Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.     

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

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.     

Accelerated evolution and loss of a domain of the sperm-egg-binding protein SED1 in ancestral primates

Proteins involved in sperm-egg binding have been shown to evolve rapidly in several groups of invertebrates and vertebrates. Mammalian SED1 (secreted protein containing N-terminal Notch-like type II epidermal growth factor (EGF) repeats and C-terminal discoidin/F5/8 C domains) is a recently identified sperm surface protein that binds the egg zona pellucida and facilitates sperm-egg adhesion. SED1-null male mice are subfertile. Here we examine the SED1 gene from 11 mammalian species and provide evidence that it underwent accelerated evolution in ancestral primates, most likely driven by positive selection. Specifically, the intensity of the positive selection across various protein domains of SED1 was heterogeneous. Although one of the 2 Notch-like EGF domains, which mediate protein-protein binding, was lost in primate SED1, the second EGF domain evolved under strong positive selection favoring polar to nonpolar amino acid replacements. By contrast, the 2 discoidin/F5/8 type C domains, which are involved in protein-cell membrane binding, do not show definite signs of positive selection. The structural modification and occurrence of directional selection in ancestral primates but not any other lineage suggest that the function of SED1 may have changed during primate evolution. These results reveal a different evolutionary pattern of SED1 from that of many other sperm-egg-binding proteins, which often show diversifying selection occurring in multiple lineages.     

Alternative promoters in expression of genetic information

Many genes are known to have several promoters. The contribution of alternative promoters to the structural and functional diversity of protein isoforms in eukaryotic cells is considered, including their role in synthesis of identical proteins from different mRNAs, generation of protein isoforms with different and even opposite functions, expression of housekeeping genes, and the variation of the recognition domains of adhesion proteins and receptors. In some cases, alternative promoters allow one gene to produce mRNAs with different open reading frames and, consequently, proteins with no amino acid sequence homology.     

The classical arabinogalactan protein gene family of arabidopsis

Arabinogalactan proteins (AGPs) are extracellular proteoglycans implicated in plant growth and development. We searched for classical AGPs in Arabidopsis by identifying expressed sequence tags based on the conserved domain structure of the predicted protein backbone. To confirm that these genes encoded bona fide AGPs, we purified native AGPs and then deglycosylated and deblocked them for N-terminal protein sequencing. In total, we identified 15 genes encoding the protein backbones of classical AGPs, including genes for AG peptides-AGPs with very short backbones (10 to 13 amino acid residues). Seven of the AGPs were verified as AGPs by protein sequencing. A gene encoding a putative cell adhesion molecule with AGP-like domains was also identified. This work provides a firm foundation for beginning functional analysis by using a genetic approach.     

Molecular evolution of lysin motif-type receptor-like kinases in plants

The lysin motif (LysM) domain is an ancient and ubiquitous protein module that binds peptidoglycan and structurally related molecules. A genomic survey in a large number of species spanning all kingdoms reveals that the combination of LysM and receptor kinase domains is present exclusively in plants. However, the particular biological functions and molecular evolution of this gene family remain largely unknown. We show that LysM domains in plant LysM proteins are highly diversified and that a minimum of six distinct types of LysM motifs exist in plant LysM kinase proteins and five additional types of LysM motifs exist in nonkinase plant LysM proteins. Further, motif similarities suggest that plant LysM motifs are ancient. Although phylogenetic signals are not sufficient to resolve the earliest relationships, plant LysM motifs may have arisen through common ancestry with LysM motifs in other kingdoms. Within plants, the gene family has evolved through local and segmental duplications. The family has undergone further duplication and diversification in legumes, where some LysM kinase genes function as receptors for bacterial nodulation factor. Two pairs of homeologous regions were identified in soybean (Glycine max) based on microsynteny and fluorescence in situ hybridization. Expression data show that most plant LysM kinase genes are expressed predominantly in the root and that orthologous LysM kinase genes share similar tissue expression patterns. We also examined synteny around plant LysM kinase genes to help reconstruct scenarios for the evolution of this important gene family.     

Molecular evolution of olfactomedin

Olfactomedin is a secreted polymeric glycoprotein of unknown function, originally discovered at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently found throughout the mammalian brain. As a first step toward elucidating the function of olfactomedin, its phylogenetic history was examined to identify conserved structural motifs. Such conserved motifs may have functional significance and provide targets for future mutagenesis studies aimed at establishing the function of this protein. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedins in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homopolymer formation such as the CXC motif near the amino terminus are conserved, as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, evolutionary analysis of a region of 53 amino acids of fish, frog, rat, mouse, and human olfactomedins indicates that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian, and mammalian lineages. Indeed, a distant olfactomedin homolog was identified in Caenorhabditis elegans. Although the amino acid sequence of this invertebrate protein is longer and highly divergent compared with its vertebrate homologs, the protein from C. elegans shows remarkable similarities in terms of conserved motifs and posttranslational modification sites. Six universally conserved motifs were identified, and five of these are clustered in the carboxyl terminal half of the protein. Sequence comparisons indicate that evolution of the N-terminal half of the molecule involved extensive insertions and deletions; the C-terminal segment evolved mostly through point mutations, at least during vertebrate evolution. The widespread occurrence of olfactomedin among vertebrates and invertebrates underscores the notion that this protein has a function of universal importance. Furthermore, extensive modification of its N-terminal half and the acquisition of a C-terminal SDEL endoplasmic-reticulum- targeting sequence may have enabled olfactomedin to adopt new functions in the mammalian central nervous system.      

A case of convergent evolution of nucleic acid binding modules

Divergent evolution can explain how many proteins containing structurally similar domains, which perform a variety of related functions, have evolved from a relatively small number of modules or protein domains. However, it cannot explain how protein domains with similar, but distinguishable, functions and similar, but distinguishable, structures have evolved. Examples of this are the RNA-binding proteins containing the RNA-binding domain (RBD), and a newly established protein group, the cold-shock domain (CSD) protein family. Both protein domains contain conserved RNP motifs on similar single-stranded nucleic acid-binding surfaces. Apart from the RNP motifs, which have a similar function, the two families show little similarity in topology or amino acid sequence. This can be considered an interesting example of convergent evolution at the molecular level. Previously, a β-sheet surface was found to interact with RNA in non-homologous proteins from yeast, phage and man, revealing that this mode of RNA binding may be a widely recurring theme.     

Molecular evolution of the cadherin superfamily

This review deals with the large and pleiotropic superfamily of cadherins and its molecular evolution. We compiled literature data and an in-depth phylogenetic analysis of more than 350 members of this superfamily from about 30 species, covering several but not all representative branches within metazoan evolution. We analyzed the sequence homology between either ectodomains or cytoplasmic domains, and we reviewed protein structural data and genomic architecture. Cadherins and cadherin-related molecules are defined by having an ectodomain in which at least two consecutive calcium-binding cadherin repeats are present. There are usually 5 or 6 domains, but in some cases as many as 34. Additional protein modules in the ectodomains point at adaptive evolution. Despite the occurrence of several conserved motifs in subsets of cytoplasmic domains, these domains are even more diverse than ectodomains and most likely have evolved separately from the ectodomains. By fine tuning molecular classifications, we reduced the number of solitary superfamily members. We propose a cadherin major branch, subdivided in two families and 8 subfamilies, and a cadherin-related major branch, subdivided in four families and 11 subfamilies. Accordingly, we propose a more appropriate nomenclature. Although still fragmentary, our insight into the molecular evolution of these remarkable proteins is steadily growing. Consequently, we can start to propose testable hypotheses for structure-function relationships with impact on our models of molecular evolution. An emerging concept is that the ever evolving diversity of cadherin structures is serving dual and important functions: specific cell adhesion and intricate cell signaling.     

Adaptive Evolution of cry Genes in Bacillus thuringiensis： Implications for Their Specificity Determination

The cry gene family, produced during the late exponential phase of growth in Bacillus thuringiensis, is a large, still-growing family of homologous genes, in which each gene encodes a protein with strong specific activity against only one or a few insect species. Extensive studies are mostly focusing on the structural and functional relationships of Cry proteins, and have revealed several residues or domains that are important for the target recognition and receptor attachment. In this study, we have employed a maximum likelihood method to detect evidence of adaptive evolution in Cry proteins, and have identified 24 positively selected residues, which are all located in Domain Ⅱ or Ⅲ. Combined with known data from mutagenesis studies, the majority of these residues, at the molecular level, contribute much to the insect specificity determination. We postulate that the potential pressures driving the diversification of Cry proteins may be in an attempt to adapt for the ＂arm race＂ between δ-endotoxins and the targeted insects, or to enlarge their target spectra, hence result in the functional divergence. The sites identified to be under positive selection would provide targets for further structural and functional analyses on Cry proteins.     

The insertion of palindromic repeats in the evolution of proteins

The current theory of protein evolution is that all contemporary proteins are derived from an ancestral subset. However, each new sequenced genome exhibits many genes with no detectable homologues in other species, leading to the paradoxical picture of a universal ancestor with more genes than any of its progeny. Standard explanations indicate that fast evolving genes might disappear into the 'twilight zone' of sequence similarity. Regardless of the size of the original ancestral subset, its origin and the potential mechanisms of its subsequent enlargement are rarely addressed. Sequencing of Rickettsia conorii genome recently led to the discovery of three families of repeat-mobile elements frequently inserted into the middle of protein coding genes. Although not yet identified in other species of bacteria, this discovery has provided the first clear evidence for the de novo creation of long protein segments (up to 50 amino acid residues) by repeat insertion. Based on previous results and theories on the coding potential of palindromic elements, we speculate that their insertion and mobility might have played a significant role in the early stages of protein evolution.     

Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells

The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.     

Evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase family of the enolase superfamily

Understanding how proteins evolve to provide both exquisite specificity and proficient activity is a fundamental problem in biology that has implications for protein function prediction and protein engineering. To study this problem, we analyzed the evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase (OSBS/NAAAR) family, part of the mechanistically diverse enolase superfamily. Although all characterized members of the family catalyze the OSBS reaction, this family is extraordinarily divergent, with some members sharing <15% identity. In addition, a member of this family, Amycolatopsis OSBS/NAAAR, is promiscuous, catalyzing both dehydration and racemization. Although the OSBS/NAAAR family appears to have a single evolutionary origin, no sequence or structural motifs unique to this family could be identified; all residues conserved in the family are also found in enolase superfamily members that have different functions. Based on their species distribution, several uncharacterized proteins similar to Amycolatopsis OSBS/NAAAR appear to have been transmitted by lateral gene transfer. Like Amycolatopsis OSBS/NAAAR, these might have additional or alternative functions to OSBS because many are from organisms lacking the pathway in which OSBS is an intermediate. In addition to functional differences, the OSBS/NAAAR family exhibits surprising structural variations, including large differences in orientation between the two domains. These results offer several insights into protein evolution. First, orthologous proteins can exhibit significant structural variation, and specificity can be maintained with little conservation of ligand-contacting residues. Second, the discovery of a set of proteins similar to Amycolatopsis OSBS/NAAAR supports the hypothesis that new protein functions evolve through promiscuous intermediates. Finally, a combination of evolutionary, structural, and sequence analyses identified characteristics that might prime proteins, such as Amycolatopsis OSBS/NAAAR, for the evolution of new activities.     

Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells. Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.     

Autotransporter proteins: novel targets at the bacterial cell surface

Autotransporter proteins constitute a family of outer membrane/secreted proteins that possess unique structural properties that facilitate their independent transport across the bacterial membrane system and final routing to the cell surface. Autotransporter proteins have been identified in a wide range of Gram-negative bacteria and are often associated with virulence functions such as adhesion, aggregation, invasion, biofilm formation and toxicity. The importance of autotransporter proteins is exemplified by the fact that they constitute an essential component of some human vaccines. Autotransporter proteins contain three structural motifs: a signal sequence, a passenger domain and a translocator domain. Here, the structural properties of the passenger and translocator domains of three type Va autotransporter proteins are compared and contrasted, namely pertactin from Bordetella pertussis, the adhesion and penetration protein (Hap) from Haemophilus influenzae and Antigen 43 (Ag43) from Escherichia coli. The Ag43 protein is described in detail to examine how its structure relates to functional properties such as cell adhesion, aggregation and biofilm formation. The widespread occurrence of autotransporter-encoding genes, their apparent uniform role in virulence and their ability to interact with host cells suggest that they may represent rational targets for the design of novel vaccines directed against Gram-negative pathogens.     

Bindin,a multifunctional sperm ligand and the evolution of new species

Sea urchin sperm species-specifically adhere to the egg surface during fertilization. The protein which mediates this adhesion is known as bindin and cDNAs have recently been cloned and sequenced from several different species. Bindin proteins contain a highly conserved central domain flanked by much more highly divergent amino- and carboxyl-terminal domains. Investigations of the structure and function relationships indicate that the conserved domains may participate in membrane fusion and sulfated fucan binding activities, which may be conserved functions of bindin. The species-specific adhesion activity appears to be duplicated in both the amino- and carboxyl-terminal domain and may correspond to repeated sequence motifs found in these domains. The duplication of these sequence motifs and the redundancy of the adhesive domains may be important for the molecular mechanism of bindin evolution during speciation.