Gains, losses and changes of function after gene duplication: study of the metallothionein family

Abstract

Metallothioneins (MT) are small proteins involved in heavy metal detoxification and protection against oxidative stress and cancer. The mammalian MT family originated through a series of duplication events which generated four major genes (MT1 to MT4). MT1 and MT2 encode for ubiquitous proteins, while MT3 and MT4 evolved to accomplish specific roles in brain and epithelium, respectively. Herein, phylogenetic, transcriptional and polymorphic analyses are carried out to expose gains, losses and diversification of functions that characterize the evolutionary history of the MT family. The phylogenetic analyses show that all four major genes originated through a single duplication event prior to the radiation of mammals. Further expansion of the MT1 gene has occurred in the primate lineage reaching in humans a total of 13 paralogs, five of which are pseudogenes. In humans, the reading frame of all five MT1 pseudogenes is reconstructed by sequence homology with a functional duplicate revealing that loss of invariant cysteines is the most frequent event accounting for pseudogeneisation. Expression analyses based on EST counts and RT-PCR experiments show that, as for MT1 and MT2, human MT3 is also ubiquitously expressed while MT4 transcripts are present in brain, testes, esophagus and mainly in thymus. Polymorphic variation reveals two deleterious mutations (Cys30Tyr and Arg31Trp) in MT4 with frequencies reaching about 30% in African and Asian populations suggesting the gene is inactive in some individuals and physiological compensation for its loss must arise from a functional equivalent. Altogether our findings provide novel data on the evolution and diversification of MT gene duplicates, a valuable resource for understanding the vast set of biological processes in which these proteins are involved.

Figure 1. Bayesian phylogenetic analysis of the MT family.

The gene tree was constructed using coding sequences from the Ensembl database (Table S1). MT1/MT2, MT3 and MT4 clusters are represented in blue, red and green, respectively. Posterior probability values are given for branch support. Scale bar stands for the number of replacements per site.

Figure 2. Illustration of the MT family in humans and mice.

In mice, the family comprises four functionally active genes (mt1 to mt4). In humans, the family harbours a single-copy of MT2, MT3 and MT4, and by a tandemly duplicated array of the MT1 duplicates spanning about 66.6 Kb, where eight active genes (MT1A to MT1J, MT1L, MT1M and MT1X) and five pseudogenes (MT1L, MT1J, MT1D, MT1C and MT1I) are known. The direction of transcription for each active gene is indicated by an arrow. Genes are coloured as follows: MT1 (dark blue), MT2 (light blue), MT3 (red) and MT4 (green). Pseudogenes are represented by dashed boxes. Numbers corresponding to the sizes of gene and intergenic regions are given in Kb.

Figure 3. Sequence comparison and structural features of MT proteins.

(A) Sequence alignment for human (Hs) and mouse (Mm) proteins. Residues spanning α and β domains are indicated as well as are the invariant metal-binding cysteine residues. Sequences were aligned with ClustalW (B) Structural representation of the mouse Mt2 (PDB 4mt2) showing the two metal-binding clusters and the detailed spatial organisation of the cysteine residues with the S-atoms oriented towards metal ions. Elements are coloured as follows: S (yellow), O (red), N (dark blue), Zn (purple) and Cd (orange).

Figure 4. The amino acid identity matrix for human and mouse proteins.

Amino acid identity for each pairwise comparison between a human and a mouse protein is given as percentage values.

Figure 5. The reconstructed open reading frame of MT1 pseudogenes.

The reconstruction was performed using the genomic sequence of human MT1 pseudogenes to a homology-based comparison with the functional MT1E. Each exon is shown in a separate row. Amino-acids are represented in single-letter code above the corresponding codon. Strong deleterious mutation candidates (loss of invariant cysteines, gain of aromatic residues, indels and splice-site mutations) are underlined.

Figure 6. Expression profile of MT genes.

(A) Expression of the MT genes in human and mouse tissues represented as log₂ of EST counts by colour coding. (B) RT-PCR analysis of human MT2, MT3 and MT4 in 15 tissues using GAPDH as control.