Primary structure of a non-secretory ribonuclease from bovine kidney

The primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRHNCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pancreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).     

First demonstration of lactoribonuclease, a ribonuclease from bovine milk with similarity to bovine pancreatic ribonuclease

The isolation of a ribonuclease designated lactoribonuclease, with a molecular weight and an N-terminal amino acid sequence identical to those of bovine pancreatic ribonuclease, was first reported from bovine milk. After removal of globulin from acid whey by precipitation with 1.8 M (NH4)2SO4, (NH4)2SO4 was added to attain a concentration of 3.6 M. Adsorption on the ion exchanger CM-Sepharose and subsequently on Mono S by fast protein liquid chromatography yielded pure lactoribonuclease. The enzyme, like pancreatic ribonuclease, was most active at pH 7.5 with yeast transfer RNA (tRNA) as substrate. Lactoribonuclease and pancreatic ribonuclease showed a strong preference for poly(C) over poly(U). However, pancreatic ribonuclease did so with a higher specific activity, suggesting that the two ribonucleases are not identical. No inhibitory effect was shown by either lactoribonuclease or pancreatic ribonuclease toward poly (A) and poly (G). The effect of lactoribonuclease and pancreatic ribonuclease on tRNA increased with the concentration of tRNA. Lactoribonuclease inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 3.5 nM while the corresponding IC50 for pancreatic ribonuclease was 0.09 nM.     

Semisynthetic studies on bovine pancreatic ribonuclease

The S-peptide of the enzyme bovine pancreatic ribonuclease has been used as a model for covalent semisynthesis. Methods for side-chain protection, enzymatic cleavage of the peptide chain at the level of the single arginine-10 and for selective deprotection of the alpha-carboxyl function of this residue, have been examined. The partially protected [1-10] sequence has been coupled to a solid-phase generated [11-15] sequence attached to the polymer. After deblocking from the solid-support, the [1-15] semisynthetic peptide was complexed with native S-protein to give a complex with high biological activity.     

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 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.     

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.     

Primary structure of a ribonuclease from bovine brain

The primary structure of a pyrimidine base-specific ribonuclease from bovine brain was determined. The sequence determined is (sequence; see text). Although the sequence homology of this RNase with bovine pancreatic RNase A is 78.2%, it consists of 140 amino acid residues, and it is 16 amino acid residues longer than RNase A at the carboxyl-terminal. In addition to an N-glycosylated long carbohydrate chain, the bovine brain RNase has two short O-glycosylated carbohydrate chains at the 129th and the 133rd serine residues. The additional C-terminal tail of the bovine brain RNase has a unique composition: 6 proline, 5 hydrophobic amino acids, and two basic amino acids, arginine and histidine.     

Observations on the carbohydrate moiety of bovine pancreatic deoxyribonuclease A

The carbohydrate side chain of bovine pancreatic deoxyribonuclease A, which is attached to asparagine residue 18, contains two residues of N-acetylglucosamine proximal to the peptide chain followed by a variable number of mannose residues (4–10). The oligosaccharide structure bears a similarity to that in bovine pancreatic ribonuclease B. The present sequence studies have made use of α-mannosidase chromatographically purified from jack bean meal.     

Interchain disulfide bridges in ribonuclease BS-1

RNAase BS-1, a dimeric ribonuclease isolated from bovine seminal plasma, is made up of two identical subunits whose amino acid sequence is homologous to the sequence of bovine pancreatic RNAase A. The dimeric structure, resistant to denaturating agents, is sensitive to thiol reagents even in the absence of denaturants. The isolation and characterization of a cystine peptide containing two adjacent $\text{1}\text{2}\text{cystine}$ residues is reported. As the peptide molecular weight is halved after reductive cleavage with dithiothreitol, a structure based on two interchain disulfide bonds between the two adjacent $\text{1}\text{2}\text{cystine}$ of each subunit is proposed. The singularity of such a structure for a small enzymatic protein is discussed.     

A strong homology exists between the active T-cell binding gp120 octapeptide of human immunodeficiency virus and the subtilisin cleavage peptide of bovine ribonuclease A

A homology has been found between an octapeptide involved in attachment of the human immunodeficiency virus to helper/inducer T cells and an octapeptide segment of bovine pancreatic ribonuclease A. This segment (residues 19-26) contains the sites for subtilisin cleavage of this enzyme into the S-peptide and S-protein. From the X-ray crystal structure of ribonuclease, this sequence is known to be exposed to solvent and interacts little with the rest of the protein. A structure for the human immunodeficiency virus attachment peptide can be deduced from this homology, as a well-defined structure has been determined for this sequence in ribonuclease. This can be readily accomplished using previously developed computer methods based upon conformational energy calculations. The calculated structure for human immunodeficiency virus peptide is identical to the ribonuclease segment (19-26) in backbone conformation. It is stabilized by internal interactions of nonpolar residues, and by exposure of polar hydroxyl groups. The results suggest that the T-cell human immunodeficiency virus receptor may be hydrophilic in nature and that conservation of the sequence in two presumably functionally unrelated proteins is related to the need for conservation of exposed structure.     

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.     

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.     

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.     

Glycosylation of ribonuclease A catalysed by rabbit liver extracts.

Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.     

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.     

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 a hybrid between ribonuclease A and bovine seminal ribonuclease--the basic surface, at 2.0 A resolution.

A variant of bovine pancreatic ribonuclease A has been prepared with seven amino acid substitutions (Q55K, N62K, A64T, Y76K, S80R, E111G, N113K). These substitutions recreate in RNase A the basic surface found in bovine seminal RNase, a homologue of pancreatic RNase that diverged some 35 million years ago. Substitution of a portion of this basic surface (positions 55, 62, 64, 111 and 113) enhances the immunosuppressive activity of the RNase variant, activity found in native seminal RNase, while substitution of another portion (positions 76 and 80) attenuates the activity. Further, introduction of Gly at position 111 has been shown to increase the catalytic activity of RNase against double-stranded RNA. The variant and the wild-type (recombinant) protein were crystallized and their structures determined to a resolution of 2.0 A. Each of the mutated amino acids is seen in the electron density map. The main change observed in the mutant structure compared with the wild-type is the region encompassing residues 16-22, where the structure is more disordered. This loop is the region where the polypeptide chain of RNase A is cleaved by subtilisin to form RNase S, and undergoes conformational change to allow residues 1-20 of the RNase to swap between subunits in the covalent seminal RNase dimer.