The speciation of conger eel galectins by rapid adaptive evolution

Many cases of accelerated evolution driven by positive Darwinian selection are identified in the genes of venomous and reproductive proteins. This evolutional phenomenon might have important consequences in their gene-products' functions, such as multiple specific toxins for quick immobilization of the prey and the establishment of barriers to fertilization that might lead to speciation, and in the molecular evolution of novel genes. Recently, we analyzed the molecular evolution of two galectins isolated from the skin mucus of conger eel (Conger myriaster), named congerins I and II, by cDNA cloning and X-ray structural analysis, and we found that they have evolved in the rapid adaptive manner to emergence of a new structure including strand-swapping and a unique new ligand-binding site. In this review article we summarize and discuss the molecular evolution, especially the rapid adaptive evolution, and the structure-function relationships of conger eel galectins. Published in 2004.     

Accelerated evolution in the protein-coding region of galectin cDNAs, congerin I and congerin II, from skin mucus of conger eel (Conger myriaster).

Two cDNAs encoding galectins named congerins I and II from the skin mucus of conger eel (Conger myriaster) were isolated and sequenced. Comparison of the nucleotide sequences of congerins I and II showed that the sequence similarities of the 5' and 3' untranslated regions (86 and 88%, respectively) were much higher than those of the protein-coding region (73%). The numbers of nucleotide substitutions per site (KN) for the untranslated regions are smaller than the numbers of nucleotide substitutions per synonymous site (KS) for the protein coding region. Furthermore, nonsynonymous nucleotide substitutions have accelerated more frequently than synonymous nucleotide substitutions in the protein coding region (KA/KS = 2.57). These results suggest that accelerated substitutions have occurred in the protein-coding regions of galectin genes to generate diverse galectins with different molecular properties. Northern blot analysis showed that both congerins were expressed not only in the skin tissues but also in the stomach of conger eel.     

Allosteric Regulation of the Carbohydrate-binding Ability of a Novel Conger Eel Galectin by D-Mannoside

Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of d-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that d-mannoside can act as a modulator of Con-P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type β-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties.     

Crystal structure of a conger eel galectin (congerin II) at 1.45A resolution: implication for the accelerated evolution of a new ligand-binding site following gene duplication

The crystal structure of congerin II, a galectin family lectin from conger eel, was determined at 1.45A resolution. The previously determined structure of its isoform, congerin I, had revealed a fold evolution via strand swap; however, the structure of congerin II described here resembles other prototype galectins. A comparison of the two congerin genes with that of several other galectins suggests acceralated evolution of both congerin genes following gene duplication. The presence of a Mes (2-[N-morpholino]ethanesulfonic acid) molecule near the carbohydrate-binding site in the crystal structure points to the possibility of an additional binding site in congerin II. The binding site consists of a group of residues that had been replaced following gene duplication suggesting that the binding site was built under selective pressure. Congerin II may be a protein specialized for biological defense with an affinity for target carbohydrates on parasites' cell surface.     

Reconstruction of a probable ancestral form of conger eel galectins revealed their rapid adaptive evolution process for specific carbohydrate recognition

Recently, many cases of rapid adaptive evolution, which is characterized by the higher substitution rates of nonsynonymous substitutions to synonymous ones, have been identified in the various genes of venomous and biodefense proteins, including the conger eel galectins, congerins I and II (ConI and ConII). To understand the evolutionary process of congerins, we prepared a probable ancestral form, Con-anc, corresponding to the putative amino acid sequence at the divergence of ConI and ConII in phylogenetic tree with 76% and 61% sequence identities to the current proteins, respectively. Con-anc and ConII had comparable thermostability and similar carbohydrate specificities in general, whereas ConI was more thermostable and showed different carbohydrate specificities. Con-anc showed decreased specificity to oligosaccharides with alpha 2,3-sialyl galactose moieties. These suggest that ConI and ConII have evolved via accelerated evolution under significant selective pressure to increase the thermostability and to acquire the activity to bind to alpha2,3-sialyl galactose present in pathogenic bacteria, respectively. Furthermore, comparative mutagenesis analyses of Con-anc and congerins revealed the structural basis for specific recognition of ConII to alpha2,3-sialyl galactose moiety, and strong binding ability of ConI to oligosaccharides including lacto-N-biosyl (Galbeta1-3GlcNAc) or lacto-N-neobiosyl (Galbeta1-4GlcNAc) residues, respectively. Thus, protein engineering using a probable ancestral form presented here is a powerful approach not only to determine the evolutionary process but also to investigate the structure-activity relationships of proteins.     

LPS induces gene expression of interleukin-1beta in conger eel (Conger myriaster) macrophages: first cytokine sequence within Anguilliformes

Our aim was to comprehensively analyze the immune response in Anguilliformes macrophages and to survey cytokine genes expressed from them. We therefore used suppression subtractive hybridization (SSH) to randomly clone molecules that are specifically expressed in conger eel (Conger myriaster) macrophages when cells are stimulated by LPS. As a result, we succeeded in identifying a conger eel IL-1beta. This is the first report on cytokines in Anguilliformes, which is the most ancient order in living teleosts.     

High-level expression and characterization of fully active recombinant conger eel galectins in Eschericia coli

An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli. Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield. Mutation of codons for Val and Leu located in the N-terminal region of Con I increased the expression efficiency. Purification of recombinant proteins were done by only two chromatographical steps from E. coli extract. The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity.     

The visual sensitivity of the retina of the conger eel

We measured the visual sensitivity of the conger eel retina by means of its electroretinogram (e.r.g.) and whole nerve responses. The spectral sensitivity of the retina closely corresponded to a prediction based on the density spectrum of the conger visual pigment, measured in situ. The pigment density in the conger eel retina is high, perhaps as high as 1.0. Thus, the predicted spectral sensitivity would be much broader than is observed if the absorption spectrum of the pigment governed the visual sensitivity. The reason why the visual spectral sensitivity corresponds to the density spectrum and not to the absorption spectrum is that the photoreceptors in the conger eye are arranged in tiers and only the inner tier contributes to vision.     

Isolation of epidermal cells and cDNA cloning of TNF decoy receptor 3 of conger eel, Conger myriaster

By using EDTA and a trypsin solution, we established a method for isolating the epidermal cells of the conger eel, Conger myriaster. We then identified TNF decoy receptor (DcR) cDNA in the species from a suppression subtractive hybridization library prepared from the epidermal cells stimulated with LPS. The full-length cDNA of conger TNF DcR (conDcR) consisted of 1479 base pairs, and the protein comprised 286 amino acid residues. Phylogenetic analysis indicated that conDcR was clustered into a DcR3 branch. ConDcR is likely to act as an important immune-regulating factor in inhibiting the apoptosis-inducing effect of TNF in the skin of conger eel.     

A new aging technique by UV light observation of burnt otoliths for the conger eel Conger myriaster (Brevoort)

A new aging method, fluorescent observation of burnt otoliths, was discovered to disclose the age and growth of the conger eel. Under UV light, bright fluorescent zones were visible in the burnt otolith but not in the unburnt otolith. An illumination wavelength around 380 nm was found to be suitable for fluorescence observation of burnt otoliths. Bright zones of the conger eel otolith formed around June–August in Sendai Bay and were validated as annuli. The conger eels caught by net pot fishery were found to be mainly aged from 1+ to 4+ years. Received: March 7, 2001 / Revised: September 12, 2001 / Accepted: October 10, 2001     

High-level Expression and Characterization of Fully Active Recombinant Conger Eel Galectins in Eschericia coli

An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli. Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield. Mutation of codons for Val and Leu located in the N-terminal region of Con I increased the expression efficiency. Purification of recombinant proteins were done by only two chromatographical steps from E. coli extract. The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity.     

Shall we dance or shall we fight? Using DNA sequence data to untangle controversies surrounding sexual selection.

Population and evolutionary genetics studies have largely benefitted from advances in DNA manipulation and sequencing, as well as DNA data analysis techniques. Molecular evolution studies of male reproductive genes show a pattern of rapid evolution shaped, in some cases, by an adaptive selective process. Despite the large body of data on male reproductive genes, the female side of the story has remained unexplored. The few cases of female egg receptors analyzed also show rapid evolution. However, to disentangle between competing hypotheses on how selection operates on male x female molecular interaction leading to fertilization, we need to find male and female molecules that are partners in fertilization. A conflict model of sexual selection (similar to a host-parasite model) assumes a male-driven system where females are being forced under suboptimal conditions. This predicts that the amount of divergence at a female receptor depends on the amount of divergence among the male reproductive proteins that it binds (i.e., males are leading). Under a classical model of runaway sexual selection, female protein receptors might be the key to the rapid molecular changes observed in male reproductive proteins and higher divergence should be expected among female receptors than among their respective male binding proteins.     

Diversifying selection of the tumor-growth promoter angiogenin in primate evolution

Diversifying selection drives the rapid differentiation of gene sequences and is one of the main forces behind adaptive evolution. Most genes known to be shaped by diversifying selection are those involved in host-pathogen or male-female interactions characterized as molecular "arms races." Here we report the unexpected detection of diversifying selection in the evolution of a tumor-growth promoter, angiogenin (ANG). A comparison among 11 primate species demonstrates that ANG has a significantly higher rate of nucleotide substitution at nonsynonymous sites than at synonymous sites, a hallmark of positive selection acting at the molecular level. Furthermore, we observed significant charge diversity at the molecular surface, suggesting the presence of selective pressures in the microenvironment of ANG, including its binding molecules. A population survey of ANG in chimpanzees, however, reveals no polymorphism, which may have resulted from a recent selective sweep of a charge-altering substitution in chimpanzee evolution. Functional assays of recombinant ANGs from the human and owl monkey indicate that primate ANGs retain angiogenic activity despite rapid evolution. Our study, together with findings of similar selection in the primate breast cancer suppressor gene, BRCA1, reveals an intriguing phenomenon of unusual selective pressures on, and adaptive evolution of, cancer-related genes in primate evolution.     

Protein engineering of conger eel galectins by tracing of molecular evolution using probable ancestral mutants

Background  

Conger eel galectins, congerin I (ConI) and congerin II (ConII), show the different molecular characteristics resulting from accelerating evolution. We recently reconstructed a probable ancestral form of congerins, Con-anc. It showed properties similar to those of ConII in terms of thermostability and carbohydrate recognition specificity, although it shares a higher sequence similarity with ConI than ConII.     

High-resolution structure of the conger eel galectin, congerin I, in lactose-liganded and ligand-free forms: emergence of a new structure class by accelerated evolution.

BACKGROUND: Congerin I is a member of the galectin (animal beta-galactoside-binding lectin) family and is found in the skin mucus of conger eel. The galectin family proteins perform a variety of biological activities. Because of its histological localization and activity against marine bacteria and starfish embryos, congerin I is thought to take part in the eels' biological defense system against parasites. RESULTS: The crystal structure of congerin I has been determined in both lactose-liganded and ligand-free forms to 1. 5 A and 1.6 A resolution, respectively. The protein is a homodimer of 15 kDa subunits. Congerin I has a beta-sheet topology that is markedly different from those of known relatives. One of the beta-strands is exchanged between two identical subunits. This strand swap might increase the dimer stability. Of the known galectin complexes, congerin I forms the most extensive interaction with lactose molecules. Most of these interactions are substituted by similar interactions with water molecules, including a pi-electron hydrogen bond, in the ligand-free form. This observation indicates an increased affinity of congerin I for the ligand. CONCLUSIONS: The genes for congerin I and an isoform, congerin II, are known to have evolved under positive selection pressure. The strand swap and the modification in the carbohydrate-binding site might enhance the cross-linking activity, and should be the most apparent consequence of positive selection. The protein has been adapted to functioning in skin mucus that is in direct contact with surrounding environments by an enhancement in cross-linking activity. The structure of congerin I demonstrates the emergence of a new structure class by accelerated evolution under selection pressure.     

Explosive radiations and the reliability of molecular clocks: island endemic radiations as a test case

The reliability of molecular clocks has been questioned for several key evolutionary radiations on the basis that the clock might run fast in explosive radiations. Molecular date estimates for the radiations of metazoan phyla (the Cambrian explosion) and modern orders of mammals and birds are in many cases twice as old as the palaeontological evidence would suggest. Could some aspect of explosive radiations speed the molecular clock, making molecular date estimates too old? Here we use 19 independent instances of recent explosive radiations of island endemic taxa as a model system for testing the proposed influence of rapid adaptive radiation on the rate of molecular evolution. These radiations are often characterized by many of the potential mechanisms for fast rates in explosive radiations--such as small population size, elevated speciation rate, rapid rate of morphological change, release from previous ecological constraints, and adaptation to new niches--and represent a wide variety of species, islands, and genes. However, we find no evidence of a consistent increase in rates in island taxa compared to their mainland relatives, and therefore find no support for the hypothesis that the molecular clock runs fast in explosive radiations.     

Effects of the piezo-tolerance of cultured deep-sea eel cells on survival rates, cell proliferation, and cytoskeletal structures

We investigated the pressure tolerance of deep-sea eel (Simenchelys parasiticus; habitat depth, 366–2,630 m) cells, conger eel (Conger myriaster) cells, and mouse 3T3-L1 cells. Although there were no living mouse 3T3-L1 and conger eel cells after 130 MPa (0.1 MPa = 1 bar) hydrostatic pressurization for 20 min, all deep-sea eel cells remained alive after being subjected to pressures up to 150 MPa for 20 min. Pressurization at 40 MPa for 20 min induced disruption of actin and tubulin filaments with profound cell-shape changes in the mouse and conger eel cells. In the deep-sea eel cells, microtubules and some actin filaments were disrupted after being subjected to hydrostatic pressure of 100 MPa and greater for 20 min. Conger eel cells were sensitive to pressure and did not grow at 10 MPa. Mouse 3T3-L1 cells grew faster under pressure of 5 MPa than at atmospheric pressure and stopped growing at 18 MPa. Deep-sea eel cells were capable of growth in pressures up to 25 MPa and stopped growing at 30 MPa. Deep-sea eel cells required 4 h at 20 MPa to finish the M phase, which was approximately fourfold the time required under atmospheric conditions.     

Possible immune functions of congerin, a mucosal galectin, in the intestinal lumen of Japanese conger eel

Congerin, a mucosal galectin of the Japanese conger eel, provides chemical fortification through its agglutinating and opsonizing activity. Congerin is produced in the epidermis, and the epithelia of the oral cavity to the esophagus, but not in the stomach or intestine. We hypothesized that congerin secreted from the upper digestive tract can reach and function in the intestinal lumen. We found that congerin possessed marked resistance against digestion by gastric and enteric enzymes of conger eel. It was not degraded until 6h of incubation with stomach extract or intestinal digestion juice. Western blotting demonstrated that congerin essentially remained in the intestinal mucus. The mucus agglutinated rabbit erythrocytes, and the agglutination was hampered by anti-congerin antibody. Furthermore, congerin could bind to some enteric bacteria. These results support the above hypothesis.     

Evolutionary expressed sequence tag analysis of Drosophila female reproductive tracts identifies genes subjected to positive selection

Genes whose products are involved in reproduction include some of the fastest-evolving genes found within the genomes of several organisms. Drosophila has long been used to study the function and evolutionary dynamics of genes thought to be involved in sperm competition and sexual conflict, two processes that have been hypothesized to drive the adaptive evolution of reproductive molecules. Several seminal fluid proteins (Acps) made in the Drosophila male reproductive tract show evidence of rapid adaptive evolution. To identify candidate genes in the female reproductive tract that may be involved in female-male interactions and that may thus have been subjected to adaptive evolution, we used an evolutionary bioinformatics approach to analyze sequences from a cDNA library that we have generated from Drosophila female reproductive tracts. We further demonstrate that several of these genes have been subjected to positive selection. Their expression in female reproductive tracts, presence of signal sequences/transmembrane domains, and rapid adaptive evolution indicate that they are prime candidates to encode female reproductive molecules that interact with rapidly evolving male Acps.