Journal of Molecular Evolution Volume 78 Number 3–4 April 2014
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JOURNAL OF MOLECULAR EVOLUTION VOLUME 78 NUMBER 3–4 APRIL 2014 Syntenic comparison of the metallothionein genes in vertebrates 123 00239 • ISSN 0022-2844 78(3–4) 171–242 (2014) J Mol Evol (2014) 78:217–233 DOI 10.1007/s00239-014-9612-5 ORIGINAL ARTICLE Molecular Evolution and Functional Divergence of the Metallothionein Gene Family in Vertebrates Nina Sere´n • Scott Glaberman • Miguel A. Carretero • Ylenia Chiari Received: 19 November 2013 / Accepted: 1 February 2014 / Published online: 21 February 2014 Ó Springer Science+Business Media New York 2014 Abstract The metallothionein (MT) gene superfamily Analyses of functional divergence identified amino acid sites consists of metal-binding proteins involved in various metal correlated with functional divergence among MT types. detoxification and storage mechanisms. The evolution of this Uncovering the number of genes and sites possibly corre- gene family in vertebrates has mostly been studied in lated with functional divergence will help to design cost- mammals using sparse taxon or gene sampling. Genomic effective MT functional and gene expression studies. This databases and available data on MT protein function and will permit further understanding of the distinct roles and expression allow a better understanding of the evolution and specificity of these proteins and to properly target specific functional divergence of the different MT types. We MT for different types of functional studies. Therefore, this recovered 77 MT coding sequences from 20 representative work presents a critical background on the molecular evo- vertebrates with annotated complete genomes. We found lution and functional divergence of vertebrate MTs to carry multiple MT genes, also in reptiles, which were thought to out further detailed studies on the relationship between have only one MT type. Phylogenetic and synteny analyses heavy metal metabolism and tolerances among vertebrates. indicate the existence of a eutherian MT1 and MT2, a tet- rapod MT3, an amniote MT4, and fish MT. The optimal Keywords CDS Á Functional analysis Á Gene duplication Á gene-tree/species-tree reconciliation analyses identified the Gene tree Á Genomic database Á Reconciliation best root in the fish clade. Functional analyses reveal vari- ation in hydropathic index among protein domains, likely correlated with their distinct flexibility and metal affinity. Introduction Gene families are a set of genes sharing sequence, and Scott Glaberman—See Disclaimer. often functional, homology. The evolution of gene families Electronic supplementary material The online version of this is considered an important driver of species evolution article (doi:10.1007/s00239-014-9612-5) contains supplementary (Ohno 1970; Demuth et al. 2006 and references therein). material, which is available to authorized users. Gene families mainly evolve as a result of duplication and loss events, often associated with gain of adaptive function N. Sere´n Á M. A. Carretero Á Y. Chiari CIBIO, Centro de Investigac¸a˜o em Biodiversidade e Recursos through neofunctionalization or subfunctionalization (e.g., Gene´ticos Campus Agra´rio de Vaira˜o, Universidade do Porto, Chang and Duda 2012; Kondrashov 2012; Zhang 2003). In 4485-661 Vaira˜o, Portugal neofunctionalization, duplicated genes may undergo an accelerated rate of mutation in one of the recently dupli- S. Glaberman U.S. Environmental Protection Agency, Office of Pesticide cated copies when freed from selective constraints, Programs (MC-7507P), Washington, DC 20460, USA potentially leading to new function (e.g., Ohno 1970; Zhang et al. 1998). In subfunctionalization, paralogs & Y. Chiari ( ) gradually take on multiple functions once maintained by Department of Biology, University of South Alabama, LSCB 123, 5871 USA Dr. N, Mobile, AL 36688, USA the original single copy gene, leading to specialized genes e-mail: [email protected] with no overlapping functions (e.g., Force et al. 1999; 123 218 J Mol Evol (2014) 78:217–233 Prince and Pickett 2002; see also Kondrashov et al. 2002). those in mammals (called class I), proteinaceous MTs that Independently of the underlying mechanisms driving the lack this close resemblance (class II), and non-proteina- process, functional divergence of genes following dupli- ceous MT-like polypeptides (class III) (Fowler et al. 1987; cation may promote increased gene diversity and novel reviewed in Blindauer and Leszczyszyn 2010; Miles et al. gene functions (Kondrashov et al. 2002; Prince and Pickett 2000). However, the currently adopted classification 2002; Zhang 2003), possibly facilitating organismal adap- scheme in available genomic and protein databases of MT tation to environmental conditions (e.g., Brown et al. 1998; genes are primarily based on the phylogenetic relationships Lenormand et al. 1998; Kondrashov 2012). among mammalian MT sequences, which are known for The metallothionein (MT) gene superfamily is known for their great functional diversity (e.g., Capdevila and Atrian its high turnover through gene duplication and loss (Cap- 2011; Vasˇa´k and Meloni 2011). According to this classi- devila and Atrian 2011). In some mammals, such as mice fication system, MTs fall into at least four subgroups: MT1, and humans, the presence of multiple MT genes has been MT2, MT3, and MT4, which are generally differentially associated with varying levels of gene expression, as well as expressed and induced, and show diverse metal-binding gains and losses of function (e.g., Garrett et al. 1998;Mo- affinities (e.g., Tı´o et al. 2004; reviewed in Davis and leirinho et al. 2011;Tı´oetal.2004; reviewed in Blindauer Cousins 2000; Miles et al. 2000; Vasˇa´k and Meloni 2011). and Leszczyszyn 2010). MTs are ubiquitous low-molecular There is a partial correspondence shown between the early weight proteins and polypeptides of extremely high metal classification and the more recent mammalian MT sub- and sulfur content (Nordberg and Nordberg 2009). These groups (e.g., MT1 and 2 to class I) (reviewed in Palacios inducible proteins exhibit essential metal-binding properties et al. 2011). Recent studies suggest that the later classifi- and have several roles in metabolism, homeostasis, and cation system based on mammalian MTs is unsuited for kinetics of metals such as transport, storage and detoxifica- molecular evolution studies at a taxonomically large scale tion of metal ions in cells (e.g., Carpene` et al. 2007;Nord- due to differences in underlying physiologies between berg and Nordberg 2009;Palmiter1998). Metal affinity mammals and other organisms (reviewed in Capdevila and varies among the different MT types (Nordberg 1989). MTs Atrian 2011). For example, studies on other taxonomic have also recently gained attention in biomedical studies, groups, including plants and bacteria, underline a departure due to their proposed involvement in cancer or neurological from the classical mammalian (humans and mice) amino diseases (reviewed in Hidalgo et al. 2009). Despite this acid composition, biochemical metal-binding characteris- increasing understanding of the physiological properties and tics, and protein folding (reviewed in Vasˇa´k and Meloni function of MTs, very little is known about the intra- and 2011; see also Villarreal et al. 2006). inter-specific variation of these genes in vertebrates. It has The increasing availability of annotated genomic dat- been shown in invertebrates that both duplication events and abases provides an incredible resource to study functional molecular changes in the regulatory and coding regions of diversification and evolution of genes and gene families (e.g., MT genes produce species-specific differences in terms of Koonin 2009; Yanai et al. 2000). In vertebrates, available expression, tertiary structure, metal-binding affinity, and gene and protein databases have recently been used to infer metal resistance (Dallinger et al. 1997;Shawetal.2007; MT gene family origin and evolution through comparative reviewed in Dallinger and Ho¨ckner 2013). analyses (e.g., Moleirinho et al. 2011; Guirola et al. 2012; Although MT genes are widely represented across all Trinchella et al. 2008, 2012). However, these studies have three domains of life, they have not been equally studied in been based either on sparse taxon or gene sampling when different taxonomic groups. In vertebrates, a large number species with fully sequenced genomes have been considered of biochemical, molecular, and chemical studies have been (e.g., Moleirinho et al. 2011) or they have been based on carried out on these multi-functional genes in mammals cDNA or protein data (Guirola et al. 2012; Trinchella et al. (mostly in humans and mice), which contrasts with the very 2008, 2012). In the latter case, proper distinction between limited data available for other groups (reviewed in different genes versus different isoforms cannot be assessed Blindauer and Leszczyszyn 2010; Hidalgo et al. 2009). without genomic sequencing of the target gene or compari- Different MT classification systems have been developed son of untranslated regions (UTRs). In addition, for cDNA for all organisms based on protein structure (e.g., Fowler data, all existing genes cannot be easily detected if they are et al. 1987; Nordberg and Kojima 1979; Palacios et al. not expressed, for example, as a result of a lack of response to 2011; Valls et al. 2001; Vasˇa´k and Armitage 1986; metal treatment or differential expression in time and space. reviewed in Nordberg and Nordberg 2009) or using both This could produce misleading estimates of duplication and protein