Evolution of mammalian chitinase(-like) members of family 18 glycosyl hydrolases

Family 18 of glycosyl hydrolases encompasses chitinases and so-called chi-lectins lacking enzymatic activity due to amino acid substitutions in their active site. Both types of proteins widely occur in mammals although these organisms lack endogenous chitin. Their physiological function(s) as well as evolutionary relationships are still largely enigmatic. An overview of all family members is presented and their relationships are described. Molecular phylogenetic analyses suggest that both active chitinases (chitotriosidase and AMCase) result from an early gene duplication event. Further duplication events, followed by mutations leading to loss of chitinase activity, allowed evolution of the chi-lectins. The homologous genes encoding chitinase(-like) proteins are clustered in two distinct loci that display a high degree of synteny among mammals. Despite the shared chromosomal location and high homology, individual genes have evolved independently. Orthologs are more closely related than paralogues, and calculated substitution rate ratios indicate that protein-coding sequences underwent purifying selection. Substantial gene specialization has occurred in time, allowing for tissue-specific expression of pH optimized chitinases and chi-lectins. Finally, several family 18 chitinase-like proteins are present only in certain lineages of mammals, exemplifying recent evolutionary events in the chitinase protein family.     

New Paralogues and Revised Time Line in the Expansion of the Vertebrate GH18 Family

Chitinase enzymes hydrolyse the polysaccharide chitin, an abundant architectural component in invertebrates and fungi. Most mammals encode at least two endochitinases (CHIT1 and CHIA/AMCase), as well as several homologues encoding catalytically inactive chitinase-like proteins or chilectins (all GH18 family proteins). It is becoming increasingly apparent that chitinases and chilectins play an important role in inflammation and their over-expression is correlated with numerous pathological conditions. We have conducted a detailed phylogenomic study of this gene family in order to understand its evolutionary history and the selection forces at work. The family has undergone extensive expansion, initiating with a duplication event at the root of the vertebrate tree generating the ancestors of CHIT1 and CHIA. Our analyses indicate that two further duplications of ancestral CHIA predate the divergence of bony fishes, one leading to a newly identified paralogous group (we have termed CHIO). In fish these sequences fall into two clades bearing the hallmarks of the teleost-specific genome duplication (referred to as 3R). In tetrapods, additional duplications predate and postdate the amphibian/mammalian split and relics of some exist as pseudogenes in the human genome. Expansion and selection of chilectins is pronounced in mammals and CHI3L1 (with a proposed function in immunity) is found in most mammals but not other vertebrates, while CHI3L2 is also evident in reptiles. Notably oviductin (OVGP1) became basic and gained a glycosylated tail with its evolving role in the mammalian reproductive system. In each case, retention of the sugar-binding barrel structure has constrained positive selection to limited sites.     

Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family

Background  

Chitinases (EC.3.2.1.14) hydrolyze the β-1,4-linkages in chitin, an abundant N-acetyl-β-D-glucosamine polysaccharide that is a structural component of protective biological matrices such as insect exoskeletons and fungal cell walls. The glycoside hydrolase 18 (GH18) family of chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Mammals are not known to synthesize chitin or metabolize it as a nutrient, yet the human genome encodes eight GH18 family members. Some GH18 proteins lack an essential catalytic glutamic acid and are likely to act as lectins rather than as enzymes. This study used comparative genomic analysis to address the evolutionary history of the GH18 multiprotein family, from early eukaryotes to mammals, in an effort to understand the forces that shaped the human genome content of chitinase related proteins.     

Molecular cloning and functional characterization of mouse chitotriosidase

Mammalian chitinase and chitinase-like proteins are members of a recently discovered gene family. Thus far, neither chitin nor chitin synthase has been found in mammals. The existence of chitinase genes in mammals is intriguing and the physiologic functions of chitinases are not clear. Human chitotriosidase, also called chitinase 1 (chit1), has been cloned. It has been found that high levels of serum chitotriosidase are associated with several diseases, but the physiologic functions of this enzyme are still unclear. To facilitate the studies in animal models we cloned and characterized a cDNA that encodes the mouse chitotriosidase. The open reading frame of this cDNA predicts a protein of 464 amino acids with a typical chitinase structure, including a signal peptide, a highly conserved catalytic domain and a chitin-binding domain. The predicted amino acid sequence is highly homologous to that of human chitotriosidase and to that of mouse acidic mammalian chitinase. Sequence analysis indicates that the mouse chitotriosidase gene has 12 exons, spanning a 40-kb region in mouse chromosome 1. The constitutive expression of mouse chitotriosidase is restricted to brain, skin, bone marrow, kidney, tongue, stomach and testis. Recombinant expression of the cloned cDNA demonstrated that the encoded protein is secreted and has chitinolytic activity that is sensitive to the specific chitinase inhibitor allosamidin and has the ability to bind to chitin particles. Substitution mutations at the conserved catalytic site completely abolished the enzymatic activity of the recombinant protein. These studies illustrate that mouse chitotriosidase is a typical chitinase that belongs to the mammalian chitinase gene family.     

Sexual Selection and the Molecular Evolution of ADAM Proteins

Rapid evolution has been identified for many reproductive genes and recent studies have combined phylogenetic tests and information on species mating systems to test sexual selection. Here we examined the molecular evolution of the ADAM gene family, a diverse group of 35 proteins capable of adhesion to and cleavage of other proteins, using sequence data from 25 mammalian genes. Out of the 25 genes analyzed, all those expressed in male reproductive tissue showed evidence of positive selection. Positively selected amino acids within the protein adhesion domain were only found in sperm surface ADAM proteins (ADAMs 1, 2, 3, 4, and 32) suggesting selection driven by male × female interactions. We tested heterogeneity in rates of evolution of the adhesion domain of ADAM proteins by using sequence data from Hominidae and macaques. The use of the branch and branch-site models (PAML) showed evidence of higher d _N/d _S and/or positive selection linked to branches experiencing high postmating selective pressures (chimpanzee and macaque) for Adams 2, 18, and 23. Moreover, we found consistent higher proportion of nonsynonymous relative to synonymous and noncoding sequence substitutions in chimpanzee and/or macaque only for Adams 2, 18, and 23. Our results suggest that lineage-specific sexual selection bouts might have driven the evolution of the adhesion sperm protein surface domains of ADAMs 2 and 18 in primates. Adams 2 and 18 are localized in chromosome 8 of primates and adjacent to each other, so their evolution might have also been influenced by their common genome localization.     

Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases

The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.     

Evidence for positive selection on the leptin gene in Cetacea and Pinnipedia

The leptin gene has received intensive attention and scientific investigation for its importance in energy homeostasis and reproductive regulation in mammals. Furthermore, study of the leptin gene is of crucial importance for public health, particularly for its role in obesity, as well as for other numerous physiological roles that it plays in mammals. In the present work, we report the identification of novel leptin genes in 4 species of Cetacea, and a comparison with 55 publicly available leptin sequences from mammalian genome assemblies and previous studies. Our study provides evidence for positive selection in the suborder Odontoceti (toothed whales) of the Cetacea and the family Phocidae (earless seals) of the Pinnipedia. We also detected positive selection in several leptin gene residues in these two lineages. To test whether leptin and its receptor evolved in a coordinated manner, we analyzed 24 leptin receptor gene (LPR) sequences from available mammalian genome assemblies and other published data. Unlike the case of leptin, our analyses did not find evidence of positive selection for LPR across the Cetacea and Pinnipedia lineages. In line with this, positively selected sites identified in the leptin genes of these two lineages were located outside of leptin receptor binding sites, which at least partially explains why co-evolution of leptin and its receptor was not observed in the present study. Our study provides interesting insights into current understanding of the evolution of mammalian leptin genes in response to selective pressures from life in an aquatic environment, and leads to a hypothesis that new tissue specificity or novel physiologic functions of leptin genes may have arisen in both odontocetes and phocids. Additional data from other species encompassing varying life histories and functional tests of the adaptive role of the amino acid changes identified in this study will help determine the factors that promote the adaptive evolution of the leptin genes in marine mammals.     

Episodic Molecular Evolution of Pituitary Growth Hormone in Cetartiodactyla

The sequence of growth hormone (GH) is generally strongly conserved in mammals, but episodes of rapid change occurred during the evolution of primates and artiodactyls, when the rate of GH evolution apparently increased substantially. As a result the sequences of higher primate and ruminant GHs differ markedly from sequences of other mammalian GHs. In order to increase knowledge of GH evolution in Cetartiodactyla (Artiodactyla plus Cetacea) we have cloned and characterized GH genes from camel (Camelus dromedarius), hippopotamus (Hippopotamus amphibius), and giraffe (Giraffa camelopardalis), using genomic DNA and a polymerase chain reaction technique. As in other mammals, these GH genes comprise five exons and four introns. Two very similar GH gene sequences (encoding identical proteins) were found in each of hippopotamus and giraffe. The deduced sequence for the mature hippopotamus GH is identical to that of dolphin, in accord with current ideas of a close relationship between Cetacea and Hippopotamidae. The sequence of camel GH is identical to that reported previously for alpaca GH. The sequence of giraffe GH is very similar to that of other ruminants but differs from that of nonruminant cetartiodactyls at about 18 residues. The results demonstrate that the apparent burst of rapid evolution of GH occurred largely after the separation of the line leading to ruminants from other cetartiodactyls.     

Comparative analysis of testis protein evolution in rodents

Genes expressed in testes are critical to male reproductive success, affecting spermatogenesis, sperm competition, and sperm-egg interaction. Comparing the evolution of testis proteins at different taxonomic levels can reveal which genes and functional classes are targets of natural and sexual selection and whether the same genes are targets among taxa. Here we examine the evolution of testis-expressed proteins at different levels of divergence among three rodents, mouse (Mus musculus), rat (Rattus norvegicus), and deer mouse (Peromyscus maniculatus), to identify rapidly evolving genes. Comparison of expressed sequence tags (ESTs) from testes suggests that proteins with testis-specific expression evolve more rapidly on average than proteins with maximal expression in other tissues. Genes with the highest rates of evolution have a variety of functional roles including signal transduction, DNA binding, and egg-sperm interaction. Most of these rapidly evolving genes have not been identified previously as targets of selection in comparisons among more divergent mammals. To determine if these genes are evolving rapidly among closely related species, we sequenced 11 of these genes in six Peromyscus species and found evidence for positive selection in five of them. Together, these results demonstrate rapid evolution of functionally diverse testis-expressed proteins in rodents, including the identification of amino acids under lineage-specific selection in Peromyscus. Evidence for positive selection among closely related species suggests that changes in these proteins may have consequences for reproductive isolation.     

The GH18 family of chitinases: their domain architectures, functions and evolutions

The glycoside hydrolase 18 (GH18) family of chitinases is a multigene family that plays various roles, such as ecdysis, embryonic development, allergic inflammation and so on. Efforts are still needed to reveal their functional diversification in an evolutionary and systematic manner. We collected 85 GH18 genes from eukaryotic representatives. The domain architectures of GH18 proteins were analyzed and several conserved patterns were identified. It was observed that some (11 proteins) GH18 members in Ecdysozoa or fungi possess repeats of catalytic domains and/or chitin-binding domains (ChtBs). The domain repeats are likely to meet requirements for higher efficiency of chitin degradation in chitin-containing species. On the contrary, all vertebrate GH18 proteins contain no more than one catalytic domain or ChtB. The results from homologous analysis, domain architectures, exon arrangements and synteny loci supported two evolutionary paths for the GH18 family. One path experienced gene expansion and contraction several times during evolution, covering most of GH18 members except CHID1 (stabilin-1 interacting partner) and its homologs. Proteins in this path underwent frequent domain gain and loss, as well as domain recombination, that could achieve versatility in function. The other path is comparatively conserved. The CHID1 gene evolved without gene duplication except in Danio rerio. Domain architectures of CHID1 orthologs are all identical. The diverse phylogeny of the GH18 family in arthropod is also presented.     

Episodic evolution of growth hormone in primates and emergence of the species specificity of human growth hormone receptor

Growth hormone (GH) evolution is very conservative among mammals, except for primates and ruminant artiodactyls. In fact, most known mammalian GH sequences differ from the inferred ancestral mammalian sequence by only a few amino acids. In contrast, the human GH sequence differs from the inferred ancestral sequence by 59 amino acids. However, it is not known when this rapid evolution of GH occurred during primate evolution or whether it was due to positive selection. Also, human growth hormone receptor (GHR) displays species specificity; i.e., it can interact only with human (or rhesus monkey) GH, not with nonprimate GHS: The species specificity of human GHR is largely due to the Leu-->Arg change at position 43, and it has been hypothesized that this change must have been preceded by the His-->Asp change at position 171 of GH. Is this hypothesis true? And when did these changes occur? To address the above issues, we sequenced GH and GHR genes in prosimians and simians. Our data supported the above hypothesis and revealed that the species specificity of human GHR actually emerged in the common ancestor of Old World primates, but the transitional phase still persists in New World monkeys. Our data showed that the rapid evolution of primate GH occurred during a relatively short period (in the common ancestor of higher primates) and that the rate of change was especially high at functionally important sites, suggesting positive selection. However, the nonsynonymous rate/synonymous rate ratio at these sites was <1, so relaxation of purifying selection might have played a role in the rapid evolution of the GH gene in simians, possibly as a result of multiple gene duplications. Similar to GH, GHR displayed an accelerated rate of evolution in primates. Our data revealed proportionally more amino acid replacements at the functionally important sites in both GH and GHR in simians but, surprisingly, showed few coincidental replacements of amino acids forming the same intermolecular contacts between the two proteins.     

哺乳动物抗凝血酶基因的分子特征、微共线性与适应性进化

抗凝血酶(AT)是哺乳动物体内重要的天然抗凝血因子之一,它隶属于丝氨酸蛋白酶抑制物家族,主要参与并调节复杂的血凝过程。基于比较基因组学手段,共挖掘出17个哺乳动物抗凝血酶基因(AT),并剖析了它们的基因结构、微共线性、保守基序、功能结构域、以及系统进化关系。基因结构与微共线分析表明,哺乳动物AT基因具有5-12个外显子,大多数是7个外显子;不同物种AT基因所处区段之间具有较好的共线性。哺乳动物AT蛋白特征分析显示,SERPIN功能结构域与保守基序1、2、3、4、5、8和9存在相互重叠的部分。AT基因进化树揭示基因进化和通常认为的物种进化几乎一致。同时,利用PAML中Codeml的位点特异模型在AT基因中发掘了1个正选择位点328D。     

Structure of human chitotriosidase. Implications for specific inhibitor design and function of mammalian chitinase-like lectins

Chitin hydrolases have been identified in a variety of organisms ranging from bacteria to eukaryotes. They have been proposed to be possible targets for the design of novel chemotherapeutics against human pathogens such as fungi and protozoan parasites as mammals were not thought to possess chitin-processing enzymes. Recently, a human chitotriosidase was described as a marker for Gaucher disease with plasma levels of the enzyme elevated up to 2 orders of magnitude. The chitotriosidase was shown to be active against colloidal chitin and is inhibited by the family 18 chitinase inhibitor allosamidin. Here, the crystal structure of the human chitotriosidase and complexes with a chitooligosaccharide and allosamidin are described. The structures reveal an elongated active site cleft, compatible with the binding of long chitin polymers, and explain the inactivation of the enzyme through an inherited genetic deficiency. Comparison with YM1 and HCgp-39 shows how the chitinase has evolved into these mammalian lectins by the mutation of key residues in the active site, tuning the substrate binding specificity. The soaking experiments with allosamidin and chitooligosaccharides give insight into ligand binding properties and allow the evaluation of differential binding and design of species-selective chitinase inhibitors.     

Adaptive Evolution of Gamete-Recognition Proteins in Birds

Gamete-recognition proteins have been shown to evolve by positive selection in diverse organism groups, such as marine invertebrates and mammals, although underlying evolutionary mechanisms driving this rapid divergence are poorly understood. However, several hypotheses have been put forward to explain the observed pattern, including different forms of sexual conflict and sperm competition. Because female gametes require more energy to produce than male gametes, female organisms suffer more when fertilisation goes wrong. One process that results in a failed mammalian fertilisation is polyspermy, when >1 sperm fertilises the egg. However in birds, there is no such sexual conflict because multiple sperm typically bind and fuse with the egg. If sexual conflict driven by polyspermy avoidance is important for the evolution of gamete-recognition proteins in vertebrates, we expect to find positive selection in the genes to be less pronounced in birds. We therefore sequenced six genes (ZP1, ZP2, ZP4, ZPAX, CD9, and Acrosin) encoding gamete-recognition proteins in several bird species to test for positive selection. For comparison, we also analysed ortologous sequences in a set of mammalian species. We found no major differences in the occurrence of adaptive evolution and the strength of selection between bird and mammal orthologs. From this we conclude that polyspermy avoidance does not act as the main underlying evolutionary force shaping the rate of evolution in these genes. We discuss other possible processes that could explain positive selection of gamete-recognition proteins in birds and mammals, such as hybridisation avoidance, cryptic female choice, and postcopulatory sperm competition.     

Overview of chitin metabolism enzymes in Manduca sexta: Identification,domain organization,phylogenetic analysis and gene expression

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.     

Patterns of positive selection in six Mammalian genomes

Genome-wide scans for positively selected genes (PSGs) in mammals have provided insight into the dynamics of genome evolution, the genetic basis of differences between species, and the functions of individual genes. However, previous scans have been limited in power and accuracy owing to small numbers of available genomes. Here we present the most comprehensive examination of mammalian PSGs to date, using the six high-coverage genome assemblies now available for eutherian mammals. The increased phylogenetic depth of this dataset results in substantially improved statistical power, and permits several new lineage- and clade-specific tests to be applied. Of approximately 16,500 human genes with high-confidence orthologs in at least two other species, 400 genes showed significant evidence of positive selection (FDR<0.05), according to a standard likelihood ratio test. An additional 144 genes showed evidence of positive selection on particular lineages or clades. As in previous studies, the identified PSGs were enriched for roles in defense/immunity, chemosensory perception, and reproduction, but enrichments were also evident for more specific functions, such as complement-mediated immunity and taste perception. Several pathways were strongly enriched for PSGs, suggesting possible co-evolution of interacting genes. A novel Bayesian analysis of the possible "selection histories" of each gene indicated that most PSGs have switched multiple times between positive selection and nonselection, suggesting that positive selection is often episodic. A detailed analysis of Affymetrix exon array data indicated that PSGs are expressed at significantly lower levels, and in a more tissue-specific manner, than non-PSGs. Genes that are specifically expressed in the spleen, testes, liver, and breast are significantly enriched for PSGs, but no evidence was found for an enrichment for PSGs among brain-specific genes. This study provides additional evidence for widespread positive selection in mammalian evolution and new genome-wide insights into the functional implications of positive selection.     

Maximum‐likelihood approaches reveal signatures of positive selection in BMP15 and GDF9 genes modulating ovarian function in mammalian female fertility

Bone morphogenetic proteins (BMPs) and the growth factors (GDFs) play an important role in ovarian folliculogenesis and essential regulator of processes of numerous granulosa cells. BMP15 gene variations linked to various ovarian phenotypic consequences subject to the species, from infertility to improved prolificacy in sheep, primary ovarian insufficiency in women or associated with minor subfertility in mouse. To study the evolving role of BMP15 and GDF9, a phylogenetic analysis was performed. To find out the candidate gene associated with prolificacy in mammals, the nucleotide sequence of BMP15 and GDF9 genes was recognized under positive selection in various mammalian species. Maximum‐likelihood approaches used on BMP15 and GDF9 genes exhibited a robust divergence and a prompted evolution as compared to other TGFβ family members. Furthermore, among 32 mammalian species, we identified positive selection signals in the hominidae clade resulting to 132D, 147E, 163Y, 191W, and 236P codon sites of BMP15 and 162F, 188K, 206R, 240A, 244L, 246H, 248S, 251D, 253L, 254F and other codon sites of GDF9. The positively selected amino acid sites such as Alanine, Lucien, Arginine, and lysine are important for signaling. In conclusion, this study evidences that GDF9 and BMP15 genes have rapid evolution than other TGFß family members and was subjected to positive selection in the mammalian clade. Selected sites under the positive selection are of remarkable significance for the particular functioning of the protein and consequently for female fertility.     

Molecular and functional evolution of class I chitinases for plant carnivory in the caryophyllales

Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. In flowering plants, class I chitinases are important pathogenesis-related proteins, functioning in the determent of herbivory and pathogen attack by acting on insect exoskeletons and fungal cell walls. Within the carnivorous plants, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Members of these two subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within the morphologically diverse traps that develop from carnivorous plant leaves. The degree of homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit has yet to be elucidated. This study focuses on understanding the evolution of carnivory and chitinase genes in one of the major groups of plants that has evolved the carnivorous habit: the Caryophyllales. We recover novel class I chitinase homologs from species of genera Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, while also confirming the presence of two subclasses of class I chitinases based upon sequence homology and phylogenetic affinity to class I chitinases available from sequenced angiosperm genomes. We further detect residues under positive selection and reveal substitutions specific to carnivorous plant class I chitinases. These substitutions may confer functional differences as indicated by protein structure homology modeling.