Metallothioneins of the South American opossum, Monodelphis domestica

Metallothioneins are small, cysteine-rich, metal-binding proteins which occur ubiquitously throughout the animal kingdom. After several decades of research, a definitive function for these metallothioneins has yet to be clarified. Many metallothioneins have been characterised in the placental mammals (subclass Eutheria) and their occurrence in complex multigene families has been confirmed. Subtle differences in protein structure and expression exist between metallothionein isoforms but it has yet to be established whether the various isoforms play differing roles physiologically. Of the vertebrates, the mammals alone manifest complex MT gene families. The birds, by contrast, express one or rarely two metallothionein genes. The marsupials represent a mammalian subclass (Metatheria) distinct from the placental mammals. Gross differences in reproductive biology distinguish the two groups, but in other respects the marsupials differ only slightly from placental mammals in fundamental physiology. From several perspectives, characterisation of a marsupial MT should extend our understanding of the physiological role and the evolution of the mammalian metallothioneins. Marsupial metallothionein is identified here for the first time. Several independent marsupial MT sequences were isolated from both liver and brain-derived cDNA libraries. The predicted marsupial MT proteins do not conform closely to eutherian isoforms when the traits of cognate proteins are compared, and cannot therefore be accommodated within the current classification system. The tissue-specific expression of the marsupial metallothioneins was investigated to further clarify their relationship to the eutherian metallothioneins. Here too, no confident correlation is possible. The specific and predictable expression profile of MT in the perinatal eutherian was compared with that of the marsupial neonate, which is relatively and strikingly underdeveloped. Marked differences in these profiles were observed. Like the eutherian mammals, the marsupial mammals are here shown to express a complex family of metallothionein genes. Phylogenetic analysis of the vertebrate metallothionein gene family indicates that the amplification of ancestral genes into multiple isoforms occurred independently in both mammalian lineages. It can be inferred that the multiplicity of MT isoforms has arisen not just in response to the physiological requirements that distinguish the mammals, but to some stimulus that acted upon their particular physiology a considerable time after its establishment. Two metallothionein cDNA sequences from the echidna, an Australian monotreme of the mammalian subclass Prototheria, are included in the analysis and confirm these conclusions. It is shown that the mammalian metallothioneins have evolved from an MT3 homologue closely related to bird MT. The eutherian MT1 and MT2 isoforms arose subsequent to the separation of the eutherian and metatherian lineages, approx. 120 myrs ago. The position of the MT4 isoform is less clear but is likely to represent the most ancient of the metallothionein proteins.