Gene 345 (2005) 21 – 26 www.elsevier.com/locate/gene Fish and mammalian metallothioneins: a comparative study Rosaria Scudieroa, Piero Andrea Temussib,c, Elio Parisid,* a Department of Evolutionary and Comparative Biology, University Federico II, via Mezzocannone 8, Napoli, Italy b Department of Chemistry, University Federico II, via Cinthia 45, Napoli, Italy c National Institute of Medical Research, Medical Research Council, The Ridgeway, Mill Hill, London, UK d CNR Institute of Protein Biochemistry, via Marconi 10, Napoli, Italy Received 14 September 2004; accepted 9 November 2004 Available online 24 December 2004 Received by M. Porto Abstract Structural studies show that fish and mammalian metallothioneins are endowed of distinctive features. In particular, the ninth cysteine residue present in the a domain of fish metallothionein is shifted of two positions with respect to the mammalian metallothionein, introducing a conformational modification in the protein structure. In addition, the fish metallothionein is less hydrophobic and more flexible than its mammalian counterpart. Our previous studies showed that the hydropathy of piscine and mammalian metallothioneins is significantly correlated with organismal temperature. In the present paper we have performed phylogenetic comparative analysis on metallothioneins of 24 species of fish and mammals. The results of such analysis failed to indicate that metallothionein hydropathy is an adaptive response to the thermal regime of the species. We concluded that metallothionein hydropathy is a trait that did not evolve in association with environmental changes. D 2004 Elsevier B.V. All rights reserved. Keywords: Contrast analysis; Phylogenetic dependence; Hydropathy; Protein flexibility; Protein structure; Temperature adaptation 1. Introduction Metallothioneins are ubiquitous low molecular mass proteins (typically 6–7 kDa), rich in cysteines (about 30% of the total residues) and heavy metals (7–10 equivalents per mol, depending on the metal). They are thought to play a variety of functions including homeostasis of the essential oligoelements zinc and copper, defense against the harmful effects of toxic metals like cadmium and mercury and protection from stress conditions (Kagi and Schaffer, 1988). Structural studies show that all metallothioneins have a common spatial scaffold consisting of two distinct protein domains, the N-terminal h domain and the C-terminal a Abbreviations: CAIC, Comparative Analysis by Independent Contrasts; NMR, Nuclear Magnetic Resonance. * Corresponding author. Tel.: +39 081 7257323; fax: +39 081 2396525. E-mail address: e.parisi@ibp.cnr.it (E. Parisi). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.11.024 domain, containing three and four metal atoms, respectively, bound to the cysteine sulfurs (Schultze et al., 1988). The peptide chain is wrapped around the metal moiety, so that ligand accessibility and release are somehow dependent on the flexibility of the whole protein. In a previous study, we showed that fish metallothioneins have a lower hydropatic character (a parameter inversely proportional to flexibility) with respect to mammalian metallothioneins (Capasso et al., 2003a). A higher flexibility can facilitate the conformational changes necessary to the polypeptide chain for maintaining functionality at the low temperatures that fish may experience in marine or freshwater environments. The present paper is an attempt to answer the question: is the evolutionary change in metallothionein hydropathy significantly correlated with the thermal regime of the organisms? To answer this question we have made use of a method capable of generating data for analysis that are phylogenetically independent. The phylogenetic comparative technique that is most com- 22 R. Scudiero et al. / Gene 345 (2005) 21–26 monly applied is the so-called bphylogenetic contrastsQ (Felsenstein, 1985; Harvey and Purvis, 1991) that removes the component of the character state resulting from common ancestry. The results of such an analysis show that metallothionein hydropathy is not significantly correlated with thermal regime; consequently this trait does not represent an adaptation of vertebrates to organismal temperature. 2. Materials and methods 2.1. Data set and phylogenetic tree Metallothionein sequences employed in the present study are shown in Fig. 1. Accession numbers, phylogenetic tree, hydropathy indexes and optimal temperatures were as in the previous paper (Capasso et al., 2003a). 10 R. norvegicus C. griseus M. musculus C. familiaris S. scrofa H. sapiens C. aethiops O. cuniculus E. lucius O. mykiss Z. viviparus P. platessa C. rastrospinosus N. coriiceps P. charcoti C. aceratus C. hamatus G. acuticeps P. borchgrevinki T. bernacchii O. mossambicus G. morhua D. rerio C. auratus D D D D D D D D D D D D D D D D D D D D D D D D P P P P P P P P P P P P P P P P P P P P P P P P N N N D N N N N - C C C C C C C C C C C C C C C C C C C C C C C C S S S S S S S S E E E E E E E E D D D Q E E E D C C C C C C C C C C C C C C C C C C C C C C C C S S S S A S A A S S S S S S S S S S S S A S A A T T T T A P T T K K K K K K K K K K K K K K K K G G G G G V G G T T T T S S S S S S S S T T T T R. norvegicus C. griseus M. musculus C. familiaris S. scrofa H. sapiens C. aethiops O. cuniculus E. lucius O. mykiss Z. viviparus P. platessa C. rastrospinosus N. coriiceps P. charcoti C. aceratus C. hamatus G. acuticeps P. borchgrevinki T. bernacchii O. mossambicus G. morhua D. rerio C. auratus S S S S S S S S S S S S S S S S S S S S S S S S C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C S S S S S S S S S P P P P P P P P P P P D E S S C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C P P P P P P P P P P P P P P P P P P P P P P P P V V V V V V V A S S S S S S S S S S S S S S S S G G G G G G G G G D G G G G G G G G G G G G G G G S G G G G V N G G G G G G G G G G G G G G G G 20 S T S S S S S S S S T T T T N N T T T T T S A A C C C C C C C C C C C C C C C C C C C C C C C C T T T T T A T T N N N N N N N N N N N N N N N N C C C C C C C C C C C C C C C C C C C C C C C C S S T A A A A A G G G G G G G G G G G G G G G G S S S G G G D S G G G G G G G G G G G G G T A A S S S S S S S S S S S S S S S S S S S S S S T T C C C C C C C C C C C C C C C C C C C C C C C C G G A K K K K K K K K T T T T T T T T T S T K K S S S A A A A T S S T P T T T T T T T T S S S S K K K K K K K K K K K K K K K K K K K K K K K K C C C C C C C C C C C C C C C C C C C C C C C C A A A A A A A A A A A A A A A A A A A A A A A A Q Q Q Q Q Q Q Q S S S S S S S S S S S S S S S S G G G G G G G G G G G G G G G G G G G G G G G G C C C C C C C C C C C C C C C C C C C C C C C C V V V I I I V I I V V V V V V V V V V V V V V V C C C C C C C C C C C C C C C C C C C C C C C C 40 C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C 30 K K K K K K K K S S T K T T T T T T T T T A T T N D N E D E E E N N N N N N N N N N N N K N N N C C C C C C C C C C C C C C C C C C C C C C C C K K K K K K K K A A S S S S S S S S S S S S Q Q C C C C C C C C C C C C C C C C C C C C C C C C T T T T T T T T T T T T K K K K K K K K K T T T S S S S S S S S S S T T S S S S S S S S S K T T C C C C C C C C C C C C C C C C C C C C C C C C K K K K K K K K K K K N K K K K K K K K K K K K G G G G G G G G G G G G G G G G G G G G G D G G A A A A A T A A K K K K K K K K K K K K K K N N S S A S S S S S T T T T T T T T T T T T T T S S D D D D D D E D C C C C C C C C C C C C C C C C K K K K K K K K D D D D D D D D D D D D D D G G C C C C C C C C T T T T T T T T T T T T T T T S T T T S S S N S S S S S S S S S S S S S S N S S C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C 50 K K K K K K K K K K K K K K K I K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K 60 A A A A A A A A Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Fig. 1. Aligned protein sequences of piscine and mammalian metallothioneins. The first 8 sequences refer to mammals, the remaining 16 to fish. R. Scudiero et al. / Gene 345 (2005) 21–26 23 2.2. Independent contrasts Independent contrast analysis was carried out using the method implemented in the CAIC software package (Purvis and Rambaut, 1995). The assumption of phylogenetic independence was tested as described by Abouheif (1999) using the software of the same author (Reeve and Abouheif, 2003). 2.3. Other methods Three-dimensional structures of fish and mouse metallothioneins were visualized using the software Rasmol v. 2.6. Statistical analysis was carried out using the software package StatView v. 5.0.1 released by SAS Institute (Cary, NC USA). 3. Results 3.1. A survey of fish and mammalian metallothioneins Previous studies carried out in our laboratory reported in details the distinctive features of mammalian and fish metallothioneins. The results of these studies are summarized in this section before moving into the section aimed to phylogenetic analysis. Fig. 1 shows the sequence alignment of representative fish and mammalian metallothioneins used in this study. With respect to mammalian metallothioneins, fish metallothioneins display a number of distinctive features in the primary structure, including the displacement of a cysteine residue located in the carboxyterminal half of the molecule (occupying position 54 in the alignment shown in Fig. 1) and a lower number of lysine residues juxtaposed to cysteines (Scudiero et al., 1997). The main consequence of the displacement of the banomalousQ cysteine residue is the drastically different orientation of the motif K50-G51K52-T53 of fish metallothionein with respect to the corresponding K50-G51-A52-A53 motif of mouse metallothionein (see Fig. 1). As shown in Fig. 2, in the fish metallothionein, the loop formed by the motif KGKT rotates down, opening a wide channel and causing a different arrangement of charged residues on the molecule surface (Capasso et al., 2003b). Another peculiarity of fish metallothionein is the presence of three short segments of secondary structure elements (one a-helix from T41 to A44 and one 310 helix from V48 to K50 in the a domain, one a-helix from E5 to S9 in the h domain; Fig. 2). In addition, NMR spectroscopy showed a selective broadening of the heteronuclear spectra of fish metallothionein, thus suggesting a higher flexibility of the molecule. The fact that the observed broadening did not affect the homonuclear spectra suggests an exchange phenomenon involving metal ions that is much more pronounced in fish metallothionein than in its murine counterpart (Capasso et al., 2003b). NMR observations are Fig. 2. NMR solution structures of fish (Notothenia coriiceps) and mouse metallothioneins. Backbone structures of fish and mouse a domains are shown in panels (A) and (B), respectively. Backbone structures of fish and mouse h domains are shown in panels (C) and (D), respectively. further supported by both circular dichroism (D’Auria et al., 2001) and dynamic fluorescence spectra (Capasso et al., 2002) of fish metallothionein that are considerably influenced by temperature, whereas the mouse protein is much less affected by heating. Moreover, fish metallothionein displays a more pronounced reactivity of the metal–thiolate clusters in the presence of the redox couple formed by reduced and oxidized glutathione (D’Auria et al., 2001) and a better metal exchange capability with respect to mouse metallothionein (Capasso et al., 2003b). 3.2. Phylogenetic comparative analysis The results reported above show that fish metallothionein are characterized by a higher plasticity and flexibility with respect to their mammalian counterpart. For proteins like metallothioneins an increase in flexibility is achieved mostly by weakening the hydrophobic interactions. The values of the hydropathy index for fish and mammalian metallothioneins reported in Fig. 3 suggest that all fish metallothionein are less hydrophobic than mammalian metallothioneins. The average value of hydropathy for fish metallothioneins is 0.225, whereas the corresponding value for mammals is 0.098. Unpaired t-test shows that the two means are significantly different (t=9.78, pb0.0001). The metallothionein phylogeny depicted in Fig. 3 shows that metallothioneins with low hydropathy index are closest relatives and the same is true for metallothioneins with high 24 R. Scudiero et al. / Gene 345 (2005) 21–26 Gadus morhua -0.329 10 ºC Pleuronectes platessa - 0.251 6 ºC Zoarces viviparous -0.243 10 ºC Oreochromis mossambicus -0.249 20 ºC Chionodraco rastrospinosus -0.246 -2 ºC Notothenia coriiceps -0.246 -2 ºC Trematomus bernacchii -0.246 -2 ºC Gymnodraco acuticeps -0.246 -2 ºC Pagothenia borchgrevinki -0.246 -2 ºC Chionodraco hamatus -0.246 -2 ºC Esox lucius Parachaenichthys charcoti -0.294 -2 ºC Chaenocephalus aceratus -0.152 -2 ºC -0.187 18 ºC Oncorhyncus mykiss -0.204 17 ºC Danio rerio -0.108 30 ºC Carassius auratus -0.109 25 ºC Mus musculus 0.0844 37ºC Rattus norvegicus 0.00278 37 ºC Cricetulus griseus -0.0022 37 ºC Homo sapiens 0.197 37 ºC Canis familiaris 0.0416 37 ºC Sus scrofa 0.127 37 ºC Cercopithecus aethiops 0.0702 37 ºC Oryctolagus cuniculus -0.0541 37 ºC Fig. 3. Phylogenetic tree of fish and mammalian metallothioneins used in contrast analysis. Hydropathy indexes and optimal temperatures are reported at the tips, near the species names. hydropathy index. At this point, it was mandatory to run a test to decide whether the changes in this character are due to phylogenetic contingency or adaptation. A 0.02 0 -0.02 B 4.5 Contrasts in temperature Contrasts in hydropathy 0.04 It has become increasingly clear that a phylogenetically based comparative method should be applied whenever the assumption of phylogenetic independence is violated; 3.5 2.5 1.5 0.5 -0.5 -0.04 -0.3 -0.2 -0.1 0 Hydropathy at nodes 0.1 -5 5 15 25 35 Temperature at nodes Fig. 4. Phylogenetic independence of contrasts. (A) Relationship between contrasts in hydropathy and hydropathy inferred at the nodes (R 2=0.002). (B) Relationship between contrasts in temperature and temperature inferred at the nodes (R 2=0.04). Contrasts and nodal values were computed by the program CAIC. R. Scudiero et al. / Gene 345 (2005) 21–26 however, before applying a phylogenetic comparative method it is advisable to test the assumption of phylogenetic independence with appropriate statistical methods. For such a purpose, we have used a phylogenetic independence method based on the serial independence test described by Abouheif (1999). The results of such a test shows that 1 out of 1000 randomized mean C_statistics calculated on the hydropathy index of the metallothioneins in the tree in Fig. 3 (tip values) was greater or equal to the observed mean C_statistics ( p=0.001). Hence, these data are significantly phylogenetically correlated, thus prompting us to carry out comparative analysis by independent contrasts in the two continuous traits, i.e. hydropathy and temperature. As contrasts may not be phylogenetic independent for a number of reasons, including non-brownian mode of evolution (Felsenstein, 1985), we have applied to the contrasts the same phylogenetic independence test reported above. The results of this test show that contrasts are not phylogenetically correlated ( pN0.2); indeed, the plots in Fig. 4 show lack of correlation between contrasts and values at the nodes. As shown in Fig. 5, the independent contrasts in the two continuous variables considered exhibit a coefficient of determination R 2=0.057, indicating that less than 6% of the dependent variable variation is explained by the independent variable. We have also searched for correlated evolution between hydropathy and a categorical trait occurring in the two states classified as bhomeothermsQ and bpoikilothermsQ. Under the assumption that the evolution of the continuous trait (hydropathy) is not linked to the categorical trait, the mean value of the contrasts should be zero. The null hypothesis cannot be rejected, because the mean 0.004 is not significantly different from zero ( p=0.31). A pitfall in the application of phylogenetic comparative methods is given by an erroneous estimate of contrasts if the phylogeny and the branch lengths are not correct (Pagel, 1993; Freckleton et al., 2002). To overcome this problem, we have repeated the whole analysis using a topology and the relative branch lengths inferred by a maximum likelihood method. The results of these analyses duplicated Contrasts in hydropathy 0.04 0.02 0 -0.02 -0.04 -0.5 0.5 1.5 2.5 3.5 4.5 Contrasts in temperature Fig. 5. Independent contrasts in hydropathy plotted versus independent contrasts in temperature. 25 those reported in the present study. Hence, we must conclude that there is no correlation between protein hydropathy and thermal regime of the species. 4. Discussion The variety of forms resulting from evolution depends on the different ways in which natural selection operates. In the study of evolutionary processes, a frequent hypothesis to test is whether specific traits arise from adaptation or reflect phylogeny. Indeed, phylogenetic related species may share certain characteristics as a consequence of their common ancestry (phylogenetic contingency) or, alternatively, because they are optimally adapted to their environmental conditions. Vertebrates can be gathered into the two large groups of homeotherms and poikilotherms. Homeotherms are warmblooded animals capable of maintaining the body temperature in the range of 32–42 8C, whereas poikilotherms comprise species in which the body temperature equilibrates with that of the external environment. In a previous paper (Capasso et al., 2003b), we reported the results of a study carried out on the metallothioneins of homeotherms, represented by mammals, and poikilotherms, represented by fish. All the putative functions attributed to metallothioneins give a considerable weight to the presence of reactive metal–thiolate clusters in the molecule. One of the most striking peculiarities of metallothioneins is the thermodynamic stability and the remarkable kinetic reactivity of the clusters. Consequently, in spite of the apparent rigidity of the molecule, metallothioneins are in a dynamic active state, with a continuous redistribution of the metal ions inside and within the clusters. The severe structural constraints put on metal–thiolate clusters, together with the moderate variability of the amino acids placed between the cysteines, suggested a highly conserved function for all metallothioneins, independently of the phylogenetic origin. In contrast, our studies on fish metallothionein demonstrate that the limited number of amino acid substitutions occurring in fish metallothionein is capable to affect thiol reactivity (D’Auria et al., 2001) and metal exchangeability (Capasso et al., 2003b). Owing to the compact spatial configuration, metallothioneins are endowed of remarkable tolerance to heat, thus precluding the possibility to test protein thermostability. However, analyses carried out with the aid of circular dichroism spectroscopy showed that the conformation of the Cd–thiolate chromophore of fish metallothionein is reversibly modified by temperature to a higher extent than mammalian metallothionein (D’Auria et al., 2001). Moreover, time-resolved dynamic fluorescence data (Capasso et al., 2002) and NMR spectra (Capasso et al., 2003b) unraveled a more flexible structure, while infrared spectra suggest a large accessibility to the solvent (Capasso et al., 2002). 26 R. Scudiero et al. / Gene 345 (2005) 21–26 It has been conjectured that flexible proteins are more adapted to cope the effects of low temperatures, whereas thermophilic proteins posses a more rigid structure. In a previous paper we showed that metallothionein hydropathy (that can be taken as a measure of their flexibility) is positively correlated with optimal body temperature (Capasso et al., 2003a). The present work is an attempt to establish whether metallothionein hydropathy is a property linked to adaptation or correlated to phylogenesis. Phylogenetic comparative methods may be very useful to investigate the association between two traits: the two alternative methods usually applied in comparative analysis are the so-called directional method, based on the reconstruction of the ancestral character states at the nodes of the inferred phylogeny, and the cross-sectional method that finds associations across taxa. These methods have been successfully employed to investigate the correlation between preferred temperatures and optimal performance temperatures in lizards (Huey and Bennet, 1987) and, more recently, the correlation between genomic GC levels and optimal growth temperatures in prokaryotes (Musto et al., 2004). On the basis of results of our analysis, we conclude that there is no significant relationship between metallothionein hydropathy and temperature after taking away the phylogenetic component, in spite of the cross species correlation observed between the raw data. Probably, the dichotomy between mammalian and fish metallothioneins originated at early stages of phylogenesis, as suggested by the fact that reconstructed ancestral metallothionein sequences exhibit lower values of the hydropathy index along the fish lineage with respect to the ancestral sequences inferred at the nodes of the homeotherms (Capasso et al., 2003a). The data of the present study are in keeping with results of contrast analysis applied to invertebrate and vertebrate enzymes, indicating the lack of correlation between protein stability and body temperature (Stillman and Somero, 2001) and no temperature-adaptive variations in G+C levels (Ream et al., 2003). Although the few data available do not allow drawing any general conclusion, all these results taken together suggest that certain protein features such as structural stability and molecular flexibility result from mutations arising independently of specific aspects of the environment. This does not mean that traits are not adapted at all, as optimal adaptation is the result of genetic mutations combined with natural selection, although it is not always possible to establish which conditions are more influential on selection. One of the objections raised against phylogenetic comparative methods is the irrelevance of phylogenetic dependence, because across-species variations of a specific trait are often associated to the fact that phylogenetic related species share the same ecological habitats. Such an objection contrasts with the fact that, in our case, hydropathy is not correlated with the two groups of bmammalsQ and bfishQ. In conclusion, the present study shows that metallothionein hydropathy is a character displaying significant phylogenetic dependence. Analysis by independent contrasts failed to support significant association between hydropathy and thermal regime of species. Acknowledgements This work is the frame of the bProgetto Nazionale per le Ricerche in AntartideQ (PNRA). References Abouheif, E., 1999. A method for testing the assumption of phylogenetic independence in comparative data. Evol. Ecol. Res. 1, 895 – 909. Capasso, C., et al., 2002. Stability and conformational dynamics of metallothioneins from the Antarctic fish Notothenia coriiceps and mouse. Proteins 46, 259 – 267. Capasso, C., et al., 2003a. Phylogenetic divergence of fish and mammalian metallothionein: relationships with structural diversification and organismal temperature. J. Mol. Evol. 57, S250 – S257. Capasso, C., et al., 2003b. Solution structure of MT_nc, a novel metallothionein from the Antarctic fish Notothenia coriiceps. Structure 11, 435 – 443. D’Auria, S., et al., 2001. Structural characterization and thermal stability of Notothenia coriiceps metallothionein. Biochem. J. 354, 291 – 299. Felsenstein, J., 1985. Phylogenies and the comparative method. Am. Nat. 125, 1 – 15. Freckleton, R.P., Harvey, P.H., Pagel, M., 2002. Phylogenetic analysis and comparative data: a test and review of evidence. Am. Nat. 160, 712 – 726. Harvey, P.H., Purvis, A., 1991. Comparative methods for explaining adaptations. Nature 351, 619 – 624. Huey, R.B., Bennet, A.F., 1987. Phylogenetic studies of co-adaptation; preferred temperatures versus optimal performance temperatures of lizard. Evolution 41, 1098 – 1115. Kagi, J.H.R., Schaffer, A., 1988. Biochemistry of metallothionein. Biochemistry 27, 8509 – 8515. Musto, H., Naya, H., Zavala, A., Romero, H., Alvarez-Valin, F., Bernardi, G., 2004. Correlations between genomic GC levels and optimal growth temperatures in prokaryotes. FEBS Lett. 573, 73 – 77. Pagel, M., 1993. Seeking the evolutionary regression coefficient: an analysis of what comparative methods measure. J. Theor. Biol. 164, 191 – 205. Purvis, A., Rambaut, A., 1995. Comparative analysis by independent contrasts (CAIC): an Apple Macintosh application for analyzing comparative data. Comput. Appl. Biosci. 11, 247 – 251. Ream, R.A., Johns, G.C., Somero, G.N., 2003. Base compositions of genes encoding a-actin and lactate dehydrogenase-A from differently adapted vertebrates show no temperature-adaptive variation in G+C content. Mol. Biol. Evol. 20, 105 – 110. Reeve, J., Abouheif, E., 2003. Phylogenetic Independence. Version 2.0. Department of Biology, McGill University. Schultze, P., et al., 1988. Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 203, 251 – 268. Scudiero, R., et al., 1997. Difference in hepatic metallothionein content in Antarctic red-blooded and haemoglobinless fish: undetectable metallothionein levels in hamoglobinless fish is accompanied by accumulation of untranslated metallothionein mRNA. Biochem. J. 332, 207 – 211. Stillman, J.H., Somero, G.N., 2001. A comparative analysis of the evolutionary patterning and mechanistic basis of lactate dehydrogenase thermal stability in porcelain crabs, genus Petrolisthes. J. Exp. Biol. 204, 767 – 776.