Sequences related to the ox pancreatic ribonuclease coding region in the genomic DNA of mammalian species

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants.In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species.We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.Correspondence to: A. Furia     

Secretory ribonuclease genes and pseudogenes in true ruminants

Mammalian pancreatic ribonucleases (RNase) form a family of extensively studied homologous proteins. Phylogenetic analyses, based on the primary structures of these enzymes, indicated that the presence of three homologous enzymes (pancreatic, seminal and brain ribonucleases) in the bovine species is due to gene duplication events, which occurred during the evolution of ancestral ruminants. In this paper the sequences are reported of the coding regions of the orthologues of the three bovine secretory ribonucleases in hog deer and roe deer, two deer species belonging to two different subfamilies of the family Cervidae. The sequences of the 3′ untranslated regions of the three different secretory RNase genes of these two deer species and giraffe are also presented. Comparison of these and previously determined sequences of ruminant ribonucleases showed that the brain-type enzymes of giraffe and these deer species exhibit variations in their C-terminal extensions. The seminal-type genes of giraffe, hog deer and roe deer show all the features of pseudogenes. Phylogenetic analyses, based on the complete coding regions and parts of the 3′ untranslated regions of the three different secretory ribonuclease genes of ox, sheep, giraffe and the two deer species, show that pancreatic, seminal- and brain-type RNases form three separate groups.     

Sequence analysis of a cloned cDNA coding for bovine seminal ribonuclease

The sequence of a cloned cDNA coding for bovine seminal ribonuclease, an enzyme secreted in the bull seminal vesicles, was determined. The cDNA starts at the amino acid residue 47 and terminates 12 nucleotides beyond the consensus sequence AAUAAA in the 3' non-coding region of the mRNA. Northern blotting analysis shows that the mRNA for bovine seminal ribonuclease consists of about 950 nucleotides, a value that is similar to that of other mRNAs coding for ribonucleases of the pancreatic type.     

Ruminant brain ribonucleases: expression and evolution

Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving rise to three paralogous genes occurred in ruminant ancestors. One of these genes encodes a ribonuclease identified in bovine brain. A peculiar feature of this enzyme and orthologous sequences in other ruminants are C-terminal extensions consisting of 17-27 amino acid residues. Evidence was obtained by Western blot analysis for the presence of brain-type ribonucleases in brain tissue not only of ox, but also of sheep, roe deer and chevrotain (Tragulus javanicus), a member of the earliest diverged taxon of the ruminants. The C-terminal extension of brain-type ribonuclease from giraffe deviates much in sequence from orthologues in other ruminants, due to a change of reading frame. However, the gene encodes a functional enzyme, which could be expressed in heterologous systems. The messenger RNA of bovine brain ribonuclease is not only expressed at a high level in brain tissue but also in lactating mammary gland. The enzyme was isolated and identified from this latter tissue, but was not present in bovine milk, although pancreatic ribonucleases A and B could be isolated from both sources. This suggests different ways of secretion of the two enzyme types, possibly related to structural differences. The sequence of the brain-type RNase from chevrotain suggests that the C-terminal extensions of ruminant brain-type ribonucleases originate from deletions in the ancestral DNA (including a region with stop codons), followed by insertion of a 5-8-fold repeated hexanucleotide sequence, coding for a proline-rich polypeptide.     

Phylogeny of ruminants secretory ribonuclease gene sequences of pronghorn (Antilocapra americana)

Phylogenetic analyses based on primary structures of mammalian ribonucleases, indicated that three homologous enzymes (pancreatic, seminal and brain ribonucleases) present in the bovine species are the results of gene duplication events, which occurred in the ancestor of the ruminants after divergence from other artiodactyls. In this paper sequences are presented of genes encoding pancreatic and brain-type ribonuclease genes of pronghorn (Antilocapra americana). The seminal-type ribonuclease gene could not be detected in this species, neither by PCR amplification nor by Southern blot analyses, indicating that it may be deleted completely in this species. Previously we demonstrated of a study of amino acid sequences of pancreatic ribonucleases of a large number of ruminants the monophyly of bovids and cervids, and that pronghorn groups with giraffe. Here we present phylogenetic analyses of nucleotide sequences of ribonucleases and other molecules from ruminant species and compare these with published data. Chevrotain (Tragulus) always groups with the other ruminants as separate taxon from the pecora or true ruminants. Within the pecora the relationships between Bovidae, Cervidae, Giraffidae, and pronghorn (Antilocapra) cannot be decided with certainty, although in the majority of analyses Antilocapra diverges first, separately or joined with giraffe. Broad taxon sampling and investigation of specific sequence features may be as important for reliable conclusions in phylogeny as the lengths of analyzed sequences.     

The molecular evolution of pancreatic ribonuclease

Summary The primary structures of pancreatic ribonucleases from 26 species (18 artiodactyls, horse, whale, 5 rodents and turtle) are known. Several species contain identical ribonucleases (cow/bison; sheep/goat), other species show polymorphism (arabian camel) or the presence of two structural gene loci (guinea pig pancreas contains two ribonucleases that differ at 31 positions). 26 different sequences (including the ribonuclease from bovine seminal plasma which is paralogous to the pancreatic ribonucleases) were used to construct a most parsimonious tree. A second tree that most closely approximates current biological opinion requires 402 whereas the most parsimonious tree requires 389 nucleotide substitutions. The artiodactyl part of the most parsimonious tree conforms quite well with the biological one of this order, except for the position of the giraffe which is placed with the pronghorn. Other parts of the most parsimonious tree agree less with the biological tree, probably as a result of the occurrence of many parallel and back substitutions. Bovine seminal ribonuclease was found to be the result of a gene duplication which occurred before the divergence of the true ruminants, but after the divergence of this group from the cameloids.The evolutionary rate of ribonuclease was found to be 390, 3.0 and 11 nucleotide substitutions per 10⁹ yrs per ribonuclease gene, codon and covarion respectively. However, there is much variation in evolutionary rate in different taxa. Values ranging from about 100 (in the bovidae) to about 700 (in the rodents) nucleotide substitutions per 10⁹ yrs per gene were found.A method for counting parallel and back mutations is presented. The 389 nucleotide substitutions in the most parsimonious tree occur at 88 codon positions; 154 of them are the result of parallel and back mutations. Parallel evolution to a similar structure, including the presence of 2 sites with carbohydrate, was demonstrated in an extensive region at the surface of pig and guinea pig ribonuclease B. The presence of carbohydrate probably is important in a number of species. A correlation between the presence of heavily glycosidated ribonucleases and coecal digestion was observed. Hypothetical sequences of ancestral ungulate ribonucleases contain many recognition sites for carbohydrate attachment; this suggests that herbivores with coecal digestion might have preceded the true ruminants in mammalian evolution.     

Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridges.

The hypothesis previously advanced that interchain disulfide bridges link the two identical subunits of bovine seminal ribonuclease BS-1 has been confirmed. The sedimentation rate and the electrophoretic mobility of the protein are not affected by denaturing agents unless thiol reagents are present in the denaturation mixtures. Reduction under controlled conditions results in the immediate cleavage of only 2 disulfide bonds out of 10 percent in the dimeric protein. Under these conditions, and the results do not change when partial reduction is followed by S-alkylation, 30% of the protein dissociates, while the remaining is found to consist of a dimeric species easily dissociable by denaturing agents without addition of thiol reagents. This indicates that the dimeric structure of seminal ribonuclease is maintained not only by disulfide bridges, but also by noncovalent forces. The protein derivative prepared by selective reduction and alkylation has been identified as monomeric bis-S-carboxymethylcysteine-31,32-ribonuclease BS-1. This is on the basis of the characterization of the 14C-labeled S-carboxymethylated peptides isolated from a thermolytic hydrolysate of the derivative prepared with iodo-2-[14C]acetic acid. Monomeric, selectively alkylated ribonuclease BS-1 is stable and catalytically active. The importance of such a derivative is discussed both in the light of the recent studies on the biological actions of seminal ribonuclease and as the fourth component of an experimental system of ribonucleases consisting of two homologous dimers (bovine seminal ribonuclease BS-1 and dimerized bovine pancreatic ribonuclease A) and two homologous monomers (ribonuclease A and the monomeric derivative of ribonuclease BS-1.     

Dissociation and reconstitution of bovine seminal RNAase: Construction of a hyperactive hybrid dimer

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.     

Origin of the duplicated ribonuclease gene in guinea-pig: Comparison of the amino acid sequences with those of two close relatives: Capybara and cuis ribonuclease

The amino acid sequences of the pancreatic ribonuclease from capybara (Hydrochoerus hydrochaeris) and cuis (Galea musteloides) were determined. Both species belong to the same superfamily of the hystricomorph rodents as the guinea-pig. In guinea-pig pancreas two ribonucleases are present as a result of a recent gene duplication, but in capybara and cuis pancreas only one single ribonuclease has been found. A most parsimonious tree of ribonucleases indicates that the gene duplication leading to both guinea-pig ribonucleases occurred before the divergence of guinea-pig from capybara and cuis. This would mean that changes in expression of the ribonuclease genes have occurred in these taxa. Cuis and capybara ribonuclease have no Asn-X-Ser/Thr sequences and are carbohydrate-free proteins. Capybara ribonuclease has leucine at position 114, a position occupied by proline in the cis-configuration in bovine pancreatic ribonuclease.     

Rapid Diversification of RNase A Superfamily Ribonucleases from the Bullfrog, Rana catesbeiana

We present sequences of five novel RNase A superfamily ribonuclease genes of the bullfrog, Rana catesbeiana. All five genes encode ribonucleases that are similar to Onconase, a cytotoxic ribonuclease isolated from oocytes of R. pipiens. With amino acid sequence data from 14 ribonucleases from three Rana species (R. catesbeiana, R. japonica, and R. pipiens), we have constructed bootstrap-supported phylogenetic trees that reorganize these ribonucleases into five distinct lineages--the pancreatic ribonucleases (RNases 1), the eosinophil-associated ribonucleases (RNases 2, 3, and 6), the ribonucleases 4, the angiogenins (RNases 5) and the Rana ribonucleases--with the Rana ribonucleases no more closely related to the angiogenins than they are to any of the other ribonuclease lineages shown. Further phylogenetic analysis suggests the division of the Rana ribonucleases into two subclusters (A and B), with positive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contributing to the rapid diversification of ribonucleases within each cluster. This pattern of evolution-rapid diversification via positive selection among sequences of a multigene cluster-bears striking resemblance to what we have described for the eosinophil-associated ribonuclease genes of the rodent Mus musculus, a finding that may have implications with respect the physiologic function of this unique family of proteins.     

Molecular evolution of pancreatic-type ribonucleases

Amino acid sequences of 39 mammalian ribonucleases have been used to construct trees by the maximum parsimony procedure. These trees are in fairly good agreement with the biological classification of the species involved. In the branching order of the six investigated eutherian mammalian orders, the edentates diverge first, followed, probably, by the primates. No definite conclusions can be drawn about the order of divergence of the perissodactyls, the rodents, and the group consisting of artiodactyls plus cetaceans. Nucleic acid sequences of part of the messenger RNAs of rat pancreatic and bovine seminal ribonuclease were compared. Both messengers have a second stop codon at position 129, which is in agreement with the addition of four residues at the C-terminus in several other ribonucleases. Turtle pancreatic ribonuclease and human angiogenin differ from each other and from the mammalian ribonucleases at 55%-70% of the amino acid positions; they share a number of structural features. Mammalian nonsecretory ribonucleases are homologous to the pancreatic ribonucleases in sequence regions where the active-site histidine residues are located.     

Crystal structure of the dimeric unswapped form of bovine seminal ribonuclease

Bovine seminal ribonuclease is a unique case of protein dimorphism, since it exists in two dimeric forms, with different biological and kinetic behavior, which interconvert into one another through three-dimensional swapping. Here we report the crystal structure, at 2.2 A resolution, of the unswapped form of bovine seminal ribonuclease. Besides completing the structural definition of bovine seminal ribonuclease conformational dimorphism, this study provides the structural basis to explain the dependence of the enzyme cooperative effects on its swapping state.     

Inclusion of Cetaceans Within the Order Artiodactyla Based on Phylogenetic Analysis of Pancreatic Ribonuclease Genes

Mammalian secretory ribonucleases (RNases 1) form a family of extensively studied homologous proteins that were already used for phylogenetic analyses at the protein sequence level previously. In this paper we report the determination of six ribonuclease gene sequences of Artiodactyla and two of Cetacea. These sequences have been used with ruminant homologues in phylogenetic analyses that supported a group including hippopotamus and toothed whales, a group of ruminant pancreatic and brain-type ribonucleases, and a group of tylopod sequences containing the Arabian camel pancreatic ribonuclease gene and Arabian and Bactrian camel and alpaca RNase 1 genes of unknown function. In all analyses the pig was the first diverging artiodactyl. This DNA-based tree is compatible to published trees derived from a number of other genes. The differences to those trees obtained with ribonuclease protein sequences can be explained by the influence of convergence of pancreatic RNases from hippopotamus, camel, and ruminants and by taking into account the information from third codon positions in the DNA-based analyses. The evolution of sequence features of ribonucleases such as the distribution of positively charged amino acids and of potential glycosylation sites is described with regard to increased double-stranded RNA cleavage that is observed in several cetacean and artiodactyl RNases which may have no role in ruminant or ruminant-like digestion. Received: 2 June 1998 / Accepted: 31 August 1998     

Ribonucleases and their antitumor activity

The antitumor effect of ribonucleases was studied with animal ribonucleolytic enzymes, bovine pancreatic RNase A, bovine seminal RNase (BS-RNase), onconase and angiogenin. While bovine pancreatic RNase A exerts a minor antitumor effect, BS-RNase and onconase exert significant effects. Angiogenin, as RNase, works in an opposite way, it initiates vascularization of tumors and subsequent tumor growth. Ribonunclease inhibitors are not able to inhibit the antitumor effectiveness of BS-RNase or onconase. However, they do so in the case of pancreatic RNases. Conjugation of BS-RNase with antibodies against tumor antigens (preparation of immunotoxins) like the conjugation of the enzyme with polymers enhances the antitumor activity of the ribonuclease. After conjugation with polymers, the half-life of BS-RNase in blood is extended and its immunogenicity reduced. Recombinant RNases have the same functional activity as the native enzymes. The synthetic genes have also been modified, some of them with gene sequences typical for the BS-RNase parts. Recent experimental efforts are directed to the preparation of ‘humanized antitumor ribonuclease’ that would be structurally similar to human enzyme with minimal immunogenicity and side effects. The angiogenesis of tumors is attempted to be minimized by specific antibodies or anti-angiogenic substances.     

Evolution of oligomeric proteins. The unusual case of a dimeric ribonuclease.

The model system made up of a monomeric and a dimeric ribonuclease of the pancreatic-type superfamily has recently attracted the attention of investigators interested in the evolution of oligomeric proteins. In this system, bovine pancreatic ribonuclease (RNase A) is the monomeric prototype, and bovine seminal ribonuclease (BS-RNase) is the dimeric counterpart. However, this evolutionary case is unusual, as BS-RNase is the only dimeric member of the whole large superfamily comprising more than 100 identified members from amphibia, aves, reptilia and mammalia. Furthermore, although the seminal-type RNase gene can be traced back to the divergence of the ruminants, it is expressed only in a single species (Bos taurus). These unusual findings are discussed, as well as previous hypotheses on the evolution of seminal RNase. Furthermore, a new 'minimalist' hypothesis is proposed, in line with basic principles of structural biology and molecular evolution.     

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.     

Comprehensive comparison of the cytotoxic activities of onconase and bovine seminal ribonuclease

Onconase (ONC) and bovine seminal ribonuclease (BS-RNase) are homologs of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, ONC and BS-RNase can evade the cytosolic ribonuclease inhibitor protein and are potent cytotoxins. Here, the endogenous cytotoxic activities of ONC and BS-RNase are compared in a wide variety of assays. Injections of ONC into one or both testes of mice and rats evokes a stronger aspermatogenic activity than does the injection of BS-RNase. Epididymides exposed to ONC lose mass and all sperm. Testicular tissue is gradually colonized by immunite and fibrocytic cells. Yet, Leydig cells are always present and functional in the ligamented parts of testicles injected with ONC or BS-RNase. ONC is likewise more toxic to mouse embryos than is BS-RNase, both in vitro and in vivo. The antiproliferative effect of ONC on human tumor cell line ML-2 and lymphocytes in a mixed lymphocyte culture is also more pronounced than is that of BS-RNase. The number of granulocyte-macrophage colony-forming units is repressed almost completely by ONC, whereas a five-fold higher dose of BS-RNase does not cause substantial inhibition. In mice, ONC is less immunogenic than BS-RNase but more immunogenic than RNase A. Together, these data indicate that ONC is a pluripotent cytotoxin, and serves as the benchmark with which to gauge the cytotoxicity of other ribonucleases.     

Intrachain disulfide bridges of bovine seminal ribonuclease

The pairing of the four intrachain disulfide bonds of bovine seminal ribonuclease, a dimeric protein isolated from bovine seminal plasma, has been established by the isolation and characterization of the cystine peptides obtained from a thermolytic-tryptic hydrolysate of the protein. These disulfide bonds involve eight half-cystine residues located in the protein subunit chain at sequence positions identical with those of the eight half-cystine residues of the strictly homologous chain of bovine pancreatic ribonuclease. The results reported show that these eight 'homologous' half-cystine residues pair in seminal ribonuclease exactly as they do in pancreatic ribonuclease. They also indirectly confirm that the remaining two half-cystine residues present in each chain of the seminal enzyme are involved in intersubunit bonds.     

Secretory ribonucleases in the primitive ruminant chevrotain (Tragulus javanicus).

Phylogenetic analyses of secretory ribonucleases or RNases 1 have shown that gene duplication events, giving rise to three paralogous genes (pancreatic, seminal and brain RNase), occurred during the evolution of ancestral ruminants. A higher number of paralogous sequences are present in chevrotain (Tragulus javanicus), the earliest diverged taxon within the ruminants. Two pancreatic RNase sequences were identified, one encoding the pancreatic enzyme, the other encoding a pseudogene. The identity of the pancreatic enzyme was confirmed by isolation of the protein and N-terminal sequence analysis. It is the most acidic pancreatic ribonuclease identified so far. Formation of the mature enzyme requires cleavage by signal peptidase of a peptide bond between two glutamic acid residues. The seminal-type RNase gene shows features of a pseudogene, like orthologous genes in other ruminants investigated with the exception of the bovine species. The brain-type RNase gene of chevrotain is expressed in brain tissue. A hybrid gene with a pancreatic-type N-terminal and a brain-type C-terminal sequence has been identified but nothing is known about its expression. Phylogenetic analysis of RNase 1 sequences of six ruminant, three other artiodactyl and two whale species support previous findings that two gene duplications occurred in a ruminant ancestor. Three distinct groups of pancreatic, seminal-type and brain-type RNases have been identified and within each group the chevrotain sequence it the first to diverge. In taxa with duplications of the RNase gene (ruminants and camels) the gene evolved at twice as fast than in taxa in which only one gene could be demonstrated; in ruminants there was an approximately fourfold increase directly after the duplications and then a slowing in evolutionary rate.