The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories

The RNase A superfamily has been important in biochemical, structural, and evolutionary studies and is believed to be the sole vertebrate-specific enzyme family. To understand the origin and diversification of the superfamily, we here determine its entire repertoire in the sequenced genomes of human, mouse, rat, and chicken. We report a previously unnoticed gene cluster in mouse chromosome 10 and a number of new genes, including mammalian RNases 11-13, which are close relatives of the recently identified RNases 9 and 10. Gene expression data imply male-reproductive functions for RNases 9-13, although their sequences suggest the lack of ribonucleolytic activities. In contrast to the presence of 13-20 functional genes in mammals, chicken has only 3 RNase genes, which are evolutionarily close to mammalian RNase 5, like other nonmammalian RNases. This and other evidence suggests that the RNase A superfamily originated from an RNase 5-like gene and expanded in mammals. Together with the fact that multiple lineages of the superfamily, including RNases 2, 3, 5, and 7, have antipathogenic activities, we suggest that the superfamily started off as a host-defense mechanism in vertebrates. Consistent with this hypothesis, all members of the superfamily exhibit high rates of amino acid substitution as is commonly observed in immunity genes.     

LOC 390443 (RNase 9) on chromosome 14q11.2 is related to the RNase A superfamily and contains a unique amino-terminal preproteinlike sequence

A new member of the human RNase A superfamily is reported. Identified in the human genome assembly as LOC 390443, this locus is located 128 kb telomeric to the established RNase A gene family cluster on chromosome 14q11.2. The amino acid sequence of this locus is sufficiently similar to the eight previously identified gene family members to warrant a designation as RNase 9. RNase 9 is expressed in a wide range of human tissues. In addition, a 30-amino acid sequence lying between a 26-amino acid putative signal peptide and the last 148 amino acids that align with the other RNases A is not seen in other members of the RNase A superfamily in any species. Nucleotide and amino acid sequences of RNase 9 in 13 nonhuman primate species were determined and indicate several conserved sites but, also, an excess of nonsynonymous substitutions, about one-third of which are radical substitutions. This suggests that RNase 9, similar to several other human RNases A, has been under diversifying selection in the primates. Data from the mouse and rat genomes indicate that RNase 9 is also present in rodents, thus making it older than most of the established members of the human RNase A superfamily. Many of the human RNases A have been shown to have antimicrobial, antiviral, or antiparasitic functions involved in host-defense mechanisms. The features of RNase 9 described here suggest that it, too, may be involved in host defense and that it, along with the rest of the superfamily, may prove to have played an important role in anthropoid evolution.     

Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity

We investigate the conservation of amino acid residue sequences in 21 DNA-binding protein families and study the effects that mutations have on DNA-sequence recognition. The observations are best understood by assigning each protein family to one of three classes: (i) non-specific, where binding is independent of DNA sequence; (ii) highly specific, where binding is specific and all members of the family target the same DNA sequence; and (iii) multi-specific, where binding is also specific, but individual family members target different DNA sequences. Overall, protein residues in contact with the DNA are better conserved than the rest of the protein surface, but there is a complex underlying trend of conservation for individual residue positions. Amino acid residues that interact with the DNA backbone are well conserved across all protein families and provide a core of stabilising contacts for homologous protein-DNA complexes. In contrast, amino acid residues that interact with DNA bases have variable levels of conservation depending on the family classification. In non-specific families, base-contacting residues are well conserved and interactions are always found in the minor groove where there is little discrimination between base types. In highly specific families, base-contacting residues are highly conserved and allow member proteins to recognise the same target sequence. In multi-specific families, base-contacting residues undergo frequent mutations and enable different proteins to recognise distinct target sequences. Finally, we report that interactions with bases in the target sequence often follow (though not always) a universal code of amino acid-base recognition and the effects of amino acid mutations can be most easily understood for these interactions.     

A teratocarcinoma glycoprotein carrying a developmentally regulated carbohydrate marker is a member of the immunoglobulin gene superfamily

Using the lambda gt11 expression library of murine teratocarcinoma cells, we isolated cDNA clones encoding a core protein carrying a developmentally regulated carbohydrate marker, namely the binding site for Dolichos biflorus agglutinin. The deduced amino acid sequence of the polypeptide (molecular weight, 37,102) revealed a leader sequence, nine potential asparagine glycosylation sites, and a transmembrane region. Sequence homology to variable domain of immunoglobulin kappa chain has been detected in a domain; homologous amino acids near cysteine residues are those conserved in many members of the immunoglobulin gene superfamily. In contrast to other members of the superfamily, the core protein gene was significantly expressed in embryonal carcinoma cells, which are similar to undifferentiated cells of early embryos.     

Residues 36-42 of liver RNase PL3 contribute to its uridine-preferring substrate specificity. Cloning of the cDNA and site-directed mutagenesis studies.

Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond. Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.     

Cloning and characterization of chicken adipocyte fatty acid binding protein gene

Fatty acid binding proteins (FABPs) are members of a superfamily of lipid-binding proteins and occur intracellularly in vertebrates and invertebrates. This study was designed to clone and characterize the adipocyte fatty acid binding protein (A-FABP) gene in the chicken. PCR primers were designed according to mammalian A-FABP gene sequence to amplify partial cDNA of A-FABP gene from chicken adipose tissues, and the full length of the gene was cloned by 5'RACE and 3'RACE. Analysis of sequence showed that the cDNA of the chicken A-FABP gene was 74 and 73% homologous with porcine and human A-FABP gene, respectively. The similarity was 77, 28, and 23% at the predicted amino acid level with human A-FABP, human L-FABP, and human I-FABP, respectively. RT-PCR and Northern blot analysis indicated that the chicken A-FABP gene, similar to that of the mammal, is only expressed in fat tissues. This is the first report to identify and characterize A-FABP gene in the chicken.     

A processed chicken pseudogene (CPS1) related to the ras oncogene superfamily.

We describe the first polyA-containing processed pseudogene reported in the chicken. It includes a 0.52 kb open reading frame which could encode a 175 amino acid protein. The putative protein shows extensive homology to the ras oncogene superfamily, being most closely related to the yeast protein YP2. It is one of the two most divergent members of the ras superfamily yet described and is the most homologous of any ras-related protein to the G-protein alpha-transducin. The chicken genome contains at least one other gene highly homologous to CPS1; at least one member of the CPS1 family is active, but only early in chicken development. This pattern of expression, and the presence of mutations in regions known to activate human c-ras genes to oncogenicity, suggest that CPS1 may represent a new oncogene family.     

Isolation and oncogenic potential of a novel human src-like gene.

We have isolated cDNA molecules representing the complete coding sequence of a new human gene which is a member of the src family of oncogenes. Nucleotide sequence analysis revealed that this gene, termed slk, encoded a 537-residue protein which was 86% identical to the chicken proto-oncogene product, p60c-src, over a stretch of 191 amino acids at its carboxy terminus. In contrast, only 6% amino acid homology was observed within the amino-terminal 82 amino acid residues of these two proteins. It was possible to activate slk as a transforming gene by substituting approximately two-thirds of the slk coding sequence for an analogous region of the v-fgr onc gene present in Gardner-Rasheed feline sarcoma virus. The resulting hybrid protein molecule expressed in transformed cells demonstrated protein kinase activity with specificity for tyrosine residues.     

The RNase PD2 gene of almond (Prunus dulcis) represents an evolutionarily distinct class of S-like RNase genes

A cDNA for an S-like RNase (RNase PD2) has been isolated from a pistil cDNA library of Prunus dulcis cv. Ferragnés. The cDNA encodes an acidic protein of 226 amino acid residues with a molecular weight of 25 kDa. A potential N-glycosylation site is present at the N-terminus in RNase PD2. A signal peptide of 23 amino acid residues and a transmembrane domain are predicted. The two active-site histidines present in enzymes of the T2/S RNase superfamily were detected in RNase PD2. Its amino acid sequence shows 71.2% similarity to RNS1 of Arabidopsis and RNase T2 of chickpea, respectively. Northern blotting and RT-PCR analyses indicate that PD2 is expressed predominantly in petals, pistils of open flowers and leaves of the almond tree. Analyses of shoots cultured in vitro suggested that the expression of RNase PD2 is associated with phosphate starvation. Southern analysis detected two sequences related to RNase PD2 in the P. dulcis genome. RFLP analysis showed that S-like RNase genes are polymorphic in different almond cultivars. The PD2 gene sequence was amplified by PCR and two introns were shown to interrupt the coding region. Based on sequence analysis, we have defined three classes of S-like RNase genes, with the PD2 RNase gene representing a distinct class. The significance of the structural divergence of S-like RNase genes is further discussed. Received: 24 January 2000 / Accepted: 17 March 2000     

A novel type of RNase III family proteins in eukaryotes

The RNase III family of double-stranded RNA-specific endonucleases is characterized by the presence of a highly conserved 9 amino acid stretch in their catalytic center known as the RNase III signature motif. We isolated the drosha gene, a new member of this family in Drosophila melanogaster. Characterization of this gene revealed the presence of two RNase III signature motifs in its sequence that may indicate that it is capable of forming an active catalytic center as a monomer. The drosha protein also contains an 825 amino acid N-terminus with an unknown function. A search for the known homologues of the drosha protein revealed that it has a similarity to two adjacent annotated genes identified during C. elegans genome sequencing. Analysis of the genomic region of these genes by the Fgenesh program and sequencing of the EST cDNA clone derived from it revealed that this region encodes only one gene. This newly identified gene in nematode genome shares a high similarity to Drosophila drosha throughout its entire protein sequence. A potential drosha homologue is also found among the deposited human cDNA sequences. A comparison of these drosha proteins to other members of the RNase III family indicates that they form a new group of proteins within this family.     

Primary structure of chicken pituitary prolactin deduced from the cDNA sequence. Conserved and specific amino acid residues in the domains of the prolactins

The perform of chicken prolactin (PRL) deduced from the cDNA sequence contains a signal peptide of 30 amino acid residues followed by a mature PRL of 199 residues. Chicken PRL shows 77, 68, 67, 58, and 31% identity of amino acid sequence with whale, human, ovine, rat, and salmon PRLs, respectively. Elucidation of the primary structure of avian PRL enabled extended analysis of the specific and conserved amino acid residues and domains of the PRL molecules. The mammalian, teleostean, and avian PRLs share 32 common residues, and these conserved residues are observed to cluster in four distinct domains (PD1 to PD4), corresponding to four of five conserved domains of the growth hormones. Of the 32 residues, 8 residues in the PD2 and PD4 domains, including 4 cysteines, are conserved by other members of the growth hormone family, which indicates that these 8 residues may be essential for common structural features of the gene family. On the other hand, 13 other residues distributed among all four domains are conserved almost exclusively in the PRLs, suggesting that these residues are indispensable for specific binding of the PRLs to their receptors.     

The lipase gene family

Development of the lipase gene family spans the change in science that witnessed the birth of contemporary techniques of molecular biology. Amino acid sequencing of enzymes gave way to cDNA cloning and gene organization, augmented by in vitro expression systems and crystallization. This review traces the origins and highlights the functional significance of the lipase gene family, overlaid on the background of this technical revolution. The gene family initially consisted of three mammalian lipases [pancreatic lipase (PL), lipoprotein lipase, and hepatic lipase] based on amino acid sequence similarity and gene organization. Family size increased when several proteins were subsequently added based on amino acid homology, including PL-related proteins 1 and 2, phosphatidylserine phospholipase A1, and endothelial lipase. The physiological function of each of the members is discussed as well as the region responsible for lipase properties such as enzymatic activity, substrate binding, heparin binding, and cofactor interaction. Crystallization of several lipase gene family members established that the family belongs to a superfamily of enzymes, which includes esterases and thioesterases. This superfamily is related by tertiary structure, rather than amino acid sequence, and represents one of the most populous families found in nature.     

Molecular cloning of a novel myeloid granule protein

Granulocytes are recognized by the presence of granules, including primary (azurophilic) and secondary types. Each granule ype contains distinct and characteristic families of enzymes. We have secreened a murine bone marrow cDNA library to obtain a series of sequences corresponding to mRNAs which are both myeloid-specific and appear to be expressed only in immature bone marrow cells. A 1, 160 bp sequence (B9) has been isolated which shows restricted expression in murine bone marrow, with the highest levels in cultures enriched for promyelocytes. Translation yields a single open reading frame of 167 amino acids and a calculated MW of 19.33 kd. A single potential N-glycosylation site is present. Evaluatin of the amino terminal sequence shows 2 polar amino acids flanking a hydrophobic region, suggesting a signal sequence and possibility of post-translational modification. An extensive search of the protein data base reveals 30% identity over 90 amino acids with porcine cathelin, a cystain-like systeine proteinase inhibitor. This sequence identity includes conservation of the 4 cysteine residues noted in all members of the cystain superfamily. In an attempt to further characterize this novel sequence, a polyclonal antiserum was prepared by immunization with a 20 amino acid synthetic peptide corresponding to a unique portion of the carboxy terminus. Immunoelectron microscopy localized B9 to neutrophilic granules. We have identified a novel myeloid-specific granule protein related to porcine cathelin, but showing important structural differences. This may represent this first isolated member of a new cystatin family. More importantly, the small size of the B9 gene and its tight pattern of early expression make B9 an excellent reporter molecule for the study of new factors important in myeloid differentiation. © 1995 Wiley-Liss, Inc.     

Identification of a new member of the dye-decolorizing peroxidase family from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Dye-decolorizing peroxidases (DyP) are atypical peroxidases showing no homology to other fungal peroxidases and lacking the typical heme binding region conserved among plant peroxidase superfamily. The gene and the corresponding cDNA encoding DyP from Pleurotus ostreatus have been identified on the basis of sequence homology analyses. The deduced amino acid sequence shares 43% identity with DyP from the ascomycete Thanatephorus cucumeris Dec 1. Analyses of the protein sequence by homology searches pointed out some properties of the DyP-type peroxidase family, which includes members from bacteria, ascomycete, and basidiomycete fungi. Some amino acids (C374, H379, and Y501 in the P. ostreatus DyP sequence) are proposed as candidates for the heme ligand, providing a basis for further investigations on the structure of the DyP type peroxidase family members.     

Characterization, primary structure, and evolution of lamprey plasma albumin.

The most abundant protein found in blood plasma from the sea lamprey (Petromyzon marinus) has the hallmarks of a plasma albumin: namely, high abundance, solubility in distilled water, a small number of tryptophans, and a high content of cysteines and charged residues. As in other vertebrate albumins, not all the cysteines are disulfide bonded. An unusual feature of this protein is its molecular weight of 175,000, roughly 2.5 times the size of other vertebrate albumins. Its amino acid sequence, deduced from a series of overlapping cDNA clones, can be aligned with other members of the gene family including plasma albumin, alpha-fetoprotein, and vitamin-D binding protein, confirming that it is indeed an oversized albumin. An unusual feature of the sequence is a 28-amino acid stretch consisting of a serine-threonine repeat with the general motif (STTT). Lamprey albumin contains a 23-amino acid putative signal peptide and a 6-residue putative propeptide, which, when cleaved, yield a mature protein of 1,394 amino acids with a calculated molecular weight of 157,000. The sequence also includes nine potential N-linked glycosylation sites (Asn-X-Ser/Thr), consistent with observation that lamprey albumin is a glycoprotein. If all the potential glycosylation sites were occupied by clusters of 2,000 molecular weight each, the total molecular weight would be 175,000. Like other members of the gene family, lamprey albumin is composed of a series of 190-amino acid repeats, there being seven such domains all together. Quantitative amino acid sequence comparisons of lamprey albumin with the other members of the gene family indicate that it diverged from an ancestral albumin prior to the gene duplications leading to this diverse group. This notion is confirmed by the pattern of amino acid insertions and deletions observed in a consideration of all domains that compose this family. Furthermore, it suggests that the invention of albumin antedates the vertebrate radiation.     

On a salmon (Oncorhynchus [corrected] keta) liver RNase, belonging to RNase T2 family: primary structure and some properties

A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS-PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes.     

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.     

Organic anion transporting polypeptides of the OATP/SLCO superfamily: identification of new members in nonmammalian species, comparative modeling and a potential transport mode

Organic anion-transporting polypeptides (human, OATPs; other animals, Oatps; gene symbol, SLCO/Slco) form a transport protein superfamily that mediates the translocation of amphipathic substrates across the plasma membrane of animal cells. So far, OATPs/Oatps have been identified in human, rat and mouse tissues. In this study, we used bioinformatic tools to detect new members of the OATP/SLCO superfamily in nonmammalian species and to build models for the three-dimensional structure of OATPs/Oatps. New OATP/SLCO superfamily members, some of which form distinct novel families, were identified in chicken, zebrafish, frog, fruit fly and worm species. The lack of OATP/SLCO superfamily members in plants, yeast and bacteria suggests the emergence of an ancient Oatp protein in an early ancestor of the animal kingdom. Structural models were generated for the representative members OATP1B3 and OATP2B1 based on the known structures of the major facilitator superfamily of transport proteins. A model was also built for the large extracellular region between transmembrane helices 9 and 10, following the identification of a novel homology with the Kazal-type serine protease inhibitors. Along with the electrostatic potential and the conservation of key amino acid residues, we propose a common transport mechanism for all OATPs/Oatps, whereby substrates are translocated through a central, positively charged pore in a rocker-switch type of mechanism. Several amino acid residues were identified that may play crucial roles in the proposed transport mechanism.     

Base Specificity and Primary Structure of Poly U-preferential Ribonuclease from Chicken Liver

The primary structure and base specificity of chicken liver RNase CL₁ which has been reported by Miura et al. [Chem. Pharm. Bull., 32,4053–4060 (1984)] as poly U-preferential RNase, were extensively studied. The sequence study of this enzyme and comparison of the amino acid sequence of the enzyme with homologous RNases from oyster and Drosophila melanogaster suggested that RNase CL₁ consists of three peptides with 17, 19, and 163 amino acid residues. The amino acid sequence of these three peptides were identified. The two small peptides are joined to the large peptide by disulfide bridges. The amino acid sequence of RNase CL₁ had 62 (31.2%) and 63 residues (31.6%) identical with oyster RNase and D. melanogaster RNase, respectively, and belongs to the RNase T₂ family RNase.

Reassessment of the base specificity of RNase CL₁ found that it is guanylic acid, then uridylic acid-preferential, and not poly U preferential.