Tunicates Illuminate the Enigmatic Evolution of Chordate Metallothioneins by Gene Gains and Losses, Independent Modular Expansions, and Functional Convergences

Abstract

To investigate novel patterns and processes of protein evolution, we have focused in the metallothioneins (MTs), a singular group of metal-binding, cysteine-rich proteins that, due to their high degree of sequence diversity, still represents a black hole in Evolutionary Biology. We have identified and analyzed more than 160 new MTs in non-vertebrate chordates (especially in 37 species of ascidians, 4 thaliaceans and 3 appendicularians) showing that prototypic tunicate MTs are mono-modular proteins with a pervasive preference for cadmium ions, whereas vertebrate and cephalochordate MTs are bi-modular proteins with diverse metal preferences. These structural and functional differences imply a complex evolutionary history of chordate MTs –including de novo emergence of genes and domains, processes of convergent evolution, events of gene gains and losses, and recurrent amplifications of functional domains–, that would stand for an unprecedented case in the field of protein evolution.

Modular Evolution and Population Variability of Oikopleura dioica Metallothioneins

“…Notice that the 12C domain of OdiMT1 is, indeed, an 11C domain due to a Cys to Ser substitution in the carboxyl terminal region in comparison with prototypical 12C domain (thus, we named this domain as 11C/12C; Figure 1). Our results showed that this 11C/12C domain was able to yield almost unique Zn 4and Cd 4 -protein species (Figure 2), in a similar way that other domains with 11 or 12 Cys: the 11C mammal and echinoderm α domains (Stillman et al, 1987;Tomas et al, 2013), the 12C mollusk α domains (Digilio et al, 2009), the insect 12C MTs (Egli et al, 2006) and the ascidian and thaliacean 12C MTs (Calatayud et al, 2021a). These results revealed a significant structural and functional autonomy of O. dioica 11C/12C domain, which was able to form stable metal-protein clusters with Zn(II) and Cd(II).…”

“…The arrangement of Cys motifs in O. dioica domains diverges from that found in the MTs of other tunicates belonging to the ascidian and thaliacean classes, but it is similar to that of other appendicularian species of the same genus, O. albicans and O. vanhoeffeni (Calatayud et al, 2021a). Comparison of the appendicularian MTs show that the original Oikopleura MT domain had twelve cysteines (12C), and that this domain corresponds to previously described C7 + C5 subunits (Calatayud et al, 2018(Calatayud et al, , 2021a. OdiMT1 would have therefore two 12C domains, but its amino-terminal domain was "trimmed" to become a t-12C domain that lacks the C5 subunit.…”

“…dioica has two MTs, a bi-modular OdiMT1 and a multimodular OdiMT2 (formerly OdMT1 and OdMT2) made of different number of domain repeats (Calatayud et al, 2018). The arrangement of Cys motifs in O. dioica domains diverges from that found in the MTs of other tunicates belonging to the ascidian and thaliacean classes, but it is similar to that of other appendicularian species of the same genus, O. albicans and O. vanhoeffeni (Calatayud et al, 2021a). Comparison of the appendicularian MTs show that the original Oikopleura MT domain had twelve cysteines (12C), and that this domain corresponds to previously described C7 + C5 subunits (Calatayud et al, 2018(Calatayud et al, , 2021a.…”

“…In chordates, for instance, vertebrate and cephalochordate MTs are bi-modular proteins with two domains that have diverse preferences and capacities for binding zinc (Zn), copper (Cu), or cadmium (Cd) ions (Capdevila and Atrian, 2011;Vasak and Meloni, 2011;Guirola et al, 2012;Artells et al, 2013). In contrast, most tunicate MTs are mono-modular proteins, whose single domain has a pervasive preference for Cd(II) ions (Calatayud et al, 2021a). The domain configuration of each MT is, indeed, functionally and structurally relevant because domains determine the formation of metalthiolate clusters: domains with 9 Cys cluster with three divalent metal ions, while 11/12 Cys domains cluster with four divalent metal ions (e.g., mammalian β and α domains, respectively, Otvos and Armitage, 1980;Schultze et al, 1988).…”

“…The domain configuration of each MT is, indeed, functionally and structurally relevant because domains determine the formation of metalthiolate clusters: domains with 9 Cys cluster with three divalent metal ions, while 11/12 Cys domains cluster with four divalent metal ions (e.g., mammalian β and α domains, respectively, Otvos and Armitage, 1980;Schultze et al, 1988). In addition, domain analyses have been shown to be helpful for elucidating the origin and evolutionary relationships of MTs in diverse groups of mollusks (Jenny et al, 2016;Nam and Kim, 2017;Calatayud et al, 2021b), and to reconstruct the complex evolutionary history of chordate MTs (Calatayud et al, 2021a).…”