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 nonvertebrate 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 bimodular 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.

Keywords: Chordata; Tunicata; ascidians/thaliaceans/appendicularians; metallothionein domains; metallothionein evolution; modular proteins.

Fig. 1.

Phylogenetic relationships in the Chordata phylum. Regarding tunicates, traditional classifications divided this subphylum in three classes, Appendicularia (green background), Ascidiacea (yellow background), and Thaliacea (blue background), but recent analyses propose Thaliacea species (salps, doliolids, and pyrosomes) are nested within the Ascidiacea class, closer to the Aplousobranchia and Phlebobranchia orders than to the Stolidobranchia order (Delsuc et al. 2018; Kocot et al. 2018). A schematic representation of the MTs of each taxonomic group, with a mono-, bi-, or multimodular organization with distinct domains (color coded) containing seven, nine, 11, or 12 cysteines, is showed at the right (see fig. 7 and text for additional details).

Fig. 2.

Schematic representation of multimodular Stelydae MTs. Internal duplications of a 12-Cys domain (grey box) generated multimodular MTs with variable number of repeats (R), from 2 to 9. The number of MTs with different number of repeats (R₁–R₉) is indicated (DgrMT7 = 2 repeats; EtiMT8part ≥4 repeats; ScaMT6part and SsoMT8part ≥6 repeats; PmiMT10part, BleMT1, and BleMT2 ≥7 repeats; PauMT10part and PspMT7 ≥8 repeats; BscMT1, PmamiMT3, and PpoMT7 = 9 repeats) (see supplementary fig. S2, Supplementary Material online, for further details).

Fig. 3.

Deconvoluted ESI-MS spectra of the Cd(II)-productions of ascidian CroMT1 (A), HroMT1 (B), HroMT2 (C), and +14 charge state ESI-MS spectra of the Zn(II)-production of BscMT1 (D), as well as the deconvoluted ESI-MS spectra of the Cd(II)-production of its fragment R₄ BscMT1 (E). Notice that BscMT1 is capable to bind up to 36 divalent ions, which nicely match with a multimodular organization made of nine repeated domains (12C)₉, each one binding four divalent ions alike the mono-modular (12C) MTs (A–C) or the R₄ (12C) domain (E).

Fig. 4.

Circular dichroism spectra of the Cd(II)-productions of CroMT1, HroMT1, HroMT2, BscMT1, and R₄ BscMT1, all exhibiting very similar CD signals. The exciton coupling centered at ∼250 nm confirms high robustness and compactness of the clusters formed.

Fig. 5.

Deconvoluted ESI-MS of the Cd(II)-production of S. thompsoni SthMT1 (A), SthMT2 (B), SthMT3 (C), and to SthMT4 (D). Thaliacean MTs are mono-modular proteins made of a single 12C domain capable to bind four divalent ions, identically to ascidian MTs.

Fig. 6.

Reconstruction of MT evolution in the Chordata phyla. The most parsimonious scenario would be that the ancestral chordate MT was a bimodular MT made of an N-terminal 11/12-Cys domain (in blue) and a C-terminal 9-Cys domain (in red), similar to the current cephalochordate MT1. In cephalochordates, this ancestral form was tandem duplicated. One copy lost the 11/12-Cys domain and expanded the 9-Cys domain, yielding the current bi/trimodular MT2 form. In vertebrates, the number of cysteines in each domain was readjusted to the current 9 Cys of the N-terminal β-domains, and 11 Cys of the C-terminal α domain. Successive lineage-specific tandem duplications of this primeval vertebrate MT led to different MT types (MT1, MT2, MT3, and MT4) with a variable degree of multiplicity in different species. In tunicates, the ancestral form lost the C-terminal 9-Cys domain in ascidian/thaliacean lineages, leading a mono-modular. This MT was frequently duplicated generating a high degree of MT multiplicity in many ascidian and thaliacean species. In contrast, internal duplications led to the emergence of multimodular MTs in the stelydae lineage. Finally, in the appendicularian clade, the ancestral MT appears to have been lost, and a new MT would have emerged de novo by the duplication of a 12-Cys domain (in green). During O. dioica evolution, the new bimodular MT lost five Cys in the N-terminal domain, yielding a 7-Cys/12-Cys arrangement (as it is found in current OdiMT1). Next, this 7-Cys/12-Cys MT was internal duplicated generating multimodular MTs (as it is OdiMT2).

Fig. 7.

Comparison of the arrangement of Cys motifs in different MT domains. The arrangement of the Cys motifs in the 11-Cys amino-terminal domain of cephalochordate MT1s are somewhat similar to that in the vertebrate 9-Cys β domains and in the ascidian/thaliacean 12-Cys MTs (blue boxes), whereas the Cys motifs in the carboxyl-terminal domains of cephalochordate MT1s and MT2s (9C and 9C_Like domains) are vaguely similar to those in vertebrate 11-Cys α domains (pink boxes). In contrast, the arrangement of the Cys motifs in the appendicularian MT1 seems totally different (green boxes).