Post-duplication charge evolution of phosphoglucose isomerases in teleost fishes through weak selection on many amino acid sites

Abstract

Background: The partitioning of ancestral functions among duplicated genes by neutral evolution, or subfunctionalization, has been considered the primary process for the evolution of novel proteins (neofunctionalization). Nonetheless, how a subfunctionalized protein can evolve into a more adaptive protein is poorly understood, mainly due to the limitations of current analytical methods, which can detect only strong selection for amino acid substitutions involved in adaptive molecular evolution. In this study, we employed a comparative evolutionary approach to this question, focusing on differences in the structural properties of a protein, specifically the electric charge, encoded by fish-specific duplicated phosphoglucose isomerase (Pgi) genes.

Results: Full-length cDNA cloning, RT-PCR based gene expression analyses, and comparative sequence analyses showed that after subfunctionalization with respect to the expression organ of duplicate Pgi genes, the net electric charge of the PGI-1 protein expressed mainly in internal tissues became more negative, and that of PGI-2 expressed mainly in muscular tissues became more positive. The difference in net protein charge was attributable not to specific amino acid sites but to the sum of various amino acid sites located on the surface of the PGI molecule.

Conclusion: This finding suggests that the surface charge evolution of PGI proteins was not driven by strong selection on individual amino acid sites leading to permanent fixation of a particular residue, but rather was driven by weak selection on a large number of amino acid sites and consequently by steady directional and/or purifying selection on the overall structural properties of the protein, which is derived from many modifiable sites. The mode of molecular evolution presented here may be relevant to various cases of adaptive modification in proteins, such as hydrophobic properties, molecular size, and electric charge.

Figure 1

Molecular phylogeny and spatial expression patterns of Pgi. (A) Bayesian tree of Pgi genes derived from 20 vertebrates. Numbers indicate percent posterior probabilities for the Bayesian tree (left) and bootstrap support values by the maximum likelihood method (right). Arrow denotes a gene duplication event. In cDNA clones, only one Pgi was identified from non-teleosts, whereas two Pgi genes were identified from teleosts. The two Pgi genes differed by about 20% in amino acid sequence, and were grouped into separate clades (Pgi-1 and Pgi-2). In both clades, the gene relationships were consistent with the evolutionary relationships of teleost species [18, 19, 21]. (B) Partial-length gel images of the RT-PCR expression analysis of Pgi genes and positive control (β-actin) genes in ray-finned fishes. The tree in the left panel shows the relationships among the Pgi genes inferred in this study. The black circle on the tree denotes the timing of the Pgi gene duplication event. Letters indicate tissues: M, muscle; L, liver; H, heart; Gi, gill; B, brain; K, kidney. Full-length gels, including negative controls and size markers, are presented in Additional file 1: Fig. S5.

Figure 2

Current states and inferred evolutionary process of electric charge change in PGI isoforms. (A) Maximum likelihood tree of Pgi genes in ray-finned fishes inferred by BASEML [34] with a known phylogeny [18]. Numbers indicate estimated pI. Arrow denotes a gene duplication event. (B) Amino acid sites that differ by the presence or absence of hydrophilic charged residues between current PGI-1 and PGI-2. Positively charged residues are blue; negatively charged residues, red; other residues, light gray. The numbers above refer to the amino acid positions of PGI [33]. The stars below indicate sites located on the molecular surface. (C) Inferred charge-changing substitution events mapped over the PGI phylogeny. Orange and brown bars denote upward and downward charge changes, respectively.

Figure 3

Spatial locations of inferred amino acid substitutions in the PGI structure. (A) Maximum likelihood-inferred charge-changing substitution sites after the Pgi duplication are colored magenta; charge-neutral substitution sites, dark gray; enzyme active sites, yellow. Full molecular models are shown on the left, and two cross sections are shown center and right. The inferred charge-changing sites localize to the surface of the PGI molecule (73 charge-changing sites/234 total surface sites, 3 charge-changing sites/316 total interior sites; P = 0.0000, two-tailed Fisher's exact test), in contrast to the inferred charge-neutral sites (106 charge-neutral sites/234 total surface sites, 183 charge-neutral sites/316 total interior sites; P = 0.1040, two-tailed Fisher's exact test) (B) Histograms of the inferred number of charge-changing and charge-neutral substitutions after the Pgi duplication. The solid green line denotes the proportion of charge-changing substitutions per total substitutions within the site classes based on solvent accessibility (horizontal axis): this proportion significantly increases with solvent-accessible surface area (P = 0.0000, Cochran – Armitage trend test, n = 584).