Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allelic polymorphism in Arabian camel ribonuclease and the amino acid sequence of bactrian camel ribonuclease

Pancreatic ribonucleases from several species (whitetail deer, roe deer, guinea pig, and arabian camel) exhibit more than one amino acid at particular positions in their amino acid sequences. Since these enzymes were isolated from pooled pancreas, the origin of this heterogeneity is not clear. The pancreatic ribonucleases from 11 individual arabian camels (Camelus dromedarius) have been investigated with respect to the lysine-glutamine heterogeneity at position 103 (Welling et al., 1975). Six ribonucleases showed only one basic band and five showed two bands after polyacrylamide gel electrophoresis, suggesting a gene frequency of about 0.75 for the Lys gene and about 0.25 for the Gln gene. The amino acid sequence of bactrian camel (Camelus bactrianus) ribonuclease isolated from individual pancreatic tissue was determined and compared with that of arabian camel ribonuclease. The only difference was observed at position 103. In the ribonucleases from two unrelated bactrian camels, only glutamine was observed at that position.Part of this work has been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.).     

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.     

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     

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.     

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.     

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.     

Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease

The cDNA of mouse pancreatic mRNA has been cloned. After the library was screened with a rat ribonuclease cDNA probe, the positive clones were isolated and sequenced. There were no differences from the previously determined protein sequence. The mRNA codes for a preribonuclease of 149 amino acid residues including a signal peptide of 25 amino acids. The 3' noncoding region has a length of 260 bp, and the total mRNA length is approximately 940 bp. Comparison with the rat pancreatic ribonuclease sequence showed a high rate of nucleotide substitution. Within the coding region, nonsynonymous and synonymous substitution rates are 4.3 X 10(-9) and 15 X 10(-9) nucleotide substitutions/site/year, respectively. The latter value is one of the highest rates observed in the molecular evolution of mammalian nuclear genes. In the signal sequences the synonymous substitution rate is much lower and about the same as the nonsynonymous rate. Signal sequences of other mouse and rat proteins also exhibit little difference between synonymous and nonsynonymous rates. The sequences of rat and mouse pancreatic ribonuclease messengers were compared with those of bovine pancreatic, seminal, and brain ribonuclease. While the 3' noncoding regions of rat and mouse are very similar, as are those of the three bovine messengers, there is no significant similarity between both rodent and the three bovine messengers for the greater part of these regions. There is a duplication of approximately 50 nucleotides in the 3' noncoding region of the bovine messengers, with a region rich in A and C in between. The presence of this structural feature may be correlated with recent gene duplications that have occurred in the bovine genome.     

The primary structure of muskrat pancreatic ribonuclease.

Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32-33) and Ser-Arg (tentative position 75-76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23-25 and 99-102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31-42 positions from other mammalian pancreatic ribonucleases, while rat ribonuclease differs at 44-52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64).     

The amino-acid sequence of kangaroo pancreatic ribonuclease

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.     

Molecular cloning of four novel murine ribonuclease genes: unusual expansion within the ribonuclease A gene family.

We have characterized four novel murine ribonuclease genes that, together with the murine eosinophil-associated ribonucleases 1 and 2, form a distinct and unusual cluster within the RNase A gene superfamily. Three of these genes (mR-3, mR-4, mR-5) include complete open reading frames, encoding ribonucleases with eight cysteines and appropriately spaced histidines (His11 and His124) and lysine (Lys35) that are characteristic of this enlarging protein family; the fourth sequence encodes a non-functional pseudogene (mR-6P). Although the amino acid sequence similarities among these murine ribonucleases varies from 60 to 94%, they form a unique cluster, as each sequence is found to be more closely related to another of this group than to either murine angiogenin or to murine pancreatic ribonuclease. Interestingly, the relationship between the six genes in this 'mR cluster' and the defined lineages of the RNase A gene family could not be determined by amino acid sequence homology, suggesting the possibility that there are one or more additional ribonuclease lineages that have yet to be defined. Although the nature of the evolutionary constraints promoting this unusual expansion and diversification remain unclear, the implications with respect to function are intriguing.     

The amino-acid sequence of pancreatic ribonuclease from the mole rat Spalax ehrenbergi, chromosomal species 2n = 60

The amino-acid sequence of pancreatic ribonuclease from the chromosomal species of Spalax ehrenbergi with karyotype 2n = 60 was determined. From the comparison of the sequence with other mammalian sequences we found that Spalax diverged from the myomorph rodent branch before the divergence of the Muridae and the Cricetidae. All myomorph rodent sequences evolved faster than those of other mammals, an effect being most pronounced for the rat sequence. Spalax ribonuclease shares several amino-acid residues with other myomorph rodent species. These are not or only rarely observed outside this rodent suborder. However, there are 6 amino-acid replacements not observed earlier in pancreatic ribonucleases, and 2 other replacements and an insertion of one residue in the variable loop 15-24 are only observed in the enzyme from turtle pancreas.     

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.     

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.     

Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.     

The primary structure of langur (Presbytis entellus) pancreatic ribonuclease: adaptive features in digestive enzymes in mammals

The primary structure of pancreatic ribonuclease from langur (Presbytis entellus) has been determined. This sequence differs from that of human pancreatic ribonuclease at 14 (11%) of the amino acid positions. Eight of these 14 differences involve changes of charge, with the langur enzyme having five fewer positive charges than the human enzyme. The difference in charge between human and langur ribonuclease may be an adaptation to the different requirements for a nondigestive and a digestive role, respectively. A number of similarities in expression, gene duplications, and properties between mammalian ribonucleases and lysozymes have been observed, indicating similar adaptations in both enzyme systems.     

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.     

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