The major soluble 19.6 kDa protein of the organic shell matrix of the freshwater snail Biomphalaria glabrata is an N-glycosylated dermatopontin

The major Biomphalaria glabrata shell matrix protein of 19.6 kDa was isolated by preparative electrophoresis and sequenced. The sequence of 148 amino acids showed 32% sequence identity to mammalian dermatopontin sequences and 34-37% identity to two invertebrate dermatopontins described previously. A unique feature of the shell matrix dermatopontin was the presence of a single N-glycosylation consensus sequence, the asparagine of which was completely modified with a pentasaccharide. Sequence analysis of this short N-glycan by mass spectrometry and carbohydrate composition analysis indicated that it was the ubiquitous N-glycan core oligosaccharide with the exception that the terminal mannoses were 3-O-methylated. Dermatopontin is widespread in mammalian extracellular matrices, including the matrix of biominerals such as bone and teeth. Its occurrence in an invertebrate biomineral indicates that such phylogenetically distant biomineral-forming systems as vertebrate bone and mollusk shell share components which have undergone surprisingly few changes during a long evolution.     

Secondary structure of vertebrate telomerase RNA

Telomerase is a ribonucleoprotein enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The essential RNA component of telomerase provides the template for telomeric repeat synthesis. To determine the secondary structure of vertebrate telomerase RNA, 32 new telomerase RNA genes were cloned and sequenced from a variety of vertebrate species including 18 mammals, 2 birds, 1 reptile, 7 amphibians, and 4 fishes. Using phylogenetic comparative analysis, we propose a secondary structure that contains four structural domains conserved in all vertebrates. Ten helical regions of the RNA are universally conserved while other regions vary significantly in length and sequence between different classes of vertebrates. The proposed vertebrate telomerase RNA structure displays a strikingly similar topology to the previously determined ciliate telomerase RNA structure, implying an evolutionary conservation of the global architecture of telomerase RNA.     

Phylogenetic analysis of vertebrate kininogen genes

Kininogens, the precursors of bradykinins, vary extremely in both structure and function among different taxa of animals, in particular between mammals and amphibians. This includes even the most conserved bradykinin domain in terms of biosynthesis mode and structure. To elucidate the evolutionary dynamics of kininogen genes, we have identified 19 novel amino acid sequences from EST and genomic databases (for mammals, birds, and fishes) and explored their phylogenetic relationships using combined amino acid sequence and gene structure as markers. Our results show that there were initially two paralogous kininogen genes in vertebrates. During their evolution, the original gene was saved with frequent multiplication in amphibians, but lost in fishes, birds, and mammals, while the novel gene was saved with multiple functions in fishes, birds, and mammals, but became a pseudogene in amphibians. We also propose that the defense mechanism against specific predators in amphibian skin secretions has been bradykinin receptor dependent. Our findings may provide a foundation for identification and structural, functional, and evolutionary analyses of more kininogen genes and other gene families.     

Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani

The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA(Thr) and tRNA(Pro) genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of caecilians (the Batrachia hypothesis) in a monophyletic amphibian clade, turtles show diapsid affinities and are placed as sister group of crocodiles+birds, and the marsupials are grouped together with monotremes and basal to placental mammals. The deduced phylogeny was used to characterize the molecular evolution of vertebrate mitochondrial proteins. Amino acid frequencies were analyzed across the main lineages of jawed vertebrates, and leucine and cysteine were found to be the most and least abundant amino acids in mitochondrial proteins, respectively. Patterns of amino acid replacements were conserved among vertebrates. Overall, cartilaginous fishes showed the least variation in amino acid frequencies and replacements. Constancy of rates of evolution among the main lineages of jawed vertebrates was rejected.     

Xenopus laevis integrins. Structural conservation and evolutionary divergence of integrin beta subunits

We report the sequences of cDNA clones for two different integrin beta subunits isolated from a Xenopus laevis neurula cDNA library. mRNAs corresponding to both genes are first detected at gastrulation. We show that these two beta subunits are very highly related (98% identity in amino acid sequence) and probably arose at the time of tetraploidization of the X. laevis genome around 50 million years ago. Comparison of these sequences with those of various other vertebrate integrin beta subunit establishes that all species analyzed to date contain a highly conserved integrin beta subunit (beta 1). The interspecies homologies within this class of integrin beta subunits (82-86% identity in amino acid sequence) are much greater than those among the three different beta subunits which are known in humans (40-48% identity in amino acid sequence). Analysis of the homologies clearly indicates duplication and divergence of this multigene family more than 500 million years ago prior to the appearance of the vertebrates. We also observe cross-hybridization between cDNA probes for chicken integrin beta subunits and genomic DNAs of several invertebrate species. Despite the divergence in sequence among different integrin beta subunits, certain features of their structure are remarkably conserved.     

Biochemical and functional characterization of cation dependent (Mr 46,000) goat mannose 6-phosphate receptor

The Mannose 6-phosphate receptor (MPR’s) proteins are important for transporting lysosomal enzymes from trans-golgi to the pre-lysosomal compartment. These are conserved in the vertebrates from fish to mammals. We have cloned the full length cDNA for the goat MPR 46 protein and compared its sequences to the other known vertebrate MPR 46 proteins. In the present study the full-length cDNA for the goat MPR 46 protein was expressed in MPR deficient cells. The expressed protein was purified on the multivalent phosphomannan gel in the presence of divalent metal ions. The apparent molecular mass of the expressed protein was found to be ∼46 kDa and also exhibits oligomeric nature as observed in the other species, by using an MSC1 antibody (that recognizes the MPR 46 from molluscs to mammals) as well as with a peptide specific antibody corresponding to amino acid residues (218–237) of the cytoplasmic tail of human MPR 46 protein. Furthermore the distribution of the expressed protein was visualized by immunofluorescence using MSC1 and LAMP1 antibody. Additionally in the goat MPR 46 expressing cells, the sorting function of the expressed protein to sort cathepsin D to lysosomes was studied by confocal microscopy using cathepsin D antiserum and LAMP1 antibody. The binding of goat MPR 46 to cathepsin D was shown in far Western blotting and the mannose 6-phosphate dependent binding was shown by co-immunoprecipitation.     

Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein.

Prion diseases are fatal neurodegenerative disorders in man and animal associated with conformational conversion of a cellular prion protein (PrPc) into the pathologic isoform (PrPSc). The function of PrPcand the tertiary structure of PrPScare unclear. Various data indicate which parts of PrP might control the species barrier in prion diseases and the binding of putative factors to PrP. To elucidate these features, we analyzed the evolutionary conservation of the prion protein. Here, we add the primary PrP structures of 20 ungulates, three rodents, three carnivores, one maritime mammal, and nine birds. Within mammals and birds we found a high level of amino acid sequence identity, whereas between birds and mammals the overall homology was low. Various structural elements were conserved between mammals and birds. Using the CONRAD space-scale alignment, which predicts conserved and variable blocks, we observed similar patterns in avian and mammalian PrPs, although 130 million years of separate evolution lie in between. Our data support the suggestion that the repeat elements might have expanded differently within the various classes of vertebrates. Of note is the N-terminal part of PrP (amino acid residues 23-90), which harbors insertions and deletions, whereas in the C-terminal portion (91-231) mainly point mutations are found. Strikingly, we found a high level of conservation of sequences that are not part of the structured segment 121-231 of PrPcand of the structural elements therein, e.g. the N-terminal region from amino acid residue 23-90 and the regions located upstream of alpha-helices 1 and 3.     

Modular Evolution of PGC-1α in Vertebrates

In mammals, the peroxisome proliferator activated receptor (PPAR)γ coactivator-1α (PGC-1α) is a central regulator of mitochondrial gene expression, acting in concert with nuclear respiratory factor-1 (NRF-1) and the PPARs. Its role as a “master regulator” of oxidative capacity is clear in mammals, but its role in other vertebrates is ambiguous. In lower vertebrates, although PGC-1α seems to play a role in coordinating the PPARα axis as in mammals, it does not appear to be involved in NRF-1 regulation of mitochondrial content. To evaluate the evolutionary patterns of this coactivator in fish and mammals, we investigated the evolutionary trajectories of PGC-1α homologs in representative vertebrate lineages. A phylogeny of the PGC-1 paralogs suggested that the family diversified through repeated genome duplication events early in vertebrate evolution. Bayesian and maximum likelihood phylogenetic reconstructions of PGC-1α in representative vertebrate species revealed divergent evolutionary dynamics across the different functional domains of the protein. Specifically, PGC-1α exhibited strong conservation of the activation/PPAR interaction domain across vertebrates, whereas the NRF-1 and MEF2c interaction domains experienced accelerated rates of evolution in actinopterygian (fish lineages) compared to sarcopterygians (tetrapod lineages). Furthermore, analysis of the amino acid sequence of these variable domains revealed successive serine- and glutamine-rich insertions within the teleost lineages, with important ramifications for PGC-1α function in these lineages. Collectively, these results suggest modular evolution of the PGC-1α protein in vertebrates that could allow for lineage-specific divergences in the coactivating capabilities of this regulator.     

Molecular evolution of proglucagon

The vertebrate proglucagon gene encodes glucagon, and the two glucagon-like peptides GLP-1 and GLP-2. To better understand the origin and diversification of the distinct hormonal roles of the three glucagon-like sequences encoded by the proglucagon gene, we have examined the evolution of this gene. The structure of proglucagon has been largely maintained within vertebrates. Duplication of the proglucagon gene or duplications of sequences within the proglucagon gene are rare. All proglucagon gene duplications are likely to be the result of genome duplication events. Examination of the rates of amino acid sequence evolution of each hormone reveals that they have not evolved in a uniform manner. Each hormone has evolved in an episodic fashion, suggesting that the selective constraints acting upon the sequence vary between, and within, vertebrate classes. Changes in selection on a sequence often reflect changes in the function of the sequence, such as the change in function of GLP-1 from a glucagon-like hormone in fish to an incretin in mammals. We found that the GLP-2 sequence underwent rapid sequence evolution in the early mammal lineage, therefore we have concluded that mammalian GLP-2 has acquired a new biological function that is not found in other vertebrates. Comparisons of the hormone sequences show that many amino acid residues that are functionally important in mammalian hormones are not conserved through vertebrate evolution. This observation suggests that the sequences involved in hormone action change through evolution.     

Ghrelin: a multifunctional hormone in non-mammalian vertebrates

In mammals, ghrelin is a non-amidated peptide hormone, existing in both acylated and non-acylated forms, produced mainly from the X/A or ghrelin cells present in the mucosal layer of the stomach. Ghrelin is a natural ligand of the growth hormone (GH) secretagogue-receptor (GHS-R), and functions primarily as a GH-releasing hormone and an orexigen, as well as having several other biological actions. Among non-mammalian vertebrates, amino acid sequence of ghrelin has been reported in two species of cartilaginous fish, seven species of teleosts, two species of amphibians, one species of reptile and six species of birds. The structure and functions of ghrelin are highly conserved among vertebrates. This review presents a concise overview of ghrelin biology in non-mammalian vertebrates.     

Evolution of the Integrin α and β Protein Families

A phylogenetic analysis of vertebrate and invertebrate α integrins supported the hypothesis that two major families of vertebrate α integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the αPS1 and αPS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including αPS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate αPS1 and αPS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of β integrins did not clearly resolve whether vertebrate β integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate β4 may have done so. In general, the phylogeny of neither α nor β integrins showed a close correspondence with patterns of α–β heterodimer formation or other functional characteristics. One major exception to this trend involved αL, αM, αX, and αD, a monophyletic group of immune cell-expressed α integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their β integrin partners. Received: 22 June 2000 / Accepted: 11 September 2000     

The phylogenetic distribution of tubulin:Tyrosine ligase

Summary The post-translational addition of tyrosine toa-tubulin, catalyzed by tubulin:tyrosine ligase, has been previously reported in mammals and birds. The present study demonstrated that significant ligase activity was present in representative organisms from several other major vertebrate classes (chondrichthyes through reptiles) and that both substrate and enzyme from all vertebrates investigated were compatible with mammalian ligase and tubulin in the tyrosination reaction. None of the invertebrate tissues examined showed incorporation of tyrosine, phenylalanine or dihydroxyphenylalanine intoa tubulin under conditions allowing significant incorporation of these compounds in vertebrate supernatant samples. The failure of invertebrate tubulin to incorporate tyrosine in vitro did not appear to be due to saturation of the carboxyl terminal position with tyrosine or the presence of a soluble inhibitor of ligase activity.Although tubulin amino acid composition has been highly conserved throughout evolution, a major evolutionary divergence is described based upon biochemical differences whereby invertebrate tubulin cannot be tyrosinated or posttranslationally modified with phenylalanine or dihydroxyphenylalanine under conditions suitable for the incorporation of these compounds by vertebratea tubulin.     

Isolation of Mhc class I cDNAs from the axolotl Ambystoma mexicanum

 Class I major histocompatibility complex (Mhc) cDNA clones were isolated from axolotl mRNA by polymerase chain reaction (PCR) and by screening a cDNA phage library. The nucleotide and predicted amino acid sequences show definite similarities to the Mhc class Iα molecules of higher vertebrates. Most of the amino acids in the peptide binding region that dock peptides at their N and C termini in mammals are conserved. Several amino acids considered to be important for the interaction of β₂-microglobulin with the Mhc α chain are also conserved in the axolotl sequence. The fact that axolotl class I A cDNAs are ubiquitously expressed and highly polymorphic in the α1 and α2 domains suggests the classical nature of axolotl class I A genes. Received: 3 June 1996 / Revised: 14 October 1996     

C6-Like and C3-Like Molecules from the Cephalochordate, Amphioxus, Suggest a Cytolytic Complement System in Invertebrates

The mammalian immune system has cytotoxic mechanisms, both cellular and humoral, that destroy the membrane integrity of target cells. The main effector molecules of these cytolytic mechanisms—perforin, used by killer lymphocytes, and the membrane attack complex (MAC) components of the complement system—share a unique module called the MAC/perforin module. Until now, both immunological cytotoxicity and the MAC/perforin module have been reported only in jawed vertebrates. Here, we report the identification of a protein containing the MAC/perforin module from the invertebrate cephalochordate, amphioxus (Branchiostoma belcheri), using expressed sequence tag (EST) analysis of the notochord. The deduced amino acid sequence of this molecule is most similar to the primary structure of human complement component C6 and is designated AmphiC6. AmphiC6 shares a unique modular structure, including the MAC/perforin module, with human C6 and other MAC components. Another EST clone predicts the presence of a thioester-containing protein with the closest structural similarity to vertebrate C3 (therefore designated AmphiC3). AmphiC3 retains most of the functionally important residues of vertebrate C3 and is shown by phylogenetic analysis to be derived directly from the common ancestor of vertebrate C3, C4, and C5. Only opsonic activity has been assigned to the invertebrate complement system until now. Therefore, this is the first molecular evidence for complement-mediated immunological cytotoxicity in invertebrates. Received: 24 August 2001 / Accepted: 12 November 2001     

Insulin-Like Growth Factors (IGFs) in Vertebrate Phylogenesis. Comparative Analysis of the IGF-II Domain Binding to the Mannose-6-Phosphate IGF-II-Receptor

Characteristic property of the mammalian IGF-II molecule is the capability for the high-affinity binding to the IGF-2-receptor. The history of the appearance of the IGF-2 receptor in vertebrate phylogenesis is rather confused. IGF-2-receptor isoforms that are able to bind IGF-II with high affinity are revealed in tissues of mammals and fish, but not in amphibians and birds. The appearance of IGF-II itself and of structural modifications of its molecule in the course of phylogenesis remains unclear. The author proposed principle of bipolar structure of the A-chain domain participating in binding the IGF-2-receptor. This principle has made it possible to analyze changes of the amino acid composition of this domain in molecules of IGF-II and related peptides at various stages of vertebrate phylogenesis. Composition of the studied domain has allowed considering the cyclostome IGF as a precursor of fish IGF-II and IGF-I; in vertebrates, the domain composition is more variable in IGF-II than in IGF-I. Based on the performed analysis, it is suggested that the species-specific character of interaction of IGF-II with the IGF-2-receptor in the lower vertebrates and birds is one of obstacles of detecting the IGF-2 receptor in their tissues; IGF-I is also suggested to be a possible ligand of the IGF-2-receptor in the lower vertebrates.     

Ghrelin: a multifunctional hormone in non-mammalian vertebrates.

In mammals, ghrelin is a non-amidated peptide hormone, existing in both acylated and non-acylated forms, produced mainly from the X/A or ghrelin cells present in the mucosal layer of the stomach. Ghrelin is a natural ligand of the growth hormone (GH) secretagogue-receptor (GHS-R), and functions primarily as a GH-releasing hormone and an orexigen, as well as having several other biological actions. Among non-mammalian vertebrates, amino acid sequence of ghrelin has been reported in two species of cartilaginous fish, seven species of teleosts, two species of amphibians, one species of reptile and six species of birds. The structure and functions of ghrelin are highly conserved among vertebrates. This review presents a concise overview of ghrelin biology in non-mammalian vertebrates.     

Comparative genomics using fugu: a tool for the identification of conserved vertebrate cis-regulatory elements

With the imminent completion of the whole genome sequence of humans, increasing attention is being focused on the annotation of cis-regulatory elements in the human genome. Comparative genomics approaches based on evolutionary conservation have proved useful in the detection of conserved cis-regulatory elements. The pufferfish, Fugu rubripes, is an attractive vertebrate model for comparative genomics, by virtue of its compact genome and maximal phylogenetic distance from mammals. Fugu has lost a large proportion of nonessential DNA, and retained single orthologs for many duplicate genes that arose in the fish lineage. Non-coding sequences conserved between fugu and mammals have been shown to be functional cis-regulatory elements. Thus, fugu is a model fish genome of choice for discovering evolutionarily conserved regulatory elements in the human genome. Such evolutionarily conserved elements are likely to be shared by all vertebrates, and related to regulatory interactions fundamental to all vertebrates. The functions of these conserved vertebrate elements can be rapidly assayed in mammalian cell lines or in transgenic systems such as zebrafish/medaka and Xenopus, followed by validation of crucial elements in transgenic rodents.