Structure and expression of mammalian metallothioneins

Metallothioneins (MTs) are metal binding proteins which have been implicated in a variety of physiological roles, including heavy metal detoxification, metal homeostasis and free radical scavenging. In both the human and the sheep there are multiple MT isoforms which may provide functional diversity. In both species differential regulation and expression patterns of MT isoforms have been reported. The work presented here investigates the expression and gene structure of MTs in these two mammalian species; firstly investigating MT in the developing sheep brain, and secondly studying a novel human MT isogene, MTIL. The underlying theme is to correlate the structure and expression profiles of specific MT isoforms with their proposed physiological functions. Since the discovery of a novel MT in humans, MT-III, which is brain specific, much interest has centred on the expression and role of all MTs in the brain. In this thesis, the sheep was used as a model system in which to study the neural expression of MT at the RNA and protein level. MT-I and -II expression was found to be correlated with the development of glial cells, and limited solely to glial cells including foetal oligodendrocytes, which were previously not suspected to be involved in MT physiology. Distinctive shifts in the regional and cell-type expression of MT were observed during neural development, and these are discussed in terms of the functional role of MT. The expression of MT-III, the brain specific isoform, is of current interest. This thesis reports the cloning of the gene encoding this isoform from a sheep brain cDNA library. Although homologous to MT-III isoforms from other species, the predicted sheep MT-III protein sequence contains several unique features, including the deletion of a highly conserved 3 amino acid sequence, and thus may have altered metal binding properties. Sheep MT-III mRNA was shown to follow the same regional and developmental expression patterns as MT-I and -II mRNA. This unique MT-III isoform may be useful to resolve pending questions about the role of MT-III in the brain, and the apparent neurotrophic actions of this isoform. This work also investigates a novel human MT isogene, MTIL, which contains a TGA ("stop") codon midway through the transcribed region. By analogy to previously characterised human MT genes, this codon interrupts the putative coding region of MTIL, suggesting the isogene is either not functional, expresses a truncated protein or potentially, produces a novel selenoprotein. In previous work, the author has shown that the gene has biological activity since it can confer resistance to cadmium to a transfected cell line. In this thesis, MTIL was shown to produce a full length mRNA transcript when cloned into expression vectors, and furthermore expression of MTIL mRNA was detected in a range of endocrine tissues and lymphocytes by northern blotting and RT-PCR. These findings confirm that MTIL has the potential to contribute to the repertoire of MT expression in the human