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.     

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.     

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     

Pancreatic-Type Ribonuclease 1 Gene Duplications in Rat Species

Mammalian pancreatic-type ribonucleases (RNases) 1 represent single-copy genes in the genome of most investigated mammalian species, including Mus musculus and other murid rodents. However, in six species belonging to the genus Rattus and closely related taxa, several paralogous gene products were identified by Southern blotting and PCR amplifications of genomic sequences. Phylogenies of nucleotide and derived amino acid sequences were reconstructed by several procedures, with three Mus species as outgroup. Duplications of the RNase 1 occurred after the divergence of Niviventer cremoriventer and Leopoldamys edwardsi from the other investigated species. Four groups of paralogous genes could be identified from specific amino acid sequence features in each of them. Low ratios of nonsynonymous-to-synonymous substitutions and the paucity of pseudogene features suggest functional gene products. One of the RNase 1 genes of R. norvegicus is expressed in the pancreas. RNases 1 were isolated from pancreatic tissues of R. rattus and R. exulans and submitted to N-terminal amino acid sequence analysis. In R. rattus, the orthologue of the expressed gene of R. norvegicus was identified, but in R. exulans, two paralogous gene products were found. The gene encoding for one of these had not yet been found by PCR amplification of genomic DNA. A well-defined group of orthologous sequences found in five investigated species codes for very basic RNases. Northern blot analysis showed expression of messenger RNA for this RNase in the spleen of R. norvegicus, but the protein product could not be identified. Evolutionary rates of RNase 1, expressed as nucleotide substitutions per site per 10(3) million years (Myr), vary between 5 and 9 in the lines leading to Mus, Niviventer, and Lepoldamys (on the basis of an ancestral date of mouse/rat divergence of 12.2 Myr) and between 20 and 50 in the lines to the other sequences after divergence from Niviventer and Leopoldamys (5.5 Myr).     

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 evolution of seminal ribonuclease: pseudogene reactivation or multiple gene inactivation events?

Two approaches, one novel, are applied to analyze the divergent evolution of ruminant seminal ribonucleases (RNases), paralogs of the well-known pancreatic RNases of mammals. Here, the goal was to identify periods of divergence of seminal RNase under functional constraints, periods of divergence as a pseudogene, and periods of divergence driven by positive selection pressures. The classical approach involves the analysis of nonsynonymous to synonymous replacements ratios (omega) for the branches of the seminal RNase evolutionary tree. The novel approach coupled these analyses with the mapping of substitutions on the folded structure of the protein. These analyses suggest that seminal RNase diverged during much of its history after divergence from pancreatic RNase as a functioning protein, followed by homoplastic inactivations to create pseudogenes in multiple ruminant lineages. Further, they are consistent with adaptive evolution only in the most recent episode leading to the gene in modern oxen. These conclusions contrast sharply with the view, cited widely in the literature, that seminal RNase decayed after its formation by gene duplication into an inactive pseudogene, whose lesions were repaired in a reactivation event. Further, the 2 approaches, omega estimation and mapping of replacements on the protein structure, were compared by examining their utility for establishing the functional status of the seminal RNase genes in 2 deer species. Hog and roe deer share common lesions, which strongly suggests that the gene was inactive in their last common ancestor. In this specific example, the crystallographic approach made the correct implication more strongly than the omega approach. Studies of this type should contribute to an integrated framework of tools to assign functional and nonfunctional episodes to recently created gene duplicates and to understand more broadly how gene duplication leads to the emergence of proteins with novel functions.     

Multiple ribonuclease H-encoding genes in the Caenorhabditis elegans genome contrasts with the two typical ribonuclease H-encoding genes in the human genome

Database searches of the Caenorhabditis elegans and human genomic DNA sequences revealed genes encoding ribonuclease H1 (RNase H1) and RNase H2 in each genome. The human genome contains a single copy of each gene, whereas C. elegans has four genes encoding RNase H1-related proteins and one gene for RNase H2. By analyzing the mRNAs produced from the C. elegans genes, examining the amino acid sequence of the predicted protein, and expressing the proteins in Esherichia coli we have identified two active RNase H1-like proteins. One is similar to other eukaryotic RNases H1, whereas the second RNase H (rnh-1.1) is unique. The rnh-1.0 gene is transcribed as a dicistronic message with three dsRNA-binding domains; the mature mRNA is transspliced with SL2 splice leader and contains only one dsRNA-binding domain. Formation of RNase H1 is further regulated by differential cis-splicing events. A single rnh-2 gene, encoding a protein similar to several other eukaryotic RNase H2L's, also has been examined. The diversity and enzymatic properties of RNase H homologues are other examples of expansion of protein families in C. elegans. The presence of two RNases H1 in C. elegans suggests that two enzymes are required in this rather simple organism to perform the functions that are accomplished by a single enzyme in more complex organisms. Phylogenetic analysis indicates that the active C. elegans RNases H1 are distantly related to one another and that the C. elegans RNase H1 is more closely related to the human RNase H1. The database searches also suggest that RNase H domains of LTR-retrotransposons in C. elegans are quite unrelated to cellular RNases H1, but numerous RNase H domains of human endogenous retroviruses are more closely related to cellular RNases H.     

Cloning, Expression, and Mapping of Ribonucleases H of Human and Mouse Related to Bacterial RNase HI

We identified two human sequences and one mouse sequence in the database of expressed sequence tags that are highly homologous to the N-terminal sequence of eukaryotic RNases H1. The cDNAs for humanRNASEH1and mouseRnaseh1were obtained, their nucleotide sequences determined, and the proteins expressed inEscherichia coliand partially purified. Both proteins have RNase H activityin vitroand they bind to dsRNA and RNA–DNA hybrids through the N-terminal conserved motif present in eukaryotic RNases H1. TheRNASEH1gene is expressed in all human tissues at similar levels, indicating that RNase H1 may be a housekeeping protein. The humanRNASEH1and mouseRnaseh1cDNAs were used to isolate BAC genomic clones that were used as probes for fluorescencein situhybridization. The human gene was localized to chromosome 17p11.2 and the mouse gene to a nonsyntenic region on chromosome 12A3. The chromosomal location and possible disease association of the humanRNASEH1gene are discussed.     

Differences in glycosylation pattern of human secretory ribonucleases.

The major secretory ribonuclease (RNase) of human urine (RNase HUA) was isolated and sequenced by automatic Edman degradation and analysis of peptides and glycopeptides. The isolated enzyme was shown to be free of other urine RNase activities by SDS/polyacrylamide-gel electrophoresis and activity staining. It is a glycoprotein 128 amino acids long, differing from human pancreatic RNase in the presence of an additional threonine residue at the C-terminus. It differs from the pancreatic enzyme in its glycosylation pattern as well, and contains about 45 sugar residues. Each of the three Asn-Xaa-Ser/Thr sequences (Asn-34, Asn-76, Asn-88) is glycosylated with a complex-type oligosaccharide chain. Glycosylation at Asn-88 has not been observed previously in mammalian secretory RNases. Preliminary sequence data on the major RNase of human seminal plasma have revealed no difference between it and the major urinary enzyme; their similarities include the presence of threonine at the C-terminus. The glycosylation pattern of human seminal RNase is very similar to that of the pancreatic enzyme. The structural differences between the secretory RNases from human pancreas, urine and seminal plasma must originate from organ-specific post-translational modifications of the one primary gene product. Detailed characterization of peptides and the results of gel filtration of tryptic and tryptic/chymotryptic digests of performic acid-oxidized RNase have been deposited as Supplementary Publication SUP 50146 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

Parallel functional changes in the digestive RNases of ruminants and colobines by divergent amino acid substitutions

A morphological or physiological trait may appear multiple times in evolution. At the molecular level, similar protein functions may emerge independently in different lineages. Whether these parallel functional changes are due to parallel amino acid substitutions has been a subject of debate. Here, I address this question using digestive ribonucleases (RNases) of two groups of foregut-fermenting mammals: ruminant artiodactyls and colobine monkeys. The RNase1 gene was duplicated twice in ancestral ruminants at least 40 MYA, and it was also duplicated in the douc langur, an Asian colobine, approximately 4 MYA. After duplication, similar functional changes occurred in the ruminant and monkey enzymes. Interestingly, five amino acid substitutions in ruminant RNases that are known to affect its catalytic activity against double-stranded (ds) RNA did not occur in the monkey enzyme. Rather, a similar functional change in the monkey was caused by a different set of nine substitutions. Site-directed mutagenesis was used to make three of the five ruminant-specific substitutions in the monkey enzyme. Functional assays of these mutants showed that one of the three substitutions has a similar effect in monkeys, the second has a stronger effect, and the third has an opposite effect. These results suggest that (1) an evolutionary problem can have multiple solutions, (2) the same amino acid substitution may have opposite functional effects in homologous proteins, (3) the stochastic processes of mutation and drift play an important role even at functionally important sites, and (4) protein sequences may diverge even when their functions converge.     

Sequence of human eosinophil-derived neurotoxin cDNA: identity of deduced amino acid sequence with human nonsecretory ribonucleases

Several clones of human eosinophil-derived neurotoxin (EDN) cDNA have been isolated from a lambda gt10 cDNA library prepared from mRNA derived from noninduced HL-60 cells. The amino acid (aa) sequence deduced from the coding sequence of the EDN cDNA is identical to the aa sequence of urinary nonsecretory RNase. Comparison of the aa and/or nucleotide (nt) sequences of EDN and other proteins possessing ribonucleolytic activity, namely bovine seminal RNase, human and rat pancreatic RNases, eosinophil cationic protein (ECP), and human angiogenin, shows extensive identity at half-cystine residues and at aa of active sites. Differences in aa sequences at the active sites are often the result of single nt changes in the codons. The data presented here support the concept of a RNase gene superfamily containing secretory and nonsecretory RNases, angiogenin, EDN and ECP.     

Molecular evolution of mammalian ribonucleases 1

There have been many studies on the chemistry of mammalian pancreatic ribonucleases (ribonucleases 1), but the functional biology of this family of homologous proteins is still largely unknown. Many studies have been performed on the molecular evolution and properties of this enzyme from species belonging to a large number of mammalian taxa, including paralogous gene products resulting from recent gene duplications. Novel ribonuclease 1 sequences were determined for three rodent species (gundi, brush-tailed porcupine, and squirrel), rabbit, a fruit bat, elephant, and aardvark, and the new sequences were used for deriving most parsimonious networks of ribonucleases from different mammalian orders, including earlier determined nucleotide sequences and also a larger set of protein sequences. Weak support for interordinal relationships were obtained, except for an Afrotheria clade containing elephant and aardvark. Results of current analyses and also those obtained 20 years ago on amino acid sequences confirm conclusions derived recently from larger data sets of other molecules. Several examples of recent gene duplications in ribonucleases 1 are discussed, with respect to illustrate the concepts of orthology and paralogy. Previously evidence was presented for extensive parallelism between sequence regions with attached carbohydrate (about one quarter of the molecule) of unrelated species with cecal digestion (pig and guinea pig). These features are also present in the sequences of elephant and fruit bat, species with cecal digestion, but with a very low ribonuclease content in their pancreas.     

The Complete Amino Acid Sequence of Green Turtle (Chelonia mydas) Egg White Ribonuclease

Egg white ribonuclease was first found in green turtle eggs. This enzyme has been purified by CM-toyopearl cation exchange. Two isoforms (GTRNase-1 and GTRNase-2) were further separated by RP-HPLC, with the same M.W. (13 kDa) and activity. These isoforms carried one amino acid exchange of Ser and Leu at the position 37. The N-terminal sequence, ETRYEKF, was determined for the transblotted protein. Internal sequences were analyzed by protein sequencer and ESI-Q-TOF mass spectrometry for tryptic peptides (Ts). The overlapping sequences were obtained from chymotryptic peptides, CNBr fragments and ISD-MS/MS analysis. The C-terminal Ile was identified by CPase-Y. The established sequence composed of 119 residues with the molecular mass of 12,942.1 Da for GTRNase-1 and 12,967.8 Da for GTRNase-2. The comparison of sequence with known pancreatic RNases, 27 positions including catalytic residues at the position 11 and 114 were conserved. Also basic residues contributed to phosphate binding residues were conserved with the exception of Lys 66. One insertion at the position 14, and 3 deletions at the position-1, between position 64–65, and 110 and 111 were found. Two Cys residues at position 65 and 72 that form a disulfide bond in mammalian RNase were deleted and exchanged. All these difference in the sequence were similar to reptile pancreatic RNase.Data deposition: The sequence reported in this paper has been submitted to the UniProt Knowledgebase under accession No. P84844.     

Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily

In most tissues, ribonucleases (RNases) are found in a latent form complexed with ribonuclease inhibitor (RI). To examine whether these so-called cytoplasmic RNases belong to the same superfamily as pancreatic RNases, we have purified from porcine liver two such RNases (PL1 and PL3) and examined their primary structures. It was found that RNase PL1 belonged to the same family as human RNase Us [Beintema et al. (1988) Biochemistry 27, 4530-4538] and bovine RNase K2 [Irie et al. (1988) J. Biochem. (Tokyo) 104, 289-296]. RNase PL3 was found to be a hitherto structurally uncharacterized type of RNase. Its polypeptide chain of 119 amino acid residues was N-terminally blocked with pyroglutamic acid, and its sequence differed at 63 positions with that of the pancreatic enzyme. All residues important for catalysis and substrate binding have been conserved. Comparison of the primary structure of RNase PL3 with that of its bovine counterpart (RNase BL4; M. Irie, personal communication) revealed an unusual conservation for this class of enzymes; the 2 enzymes were identical at 112 positions. Moreover, comparison of the amino acid compositions of these RNases with that of a human colon carcinoma-derived RNase, RNase HT-29 [Shapiro et al. (1986) Biochemistry 25, 7255-7264], suggested that these three proteins are orthologous gene products. The structural characteristics of RNases PL1 and PL3 were typical of secreted RNases, and this observation questions the proposed cytoplasmic origin of these RI-associated enzymes.     

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.     

Purification and properties of bovine kidney ribonucleases

Two RNases (RNases K1 and K2) were purified from bovine kidney by means of column chromatography on phospho-cellulose, Sephadex G-50, CM-cellulose, heparin-Sepharose, nd agarose-APUP. They were named RNase K1 and RNase K2 in order of elution from the heparin-Sepharose column. The purity of RNase K1 thus obtained was about 90% by SDS-disc electrophoresis. RNase K2 was purified to homogeneity by SDS- and pH 4.3 disc electrophoresis. The yield of RNase K2 was 3.4 mg from 11 kg of kidneys. The antigenic properties of the two bovine renal RNases were studied by Ouchterlony's double diffusion analysis. RNase K1 and RNase A were serologically indistinguishable. RNase K2 did not cross-react immunologically with RNase K1 or RNase A. The molecular weights of these RNases determined by gel-filtration on Sephadex G-50 were 13,400 and 14,600 for RNase K1 and RNase K2, respectively. The pH optima for RNase K1 and RNase K2 were 8.5 and 6.5, respectively. Both RNase K1 and RNase K2 were as acid stable as RNase A. RNase K2 was less heat-stable than RNase K1 and RNase A. Although both renal RNases were pyrimidine nucleotide-specific enzymes, RNase K1 and RNase A were more preferential or cytidylic acid than RNase K2. The chemical composition of RNase K2 was determined. RNase K2, like human urinary RNase Us, contained one tryptophan residue. The N-terminal sequences of RNase K2 and RNase Us were determined by Edman degradation. Rnase K2 had a homologous sequence of about 10 amino acid residues with the sequence of RNase Us, a typical non-secretory RNase, within the N-terminal 30 residues.     

Multiple tandem gene duplications in a neutral lipase gene cluster in Drosophila

We have examined a highly dynamic section of the Drosophila melanogaster genome which contains neutral lipase family genes that have undergone multiple tandem duplication events. We have identified the orthologous clusters, encoding between five and eight apparently functional lipases, in other Drosophila genomes: yakuba, ananassae, pseudoobscura, virilis, mojavensis, persimilis, grimshawi and willistoni. We examined their gene structure, duplication and pseudogene formation, and the presence of transposable elements. Based on phylogenetic comparisons, the lipase genes contained in each of the clusters fall into four distinct clades. Clades I and II have distinct evolutionary constraints to clades III and IV. Multiple gene duplications have occurred in different lineages of clades I and II while clades III and IV contain a single lipase gene from each species. Compared with lipases from other clades, clade IV genes contain an additional 3' domain of tandemly repeated sequence of varying length and composition, and a substitution in the residue adjacent to the key catalytic serine in the encoded proteins. A comparison of non-synonymous to synonymous nucleotide substitution (dN/dS) rates within each clade showed the highest rate of divergence was between paralogous lipase gene pairs suggesting selection pressure on duplicated genes. Analysis of the encoded lipase protein sequences within each species using PAML identified positively selected sites; structure homology modeling based on human pancreatic lipase indicated many of these residues formed part of the active site of the enzyme. As some of the cluster lipase genes are known to be expressed in the insect midgut and respond to changes in dietary components, we propose that the lipase cluster has undergone dynamic evolutionary changes to maximize absorption of lipid nutrients from the diet.