A New Insight into Metallothionein (MT) Classification and Evolution

Abstract

We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous Escherichia coli expression in zinc-and copper-supplemented medium was achieved for MTH, the two domains ␤␤MTH and ␤␣MTH and three site-directed mutants, ␤␤C9H, ␤␣C37H, and ␤␣E31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of M The metallothionein (MT) 1 superfamily comprises a wide range of low molecular weight polypeptides, sharing the ability to chelate heavy metal atoms (1). MT are present in all animal phyla and in most fungi and plants and have been related to metal homeostasis and detoxification, although their definite physiological function is still a matter of debate (2-5). To classify these extremely heterogeneous amino acid sequences, three different classes were proposed: Class I, comprising all the MT with clear sequence similarity to horse liver MT 1; Class II, including all the sequences with no clear similarity to horse liver MT 1; and Class III for the phytochelatins and vegetal ␥-glutamyl peptides of enzymatic origin (1). More recently, a new classification consisting of a singular set for each individual eukaryotic taxon has been proposed (www.unizh.ch/ ϳmtpage/classif.html). Both systematizations are based on primary structure data but do not include any functional or evolutionary information. Conventionally, terminology and standards corresponding to the canonical mammalian MT molecule have been extended to describe amino acid sequence and metal cluster features of newly described MT forms. Mammalian MT folds into a twodomain structure when coordinating divalent metal ions. The N-terminal segment (␤ domain), with 9 cysteines in Cys-XaaCys arrays, binds three M(II) ions, and the C-terminal segment (␣ domain), with 11 cysteines, some of which in Cys-Cys tandems binds four M(II) ions (6, 7). A higher binding affinity for monovalent (copper) than for divalent (zinc and cadmium) ions was described for the mammalian ␤ domain, with an opposite trend for the ␣ domain (8). Contemporarily, Saccharomyces cerevisiae MT CUP1 was characterized. The synthesis of this protein was induced by copper, and the MT recovered contained copper ions exclusively (9). Because the CUP1 cysteine distribution aligned well to the ␤ domain of mammalian MT, it was assumed that proteins with a primary structure similar to the mammalian ␤ domain would exhibit copper binding rather than zinc binding preferences. On the basis of their amino acid alignments, further application of the ␤/␣ domain terminology led to the consideration of crustacean peptides as ␤-␤ MT (10) and thus putative copper thioneins. This was corroborated by the three divalent ions contained in each domain of the crustacean Cd-MT structures solved by NMR (11, 12) and thus comparable with the Cd 3 -␤MT fragment in mammalian MT.A survey of the literature on naturally occurring and metalinduced crustacean MT ...

Functional Differentiation in the Mammalian Metallothionein Gene Family

“…This independent behavior could be extended to the folding of each domain in Zn 7 -MT, since comparison of the CD fingerprint of Zn 7 -MT4 with that of the sum of Zn 4 -␣MT4 and Zn 3 -␤MT4 revealed only minor differences (Fig. 2), even less significant than those reported for MT1 (21) or Homarus americanus metallothionein (20). Thus far, MT4 Zn(II) binding behavior fully mimics that of MT1, although comparison of the CD spectra revealed lower chiralities for the Zn-MT4 peptides with respect to the paralogous Zn-MT1 forms.…”

“…In both cases, the transformed E. coli cells were grown as described in Ref. 20, supplementing the medium either with ZnCl 2 or CdCl 2 to a final concentration of 300 M. In order to characterize the MT4 copper coordination behavior, and due to the special significance of these data, synthesis of MT4 and its independent domains in coppersupplemented medium were assayed in different culture conditions. In short, induction times assayed were 3 h for ␣MT4 and ␤MT4 and 2 and 3 h for the entire MT4; isopropyl-1-thio-␤-D-galactopyranoside final concentration assayed was 100 mM for ␤MT4 and 100 and 200 mM for ␣MT4 and the entire MT4; culture volumes were 1.5 and 5 liters for ␣MT4 and ␤MT4 and 1, 1.5, and 5 liters for the entire MT4.…”

“…The protein distance and treebuilding applications are included in the Phylip software package (37). The protein sequence similarity analyses were performed using the clusters of zinc-thionein and copper-thionein MT sequences previously characterized (20,27).…”

“…This, together with the following observations, led us to propose MT4 as a Cu-thionein composed of two Cu-thionein domains, whereas MT1 was previously classified as a Zn-thionein formed by a Cu-thionein (␤MT1) and a Zn-thionein (␣MT1) domain (20). First, recombinant syntheses in copper-enriched medium afforded homometallic Cu-MT4 species, in contrast to heterometallic Zn 3 Cu 7 -MT1 and Zn 1 Cu 4 -␣MT1 (23).…”

“…Subsequent analyses of the in vitro affinity of the MT1 fragments to physiological ions (zinc and copper) showed that monovalent metal ions preferentially bind to the ␤MT1 domain, whereas divalent ions prefer to occupy the ␣MT1 counterpart (15)(16)(17)(18)(19). Further characterization of the metal aggregates obtained by recombinant synthesis of several MTs provided knowledge on their in vivo metal binding preferences, which allowed us to set up a new comprehensive frame for MT classification and evolution (20). Thus, on the basis of structure/function relationships, we identified MT1 and ␣MT1 as Zn-thioneins (21-23) and the independent ␤MT1 as a Cu-thionein (24,25).…”