Two Unconventional Metallothioneins in the Apple Snail Pomacea bridgesii Have Lost Their Metal Specificity during Adaptation to Freshwater Habitats.

Mario García-Risco,Ricard Albalat,Michael Niederwanger,Mercè Capdevila,Òscar Palacios,Reinhard Dallinger,Sara Calatayud

doi:10.3390/ijms22010095

Abstract

Metallothioneins (MTs) are a diverse group of proteins responsible for the control of metal homeostasis and detoxification. To investigate the impact that environmental conditions might have had on the metal-binding abilities of these proteins, we have characterized the MTs from the apple snail Pomacea bridgesii, a gastropod species belonging to the class of Caenogastropoda with an amphibious lifestyle facing diverse situations of metal bioavailability. P. bridgesii has two structurally divergent MTs, named PbrMT1 and PbrMT2, that are longer than other gastropod MTs due to the presence of extra sequence motifs and metal-binding domains. We have characterized the Zn(II), Cd(II), and Cu(I) binding abilities of these two MTs after their heterologous expression in E. coli. Our results have revealed that despite their structural differences, both MTs share an unspecific metal-binding character, and a great ability to cope with elevated amounts of different metal ions. Our analyses have also revealed slight divergences in their metal-binding features: PbrMT1 shows a more pronounced Zn(II)-thionein character than PbrMT2, while the latter has a stronger Cu(I)-thionein character. The characterization of these two unconventional PbrMTs supports the loss of the metal-binding specificity during the evolution of the MTs of the Ampullariid family, and further suggests an evolutionary link of this loss with the adaptation of these gastropod lineages to metal-poor freshwater habitats.

Highlights

Metallothioneins (MTs) are usually small proteins known for their singularly high content of cysteine residues (Cys), which provide them the capacity for binding essential and non-essential heavy metal ions [1,2,3]
Since the expression of CdMTs is upregulated by Cd2+ ions, these MTs have been associated with the physiological control of Cd, with Cd detoxification [8,9,10], whereas the expression of CuMTs appears to be activated for controlling Cu homeostasis, which in the case of gastropods is related to the synthesis of the oxygen transporter hemocyanin [5,11]
P. bridgesii is a freshwater snail belonging to the gastropod class of Caenogastropoda and to the apple snail family of Ampullariidae

Summary

Introduction

Metallothioneins (MTs) are usually small proteins known for their singularly high content of cysteine residues (Cys), which provide them the capacity for binding essential and non-essential heavy metal ions [1,2,3]. All our preparations were characterized by UV-Vis and circular dichroism spectroscopies and mass spectrometry in order to establish the metal-binding features of the P. bridgesii MTs, providing experimental evidence to further confirm or reject the inferences made on the basis of our phylogenetic approach This information will allow us to correlate the binding abilities of these proteins with the requirements of metal handling of P. bridgesii, and to speculate how the evolution of unspecific MTs might have provided an adaptive advantage to this species for tolerating variable and fluctuating metal availabilities, improving its adaptation capacity to an amphibious lifestyle

Sequence Features of PbrMTs

Production of Metal–PbrMT Complexes

Cloning Pomacea bridgesii Metallothioneins for Heterologous Expression

Production and Purification of Recombinant Metal–PbrMT Complexes

Characterization of the Metal–PbrMT1 and Metal–PbrMT2 Complexes

In Vitro Metal–Protein Binding Studies