Metal Dealing at the Origin of the Chordata Phylum: The Metallothionein System and Metal Overload Response in Amphioxus

Maria Guirola,Sílvia Atrian,Òscar Palacios,Sílvia Pérez-Rafael,Mercè Capdevila

doi:10.1371/journal.pone.0043299

Maria Guirola, Sílvia Atrian + Show 3 more

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https://doi.org/10.1371/journal.pone.0043299

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Abstract

Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd-thioneins or Cu-thioneins, according to the ecophysiological needs of each kind of organisms.

Highlights

Metallothioneins (MTs) constitute a heterogeneous superfamily of ubiquitously occurring, low molecular weight, cysteine rich proteins, which coordinate divalent (Zn2+, Cd2+) or monovalent (Cu+) metal ions through metal-thiolate bonds that impose a definite polypeptide folding
This is compatible with an essential cadmium detoxification role, which is only induced in emergency situations
The corresponding PCR products were purified, cloned and sequenced. Their sequences fully matched those of the cDNA initially retrieved from the in silico searches in the B. floridae data bank, and these results served to verify the identity of the BfMT1 and BfMT2 protein sequence between the two Branchiostoma species

Summary

Introduction

Metallothioneins (MTs) constitute a heterogeneous superfamily of ubiquitously occurring, low molecular weight, cysteine rich proteins, which coordinate divalent (Zn2+, Cd2+) or monovalent (Cu+) metal ions through metal-thiolate bonds that impose a definite polypeptide folding (see [1,2] for recent revisions). Bioc.unizh.ch/mtpage/classif.html, including all the isoforms identified in mammals, birds, reptiles, amphibians and fishes These MTs are 60- to 68- amino acid long polypeptides, encompassing 20 cysteines (19 of which are totally conserved). Little information is available regarding the MT system in reptiles, some isoforms have been identified that bear a strong similarity to avian MTs, including preservation of cysteine alignment and structure [13] All this information has drawn a fairly clear picture of the evolution of the MT system inside the vertebrate subphyla (cf http://www.bioc.unizh.ch/mtpage/trees.html), but there is still no clue about the point (or points) of origin of MT molecular diversification of vertebrate MTs regarding other organism groups. The situation is so unclear that the relationship between the prochordate taxa and vertebrates is still a matter of much debate [18]

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