ORIGINAL RESEARCH published: 02 July 2021 doi: 10.3389/fcell.2021.702688 Modular Evolution and Population Variability of Oikopleura dioica Metallothioneins Sara Calatayud 1† , Mario Garcia-Risco 2† , Mercè Capdevila 2 , Cristian Cañestro 1 , Òscar Palacios 2* and Ricard Albalat 1* 1 Departament de Genètica, Microbiologia i Estadística, Facultat de Biologia, Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Barcelona, Spain, 2 Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain Chordate Oikopleura dioica probably is the fastest evolving metazoan reported so far, and thereby, a suitable system in which to explore the limits of evolutionary Edited by: processes. For this reason, and in order to gain new insights on the evolution of Juan Pascual-Anaya, protein modularity, we have investigated the organization, function and evolution of Malaga University, Spain multi-modular metallothionein (MT) proteins in O. dioica. MTs are a heterogeneous group Reviewed by: María Ayelén Pagani, of modular proteins defined by their cysteine (C)-rich domains, which confer the capacity Consejo Nacional de Investigaciones of coordinating different transition metal ions. O. dioica has two MTs, a bi-modular Científicas y Técnicas (CONICET), OdiMT1 consisting of two domains (t-12C and 12C), and a multi-modular OdiMT2 with Argentina Filipe Castro, six t-12C/12C repeats. By means of mass spectrometry and spectroscopy of metal- University of Porto, Portugal protein complexes, we have shown that the 12C domain is able to autonomously bind *Correspondence: four divalent metal ions, although the t-12C/12C pair –as it is found in OdiMT1– is Òscar Palacios Oscar.Palacios@uab.cat the optimized unit for divalent metal binding. We have also shown a direct relationship Ricard Albalat between the number of the t-12C/12C repeats and the metal-binding capacity of the ralbalat@ub.edu MTs, which means a stepwise mode of functional and structural evolution for OdiMT2. † These authors have contributed Finally, after analyzing four different O. dioica populations worldwide distributed, we have equally to this work and share first authorship detected several OdiMT2 variants with changes in their number of t-12C/12C domain repeats. This finding reveals that the number of repeats fluctuates between current Specialty section: O. dioica populations, which provides a new perspective on the evolution of domain This article was submitted to Evolutionary Developmental Biology, repeat proteins. a section of the journal Frontiers in Cell and Developmental Keywords: appendicularian tunicate chordates, modular protein evolution, metallothionein domains, tandem domain repeats, population variants, intra-species variability Biology Received: 29 April 2021 Accepted: 09 June 2021 Published: 02 July 2021 INTRODUCTION Citation: Oikopleura dioica is a tunicate species of the appendicularian class in the chordate phylum. This Calatayud S, Garcia-Risco M, species is emerging as a non-classical animal model in the field of evolutionary developmental Capdevila M, Cañestro C, Palacios Ò biology (a.k.a. evo-devo) especially attractive for its unusually dynamic gene and genome evolution and Albalat R (2021) Modular Evolution and Population Variability (reviewed in Ferrández-Roldán et al., 2019). At genome level, O. dioica has suffered numerous of Oikopleura dioica Metallothioneins. chromosomal rearrangements accompanied by a drastic process of compaction, becoming the Front. Cell Dev. Biol. 9:702688. smallest known chordate genome (Denoeud et al., 2010). At gene level, besides an extraordinary doi: 10.3389/fcell.2021.702688 amount of gene duplications and losses, O. dioica sequences show high evolutionary rates, which Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins are on average two-three times higher than in ascidians and different number of domain repeats (Calatayud et al., 2018). vertebrates (Berna et al., 2012; Berna and Alvarez-Valin, 2014). The arrangement of Cys motifs in O. dioica domains diverges O. dioica probably is the fastest evolving metazoan reported so from that found in the MTs of other tunicates belonging far (Edvardsen et al., 2005; Denoeud et al., 2010). In addition, to the ascidian and thaliacean classes, but it is similar to its pattern of amino acid substitution also shows some unusual that of other appendicularian species of the same genus, traits in comparison with other chordates. Cysteines (Cys, C), O. albicans and O. vanhoeffeni (Calatayud et al., 2021a). for instance, are the less conserved amino acids in O. dioica Comparison of the appendicularian MTs show that the original proteins (Berna et al., 2012; Berná and Alvarez-Valin, 2015), Oikopleura MT domain had twelve cysteines (12C), and that whereas Cys are one of the most conserved amino acids this domain corresponds to previously described C7 + C5 according with classical analyses of protein evolution (Henikoff subunits (Calatayud et al., 2018, 2021a). OdiMT1 would have and Henikoff, 1992; Marino and Gladyshev, 2010). These therefore two 12C domains, but its amino-terminal domain exceptional evolutionary features make comparative studies was “trimmed” to become a t-12C domain that lacks the C5 between O. dioica and other chordate species suitable for subunit. OdiMT2 would be a multi-modular MT derived from understanding the functional and structural limits to which an ancestral copy with a t-12C/12C domain organization, similar chordate genes and proteins can evolve. to the current OdiMT1, that was tandem duplicated five times In protein evolution, domains are considered evolutionary yielding its domain repeat organization (Calatayud et al., 2018). modules, and the majority of proteins of all living beings are In this work, we have pursued the analysis of O. dioica MTs, multi-modular proteins that consist of several domains (Apic paying special attention to their modular configuration. First, et al., 2001). While the creation of multi-modular proteins we have characterized the metal-binding features of the original through shuffling of different domain types has been extensively Oikopleura 12C domains –both the full-length (12C) and the analyzed (Apic et al., 2001; Björklund et al., 2005; Dohmen trimmed (t-12C)–, revealing that although the 12C domain et al., 2020), the evolution of proteins made of tandem domain autonomously coordinates divalent metal ions, the t-12C/12C repeats is less understood (Björklund et al., 2006). To get new pair seems an improved form for divalent metal binding. Second, insights into the functional and structural evolution of these we have shown a direct relationship between the number of the domain repeat proteins, we have focused on the multi-modular t-12C/12C domain repeats and the metal-binding capacity of metallothioneins (MTs), using those of the fast evolving O. dioica OdiMTs. Finally, taking advantage of the high level of genetic species as case study. variation among O. dioica populations (Wang et al., 2020; Metallothioneins are proteins known for their high percentage Bliznina et al., 2021), we have detected population variants of of cysteines (Kojima et al., 1999), which confers them the OdiMT2 with changes in their number of t-12C/12C domains. capability of binding both essential and non-essential transition Our data expose a high structural plasticity of MTs in O. dioica metals (reviewed in Capdevila et al., 2012; Blindauer, 2014). that, as if it was a natural test-bench, seems to be exploring the The Cys residues of MTs are arranged in distinctive motifs (i.e., chordate limits of MT modularity. CxC, CC, and CCC), whose number and distribution led to define different functional domains, originally for mammalian MTs (Braun et al., 1986), and later, in other animal MTs (Riek MATERIALS AND METHODS et al., 1999; Munoz et al., 2002; Baumann et al., 2017; Beil et al., 2019; Calatayud et al., 2021b). In chordates, for instance, Production and Purification of vertebrate and cephalochordate MTs are bi-modular proteins Recombinant Metal-MT Complexes with two domains that have diverse preferences and capacities Production and purification of recombinant metal-MT for binding zinc (Zn), copper (Cu), or cadmium (Cd) ions complexes of proteins with different number and combinations (Capdevila and Atrian, 2011; Vasak and Meloni, 2011; Guirola of t-12C and 12C domains (see Table 1 for details; sequences et al., 2012; Artells et al., 2013). In contrast, most tunicate MTs of the domains are from Norwegian OdiMT1 and OdiMT2 are mono-modular proteins, whose single domain has a pervasive sequences) was performed as described elsewhere (Calatayud preference for Cd(II) ions (Calatayud et al., 2021a). The domain et al., 2018, 2021b). In brief, synthetic cDNAs codifying the configuration of each MT is, indeed, functionally and structurally different constructs of 12-Cys domains were provided by Synbio relevant because domains determine the formation of metal- Technologies (Monmouth Junction, NJ, United States), cloned thiolate clusters: domains with 9 Cys cluster with three divalent in the pGEX-4T-1 expression vector (GE Healthcare, Chicago, metal ions, while 11/12 Cys domains cluster with four divalent IL, United States) and transformed in protease-deficient E. coli metal ions (e.g., mammalian β and α domains, respectively, Otvos BL21 strain. Metal-MT complexes were produced in E. coli and Armitage, 1980; Schultze et al., 1988). In addition, domain BL21 cultures expressing the recombinant plasmids, after analyses have been shown to be helpful for elucidating the induction with isopropyl-β-D-thiogalactopyranoside (100 µM) origin and evolutionary relationships of MTs in diverse groups of and supplementation with ZnCl2 (300 µM), CdCl2 (300 µM), mollusks (Jenny et al., 2016; Nam and Kim, 2017; Calatayud et al., or CuSO4 (500 µM). Metal-MT complexes were purified from 2021b), and to reconstruct the complex evolutionary history of the soluble protein fraction of sonicated bacteria by affinity chordate MTs (Calatayud et al., 2021a). purification of the GST-tagged proteins, and digestion with O. dioica has two MTs, a bi-modular OdiMT1 and a multi- thrombin. Notice that the digestion with thrombin added two modular OdiMT2 (formerly OdMT1 and OdMT2) made of additional residues, Gly and Ser, at the N-terminal end of all Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins purified proteins. These two amino acids do not interfere with of Barcelona (Martí-Solans et al., 2015). Primers and PCR the metal-binding features of recombinant MTs (Cols et al., conditions are listed in Supplementary Table 1. PCR products 1997). The metal-MT complexes were concentrated with a 3 kDa were cloned using Topo TA Cloning Kit of Invitrogen and R Centripep Low Concentrator (Amicon, Merck), and fractionated sequenced at the Scientific and Technological Centers of the on a Superdex-75 FPLC column (GE Healthcare) equilibrated University of Barcelona, using the Big Dye Terminator v3.1 Cycle with 20 mM Tris-HCl, pH 7.0 or with fresh 50 mM ammonium Sequencing Kit (Applied Biosystems) in an automatic sequencer acetate, pH 7.0, and run at 0.8 mL min−1 . The protein-containing (ABIPRISM 310, Applied Biosystems). For Oregonian sequences, fractions, identified by their absorbance at 254 nm, were pooled we took advantage of a data of a genomic shotgun approach. All and stored at −80◦ C until use. new MT sequences have been deposited in Genbank database at NCBI4 . Analysis of Metal-MT Complexes For comparisons, MT sequences from different O. dioica All designed constructs of OdiMTs were characterized by populations were aligned with Aliview program (Larsson, 2014) means of mass spectrometry (ESI-MS) and spectroscopy (ICP- and reviewed manually. Phylogenetic reconstructions were AES). An electrospray ionization mass spectrometry (ESI-MS) based on ML inferences calculated with PhyML v3.0 and Micro Tof-Q Instrument (Brucker Daltonics Gmbh, Bremen, automatic mode of selection of substitution model (Guindon Germany) interfaced with a Series 1100 HPLC pump (Agilent et al., 2010) using protein sequence alignments. Sequences and Technologies) was used to determine the molecular mass of the accession numbers used in this study are provided in the recombinant proteins. The instrument was calibrated with Supplementary Table 2. ESI-L Low Concentration Tuning Mix (Agilent Technologies, United States) and the experimental conditions were set up as follows: injection of 10–20 µL of sample through a PEEK long RESULTS AND DISCUSSION tube (1–1.5 m × 0.18 mm i.d.) at 30–50 µL min−1 ; capillary- counterelectrode voltage, 3.5–5.0 kV; desolvation temperature, Functional Analysis of the Modularity in 90–110◦ C; dry gas, 6 L min−1 . Data was acquired over an m/z OdiMTs range of 800–3,000. The liquid carriers were a 90:10 mixture of Metal-Binding Capabilities of 12C and t-12C Domains 15 mM ammonium acetate and acetonitrile at pH 7.0 and a 95:5 OdiMT1 is a 72 amino acid long protein (20 Cys, 28%) that mixture of formic acid and acetonitrile at pH 2.4. can be divided in three parts: an N-terminal peptide made Element concentrations of S, Zn, Cd, and Cu in the of six amino acids; next, an amino-terminal t-12C domain; sample were determined by Inductively Coupled Plasma Atomic and finally, a carboxyl-terminal 12C domain. OdiMT2 is a Emission Spectroscopy (ICP-AES) by means of a Perkin-Elmer protein of 399 amino acids (123 Cys, 31%) that consists of Optima 4300 DV (Waltham, United States) at the correct the N-terminal peptide, and six tandem repeat units (RU1– wavelength (S, 182.04 nm; Zn, 213.86 nm; Cd, 228.80 nm; Cu, RU6), each one made of a t-12C domain and a 12C domain 324.80 nm) under conventional conditions (Bongers et al., 1988). (Figure 1; Calatayud et al., 2018). To investigate the functionality MTs concentration was calculated based on the S concentration of this modular organization, we studied the formation of metal- obtained by ICP-AES, assuming that all the sulfur measured MT complexes of recombinant proteins containing different comes from peptides’ Cys and Met residues. combinations of t-12C and 12C domains (Table 1). The metal- binding capability of each construct was analyzed by inductively Analysis of MT Variation coupled plasma atomic emission spectroscopy (ICP-AES), and We analyzed the MT sequence variations of four geographically the species recovered identified by electrospray ionization mass distant O. dioica populations: Norway, Japan (Osaka and spectrometry (ESI-MS) (Capdevila et al., 2012). ICP-AES was also Okinawa), Oregon, and Catalonia. We used Norwegian OdiMT1 used for metal-to-protein stoichiometry determination through (NCBI accession number CABV01001936.1) and OdiMT2 (NCBI the measurement of element composition of the samples (S, Zn, accession number CABV01001042.1) sequences retrieved from Cd, and Cu) (Bongers et al., 1988). the Oikobase genome database1 (Denoeud et al., 2010) as We first characterized the metal-binding features of the 12C reference for blast searches in ANISEED2 and NCBI Sequence domain by analyzing its ability to form metal complexes when Read Archives3 for Japanese population (Osaka and Okinawa, expressed alone. Based on OdiMT1 sequence, we designed respectively). Raw sequence data from SRA searches was construct 1.1, which encoded a 42 amino acid peptide (residues assembled using SeqMan 8.0.2 (Pro Assembler) software from the from 31 to 72) comprising the 12C domain of OdiMT1 (Table 1). DNASTAR Lasergene package, and manually inspected in order Notice that the 12C domain of OdiMT1 is, indeed, an 11C to reconstruct the MT sequences. domain due to a Cys to Ser substitution in the carboxyl terminal For Catalonian sequences, we PCR amplified the MT region in comparison with prototypical 12C domain (thus, we genes from specimens captured in the Mediterranean coast of named this domain as 11C/12C; Figure 1). Our results showed Barcelona and cultured in our animal facility at the University that this 11C/12C domain was able to yield almost unique Zn4 - and Cd4 -protein species (Figure 2), in a similar way that other 1 http://oikoarrays.biology.uiowa.edu/Oiko domains with 11 or 12 Cys: the 11C mammal and echinoderm 2 https://www.aniseed.cnrs.fr/ 3 4 https://www.ncbi.nlm.nih.gov/sra/ https://www.ncbi.nlm.nih.gov/genbank/ Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins TABLE 1 | Heterogously produced proteins containing different number and combinations of t-12C and 12C domains. Construct Schemea Domains Amino acid included (Num. C) Num.aab Major metal-protein species OdiMT (full protein)c N-term + t-12C + 12C From 1 to 72 (20) 72 Cd7 /Zn7 /Cu12 OdiMT1-Construct 1.1 12C From 31 to 72 (11) 42 Cd4 /Zn4 /Cu9 OdiMT1-Construct 1.2 N-term + 12C From 1 to 6 (1) + from 31 to 72 (11) 48 Cd5 /Zn2 /Cu4 OdiMT1-Construct 1.3 N-term + 12C From 1 to 30 (9) + from 55 to 72 (4) 48 Cd5 /Zn4 /Cu11 OdiMT1-Construct 1.4 t-12C From 7 to 30 (8) 24 Cd3 /Zn3 /ndd OdiMT1-Construct 1.5 t-12C From 31 to 54 (7) 24 Cd3/ Zn3 /nd OdiMT2 (full protein)c From 1 to 399 (123) 399 nd/nd/nd N-term + [t-12C+12C] ×6 OdiMT2-Construct 2.1 From 1 to 199 (61) + from 396 to 399 (2) 203 nd/Cd22 /nd N-term + [t-12C+12C] ×3 OdiMT2-Construct 2.2 From 201 to 399 (62) 199 Zn20 /Cd22 /nd [t-12C+12C] ×3 a Colored boxes represents the six amino acids of the N-terminus (black), a “trimmed” 12C domain (green) and a complete 12C domain (orange). b This number does not include two additional amino acids (Gly and Ser) added at the N-terminus of the recombinant proteins as consequence of the experimental design for the expression and purification of the metal-protein complexes (see section “Materials and Methods” for details). c OdiMT1 and OdiMT2 full proteins were analyzed in Calatayud et al. (2018). d nd, no detected. α domains (Stillman et al., 1987; Tomas et al., 2013), the 12C of the protein (from 55 to 72) (Table 1). Although both mollusk α domains (Digilio et al., 2009), the insect 12C MTs (Egli constructs rendered Cd5 -protein complexes as major species et al., 2006) and the ascidian and thaliacean 12C MTs (Calatayud according to ESI-MS data (Figure 2), the samples recovered et al., 2021a). These results revealed a significant structural from both, Zn- and Cd-supplemented cultures, resulted to be and functional autonomy of O. dioica 11C/12C domain, which a significant mixture of metal-protein complexes and some was able to form stable metal-protein clusters with Zn(II) and Cd-protein species contained sulfide labile ligands (Figure 2). Cd(II). Regarding the metal preference of this domain, the The presence of S2− ions is probably due to the incapability mixture of multiple Cun -protein complexes (n ranging from of these artificially designed peptides to build a stable metal 4 to 10) in Cu(II) surplus productions (Figure 2) discarded cluster as these “extra” ligands can aid in the stabilization of a preference of this domain for monovalent Cu(I) ions. In the metal clusters (Capdevila et al., 2005). This, together with addition, a preference for Cd(II) over Zn(II) might be indirectly the heterogeneity of the samples suggested, therefore, that the inferred not only from the neatness of the ESI-MS spectra of 1.2 and 1.3 constructs have not improved the metal binding the Cd-preparation but also from the fact that the domain abilities of construct 1.1, questioning the contribution of the extra rendered homometallic Cu(I) species in the Cu-preparations Cys in the N-terminal peptide to the metal coordination, and (Figure 2), which is characteristic of Cd-thioneins, whereas reinforcing the functional entity of the 11C/12C domain as an Zn-thioneins yield heterometallic Zn/Cu-MT complexes when efficient solution for coordinating divalent metal ions emerged expressed Cu-enriched media (Palacios et al., 2011). Overall, we during the evolution of the Oikopleura lineage. concluded that the 11C/12C domain of OdiMTs formed stable Next, we investigated the functionality of the t-12C domain, clusters with four Cd(II) ions, which was in agreement with which is a 12C domain lacking the carboxyl C5 subunit, and the biochemical features reported for the full OdiMT1 protein thereby, containing only 7/8 cysteines. We analyzed the metal- (Calatayud et al., 2018). binding features of two different t-12C domains expressed by Although we obtained reliable results for the 11C/12C domain two constructs. Construct 1.4 produced the t-12C domain of of OdiMT1, we wondered if the extra Cys found the N-terminal OdiMT1, from residues 7 to 30. Construct 1.5 produced the t- peptide of OdiMT1 could compensated the loss of one Cys 12C domain resulting from the truncation of the last 18 residues in this domain, significantly improving its metal coordination of the 11/12C domain of OdiMT1, from residues 31 to 54 features (i.e., enhancing specificity, increasing stability or metal- (Table 1). Our analyses showed that both constructs mainly bind to-protein stoichiometries). To test this possibility, we designed three divalent metal ions, either Zn(II) or Cd(II) (Figure 2). two constructs: construct 1.2, which expressed a 48 amino acid This metal-to-protein stoichiometry agreed with the possibility peptide comprising the six N-term residues of OdiMT1 (which that the seven divalent metal ions (M7 ) coordinated by the full included the extra Cys) fused to the 11C/12C domain (Table 1); OdiMT1 protein are organized in two metal clusters: an 11C/12C and construct 1.3, which also expressed a 48 amino acid peptide domain cluster with four metal ions (MII 4 ) at the carboxyl-end comprising the six N-term residues fused to a 12C domain of the MT, and a t-12C domain cluster with three metal ions resulting from the combination of the t-12C domain of OdiMT1 (MII 3 ) at the amino-term region. The t-12C domains rendered, (from 7 to 30) with the 18 last residues of the carboxyl-terminus however, multiple metallospecies and some Cd-protein species Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins FIGURE 1 | Domain organization of O. dioica metallothioneins. (A) Amino acid alignment of O. dioica OdiMT1 and OdiMT2 split in its repeat units (RU1–RU6). The sequence of O. vanhoeffeni MT (OvaMT1) split in its repeat units (RU1–RU3) has been included to show that the amino-terminal 12C domains of OdiMT1 and of the RU of OdiMT2 are “trimmed” (t-12C; green background), and lacks the C5 subunit. Conserved cysteines are highlighted in gray. The arrangement of Cys motifs of each 12C domain is showed below the alignment. Notice that the 12C domain (orange background) corresponds to previously described C7 + C5 subunits (Calatayud et al., 2018). (B) Schematic representation of the modular organization of OdiMT1 and OdiMT2 proteins. OdiMT1 consists of 6 amino acids of the N-terminus (black box) followed by a single repeat unit (RU) with a t-12C domain (green box) and a 12C domain (orange box), while OdiMT2 is made of six RU, RU1–RU6. with sulfide ligands (Figure 2), which suggested that the t-12C metallospecies are common in multi-modular MT productions domain expressed alone did not efficiently bind the metals by (Palacios et al., 2014; Iturbe-Espinoza et al., 2016) could account itself, and that it would require the neighboring 11C/12C domain for the slightly high metal content (up to 24 ions) observed in to properly coordinate the seven metal ions in the full MT protein some of them. Interestingly, ESI-MS spectra of both constructs (Calatayud et al., 2018). at pH 2.4 showed species loaded with 4 and 8 divalent metal ions (Figure 3). These species might be reflecting the t-12C/12C Metal-Binding Capabilities of t-12C/12C Repeats organization of each RU, in which the “standard” 12C domain We also analyzed the metal-binding features of tandem repeats of would be more reluctant to release its four metal ions than the t-12C/12C domains as they are found in OdiMT2. We designed “trimmed” t-C12 domain. Overall, the metal stoichiometry of two constructs. Construct 2.1, encoding a 203 amino acid protein, the partial MTs pointed to a direct relationship between the comprised the three amino-terminal RU (i.e., RU1 RU2 RU3 ; number of the t-12C/12C repeats and the metal-binding capacity residues 1–199), plus the four last amino acids (two of them Cys) of multi-modular OdiMTs. of the carboxyl-end (from 396 to 399). Construct 2.2, encoding In conclusion, our analyses suggested that during the a 199 amino acid protein, comprised the three carboxyl-terminal evolution of MTs in O. dioica, an ancestral 12C domain was RU (i.e., RU4 RU5 RU6 ) plus the four last amino acids (from 201 tandem duplicated. The N-terminal 12C copy was trimmed (t- to 399) (Figure 1 and Table 1). Recombinant synthesis of the 12C domain), partially losing its autonomy for metal binding, two partial OdiMT2 proteins –RU1 RU2 RU3 and RU4 RU5 RU6 – and the t-12C/12C pair became the optimized functional in Zn(II)- and Cd(II)-enriched E. coli cultures rendered a low unit. Afterward, this primeval t-12C/12C OdiMT gene was yield of metal-protein complexes, but ICP-AES analyses showed duplicated. While one of the duplicates remained unaltered as that both produced complexes with divalent metal ions, mostly the current OdiMT1, the other copy suffered several internal with Cd(II) ions. ESI-MS analyses (Figure 3) showed mixtures of tandem duplications of the functional t-12C/12C pair in an metal-protein complexes with different stoichiometries, and Cd- evolutionary process that stepwise changed the number of protein complexes with sulfide ligands, being Zn20−21 , Cd22−23 , domain repeats affecting the metal binding capacity of the and Cd23−24 S-complexes the major species (Figure 3). The new multi-modular OdiMT2. Domain expansions that generate metal-to-protein stoichiometry of these species was close to what high metal-binding capacity MTs such as OdiMT2, gene was expected considering that the partial proteins consist of duplications that lead to the amplification of the number of three RU, each one equivalent to a full-length OdiMT1 that MT genes such as those of CUP1 in yeast (Adamo et al., binds seven Zn(II)/Cd(II) ions, i.e., 3 RU × 7 MII = 21 total 2012), and mutations in regulatory regions that lead to the divalent metal ions. The two additional Cys at the carboxyl- overexpression of MTs in insects (Costa et al., 2012; Catalan end, the presence of sulfide ligands, and the fact that multiple et al., 2016) appear to be different ways of increasing the Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins FIGURE 2 | Deconvoluted ESI-MS spectra recorded at pH 7.0 of OdiMT1 constructs 1.1 (A–C), 1.2 (D–F), 1.3 (G–I), 1.4 (J–L), and 1.5 (M,N) bioproduced with Cd (A,D,G,J,M), Zn (B,E,H,K,N), or Cu (C,F,I,L). Y -axis represents the relative intensities of the peaks while the X-axis represents the m/z ratio equivalent to the MW (Da). The metal-to-protein ratio obtained by ICP-AES is included on the top left of each panel. It is indicated those cases in which the sample concentration was lower than the limit of detection (LOD). Glycosylated species are marked with an asterisk (*). physiological capabilities of the organisms to adapt to diverse Berna and Alvarez-Valin, 2014), and significant level of conditions of metal bioavailability and other environmental sequence variation detected between O. dioica populations stress situations. (Wang et al., 2015, 2020; Bliznina et al., 2021), prompted us to investigate the OdiMT sequences in several O. dioica Genetic Variation in O. dioica MTs populations worldwide distributed. We analyzed the MT The peculiar structural and evolutionary characteristics sequences from animals from Norway (NOR), Oregon (ORE), of OdiMTs together with the high evolutionary rate Japan (Japanese specimens were from two different localizations, of O. dioica (Denoeud et al., 2010; Berna et al., 2012; Osaka (OSA) and Okinawa (OKI), and we have analyzed Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins FIGURE 3 | Deconvoluted ESI-MS spectra recorded at pH 7.0 (A,B,E,F) and pH 2.4 (C,D,G,H) of OdiMT2 constructs 2.1 (A–D) and 2.2 (E–H) bioproduced with Zn (A,C,E,G) or Cd (B,D,F,H). Y -axis represents the relative intensities of the peaks while the X-axis represents the m/z ratio equivalent to the MW (Da). The metal-to-protein ratio obtained by ICP-AES is included on the top right of panels (A,B,E,F). It is indicated those cases in which the sample concentration was lower than the limit of detection (LOD). them separately), and Catalonia (CAT), representing four protein sequence comparisons reveal amino-acid identities geographically distant O. dioica populations: north Atlantic, ranging from 94.4% between Norway and Barcelona, up to eastern Pacific, western Pacific and Mediterranean populations, 63.9% when compared with the sequence of Okinawa, which respectively. We used as guiding reference the Norwegian appeared as the more distant population to any other one sequences (OdiMTNOR ) (Figure 4 and Supplementary (Table 2). In contrast to the overall sequence variation – Figure 1) because they were the first sequences identified 82.6 and 87.7% of average amino acid identity (excluding (Calatayud et al., 2018). the divergent Okinawa sequences) for OdiMT1 and OdiMT2, Both OdiMT1 and OdiMT2 genes were present in all respectively, Cys residues were nearly invariants, with 98.3% analyzed populations (Figure 3), but comparison among preservation. This high Cys conservation contrasted with the populations revealed important differences affecting three fact that Cys are the less conserved amino acids in O. dioica aspect: (i) sequence variability, (ii) presence of non-functional proteins (Berna et al., 2012; Berná and Alvarez-Valin, 2015), allelic variants, and (iii) differences in the number of RU suggesting that Cys substitutions are negatively selected due in the case of OdiMT2. Regarding sequence variability, to the functional restrictions imposed by metal coordination. Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins FIGURE 4 | Amino acid alignments of OdiMT1 and OdiMT2 variants form Norwegian (NOR), Catalonian (CAT), Oregonian (ORE), Osaka (OSA), and Okinawa (OKI) O. dioica populations. Norwegian sequences are used as reference; dots and dashes indicate amino acid identity and gaps, respectively. Black arrowheads indicate the intron positions relative to the amino acid sequences. The sequences of two hypothetical OdiMT pseudogenes –i.e., 9 OdiMT1CAT , lacking the first 12 residues, and 9 OdiMT2CAT , with a premature stop codon (*)– are also included. Trimmed 12-Cys domains (green box) and full-length 12-Cys domains (orange box) at the N- and C-terminal regions of the RU, respectively, are depicted. Moreover, the fact that amino acid identities persistently were that MT variability among populations might probably be under slightly lower than nucleotide identities (Table 2) indicated positive selection. that nucleotide substitutions are significantly affecting non- Noteworthy, during the identification of the Catalonian MTs, synonymous positions, which can be considered an indication we detected some non-functional allelic variants of OdiMT1 Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins TABLE 2 | Percentage identitya of nucleotide (above diagonal) and amino acid sequences (below diagonal) of the OdiMT coding regions from five different O. dioica populations, Norway (NOR), Catalonia (CAT), Osaka (OSA), Okinawa (OKI), and Oregon (ORE). OdiMT1NOR OdiMT1CAT OdiMT1OSA OdiMT1OKI OdiMT1ORE OdiMT1NOR – 97.26 84.02 73.87 83.56 OdiMT1CAT 94.44 – 84.47 72.97 83.56 OdiMT1OSA 76.38 76.38 – 70.58 93.52 OdiMT1OKI 67.12 64.32 63.88 – 69.09 OdiMT1ORE 79.16 79.16 90.27 66.66 – OdiMT2NOR OdiMT2CAT OdiMT2OSA OdiMT2OKI OdiMT2ORE OdiMT2NOR – 96.42 86.62 70.40 86.68 OdiMT2CAT 94.07 – 85.81 70.72 87.42 OdiMT2OSA 85.42 81.16 – 73.95 92.77 OdiMT2OKI 67.79 68.75 67.59 – 73.76 OdiMT2ORE 87.00 86.67 91.69 69.23 – a For the calculation of sequence identities, a gap between two sequences has been considered as a single difference. FIGURE 5 | Schematic representation of the organization of RU in OdiMTs. The t-12C/12C RU in OdiMT1s (top) and in OdiMT2 variants with six (OdiMT2NOR ), four (OdiMT2CAT ), seven (OdiMT2ORE ), five (OdiMT2OSA ), and three (OdiMT2OKI ) RU are depicted following the color code of Figure 1. Repeats are positioned on the basis of intron positions (black arrowheads; dashed arrowheads denote absent introns) and phylogenetic analysis (Supplementary Figure 2). Exon and intron sizes are indicated. The high divergence of the RU of OdiMT2OKI is highlighted by the dotted pattern. and OdiMT2 genes. One variant displayed a 35 nucleotide by 9-OdiMT2CAT was unlikely because only 44% (41 out of (nt) deletion at the 5’-end of the OdiMT1 genomic region 92) of the amino acids were similar to the OdiMT2 sequence that eliminated the first exon, and therefore likely resulted in (Figure 4). The presence of these non-functional alleles for both a pseudogene, 9-OdiMT1CAT (Figure 4 and Supplementary MTs,9-OdiMT1CAT , and 9-OdiMT2CAT , in the Mediterranean Figure 1). In another variant, we found a 7 nt deletion at the population opened the possibility that some O. dioica specimens beginning of the exon 2 of the OdiMT2 that caused a frame-shift might lack functional MTs. Further analyses of the presence and mutation that resulted in a change of the amino acid sequence frequencies of non-functional variants in different populations and a premature stop codon (Figure 4 and Supplementary could reveal different selective pressures related to variations in Figure 1). The functionality of the truncated protein codified heavy-metal amounts of different environments. Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 July 2021 | Volume 9 | Article 702688 Calatayud et al. Domain Repeats in Modular Metallothioneins The most conspicuous difference of OdiMT genes between although we still do not know if adaptive selection to different O. dioica populations was the identification of OdiMT2 encoding environmental conditions would be driving the changes in the proteins with variable number of RUs (i.e., t-12C/12 pairs) number of the t-12C/12C repeats, the more genetic variation (Figures 4, 5). OdiMT2ORE with seven repeats (RU1 –RU7 ) there is, the greater the capacity for adaptive evolution of a was the longest one, followed by OdiMT2NOR , with six biological system. repeats, OdiMT2OSA , with five repeats, OdiMT2CAT , with four repeats, and OdiMT2OKI , with three repeats. The conservation in the OdiMT2 alleles of the introns both at the 50 - and DATA AVAILABILITY STATEMENT 30 -ends of the gene (Figure 5), together with phylogenetic reconstructions based on the sequence alignments of the repeats The datasets presented in this study can be found in (Supplementary Figure 2), suggested that the increase/decrease online repositories. The names of the repository/repositories in the number of repeats was the result of internal tandem and accession number(s) can be found in the article/ duplications/losses due to recombination events. Phylogenetic Supplementary Material. analysis suggested that some expansions of the RU preceded the divergence of populations, while some independent gains or losses also occurred (Supplementary Figure 2). In OdiMT2OKI , AUTHOR CONTRIBUTIONS for instance, duplications of RU seemed to have taken place independently form other OdiMT2s, suggesting again that this RA was responsible for the project coordination. RA, CC, population is the most divergent among all populations. Finally, ÒP, and MC conceived and designed the experiments. SC, the fact that in all variants the repeat units comprised complete CC, and RA collected the MT sequences from databases and t-12C/12C pairs supported a modular, step-wise evolution for elaborated the evolutionary inferences. SC performed the cloning OdiMTs, and agreed with the functional analysis of the domains and recombinant synthesis of the analyzed proteins. MG-R that demonstrated that despite the autonomy of the 12C domain, performed the ICP-AES, CD, UV-vis, and ESI-MS experiments. the t-12C/12C pair appeared as an improved functional unit for MG-R, ÒP, and MC analyzed the metal-binding data. SC and divalent metal binding (see section “Functional Analysis of the MG-R were responsible of the figures and tables. All authors Modularity in OdiMTs”). discussed the experimental results, were responsible for writing Proteins with domain repeats have been observed to be the manuscript, commented on the manuscript, and agreed to particularly common in multicellular species (Apic et al., 2001), its final version. especially in vertebrates (Björklund et al., 2006). The exact mechanism for repeat expansion remains to be discovered, but evidence of the expansion of repeats come from the fact that FUNDING orthologous proteins might have different numbers of domain repeats in different species (Björklund et al., 2006). The number RA was supported by BIO2015-67358-C2-1-P. CC was supported of domain repeats is, however, well conserved within a species, by BFU2016-80601-P and PID2019-110562GB-I00. MC and ÒP and it does not present intraspecific variability in terms of were supported by BIO2015-67358-C2-2-P from the Spanish repeat unit gains/losses (Schaper et al., 2014). The population Ministerio de Ciencia e Innovación. MC and ÒP were members variability of OdiMT2s here exposed is, therefore, surprising, of the “Grup de Recerca de la Generalitat de Catalunya,” ref. and O. dioica challenges again the standard patterns of gene and 2017SGR-864, and RA and CC of ref. 2017SGR-1665. MG-R protein evolution, opening a new perspective in the functional acknowledges to the UAB the PIF grant. and structural evolution of domain repeat proteins. In summary, our results suggested that the modular organization provides MTs with a high structural and functional ACKNOWLEDGMENTS plasticity and dynamism, as it demonstrates the detection of variants with variable number of t-12C/12C repeats. These We acknowledge the Centres Científics i Tecnològics (CCiT) features seem to have facilitated the creation of large multi- de la Universitat de Barcelona (DNA sequencing) and the modular MTs with high cysteine content and a high capacity of Servei d’Anàlisi Química (SAQ) de la Universitat Autònoma de metal binding. Large multi-modular MTs have been described Barcelona (ICP-AES, ESI-MS) for allocating instrument time. We in other organisms (Palacios et al., 2014; Iturbe-Espinoza et al., also thank to Sebastian Artime for experimental support. 2016; Dallinger et al., 2020; Calatayud et al., 2021a,b), some of them associated with biological adaptations (Palacios et al., 2014; Iturbe-Espinoza et al., 2016; Jenny et al., 2016; Baumann et al., SUPPLEMENTARY MATERIAL 2017). These large MTs did not show, however, the complexity level reached by O. dioica proteins, nor the inter-population The Supplementary Material for this article can be found variability described here. 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This is an open-access article distributed under the terms of the Creative Schaper, E., Gascuel, O., and Anisimova, M. (2014). Deep Conservation of Human Commons Attribution License (CC BY). The use, distribution or reproduction in Protein Tandem Repeats within the Eukaryotes. Mole. Biol. Evol. 31, 1132–1148. other forums is permitted, provided the original author(s) and the copyright owner(s) doi: 10.1093/molbev/msu062 are credited and that the original publication in this journal is cited, in accordance Schultze, P., Wörgötter, E., Braun, W., Wagner, G., Vašák, M., Kägi, J. H. R., et al. with accepted academic practice. No use, distribution or reproduction is permitted (1988). Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous which does not comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 July 2021 | Volume 9 | Article 702688