported in our paper (1). The obvious similarity of the two data sets conclusively shows that the basic structure of the mercury site does not fundamentally change between the two solutions, although small changes corresponding to long distances clearly indicate subtle differences, probably due to solvent effects. It is true that neither of these solutions accurately represents living tissue (although the latter is at least at physiological pH). We note that unlike other spectroscopies, XAS has no sample limitations and is capable of in situ and even in vivo measurements (7), but we prefer to report these data in future publications rather than reproduce them here. To summarize, we believe that the complaints leveled by Bridges and Zalups in their letter are substantially without basis. Having said this, it is true certainly that our methods could be enhanced; for example, the Hodgkin indices (8) could be improved upon, perhaps by constrained density functional theory refinements of each conformation being tested. Nevertheless, we believe our calculations to be substantially sound, and until
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any substantial flaws are discovered, we stand by them and our conclusions.
Letters to the Editor
Ruth E. Hoffmeyer Satya P. Singh Christian J. Doonan
References (19) Hoffmeyer, R. E., Singh, S. P., Doonan, C. J., Ross, A. R. S., Hughs, R. J., Pickering, I. J., and George, G. N. (2006) Molecular mimicry in mercury toxicology. Chem. Res. Toxicol. 19, 753-759. (20) Clarkson, T. W. (1993) Molecular and ionic mimicry of toxic metals. Annu. ReV. Pharmacol. Toxicol. 32, 545-571. (21) Simmons-Willis, T. A., Koh, A. S., Clarkson, T. W., and Ballatori, N. (2002) Transport of a neurotoxicant by molecular mimicry: The methylmercury-L-cysteine complex is a substrate for human L-type large neutral amino acid transporter (LAT) 1 and LAT2. Biochem. J. 367, 239-246. (22) Bridges, C. C., and Zalups, R. K. (2005) Moleculer and ionic mimicry and the transport of toxic metals. Toxicol. Appl. Pharmacol. 204, 274308. (23) Zalups, R. K. (2000) Molecular interactions with mercury in the kidney. Pharmacol. ReV. 52, 113-143. (24) Zalups, R. K., and Ahmad, S. (2004) Homocysteine and the renal epithelial transport and toxicity of inorganic mercury: Role of basolateral transporter organic anion transporter 1. J. Am. Soc. Nephrol. 15, 2023-2031. (25) Harris, H. H., Pickering, I. J., and George, G. N. (2003) The chemical form of mercury in fish. Science 301, 1203. (26) Hodgkin, E. E., and Richards, W. G. (1987) Molecular similarity based on electrostatic potential and electric field. Int. J. Quant. Chem.: Quant. Biol. Symp. 14, 105-110.
Ingrid J. Pickering Graham N. George* Department of Geological Sciences University of Saskatchewan 114 Science Place Saskatoon, Saskatchewan S7N 5E2 Canada Andrew R. S. Ross Richard J. Hughes National Research Council Canada Plant Biotechnology Institute 110 Gymnasium Place Saskatoon, Saskatchewan S7N 0W9 Canada TX060177S
* Author to whom correspondence should be addressed.
