Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have traced the rapid molecular evolution of eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), two host defense proteins that are members of the mammalian ribonuclease gene family. The EDN/ECP gene pair arose from a recent duplication event that occurred after the divergence of New World and Old World monkeys. Since duplication, the genes encoding EDN and ECP have accumulated non-silent mutations at rates exceeding those of all other functional coding sequences studied in primates, while retaining both the structural and catalytic components required for ribonuclease activity. These results suggest that both EDN and ECP may be responding to unusual evolutionary constraints, which has prompted a reexamination of their physiologic function.
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PMID:Rapid evolution of a unique family of primate ribonuclease genes. 766 19

Human eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are members of a unique subfamily of rapidly evolving primate ribonuclease genes that emerged via a gene duplication event occurring after the divergence of Old World from New World monkeys (Rosenberg, H. F., Dyer, K. D., Tiffany, H. L., and Gonzalez, M. (1995) Nature Genet. 10, 219-223). In this work, we studied the activity of the protein encoded by the EDN/ECP homolog of the New World monkey, Saguinus oedipus (marmoset), a representative of the "ancestral" single sequences. Although the nucleotide sequence of the single marmoset gene (mEDN) was equally homologous (82%) to both human genes, the encoded amino acid sequence, calculated isoelectric point, and immunoreactivity all suggested a closer relationship with EDN. Furthermore, mEDN (at 0.3-1.0 microM concentrations) had no measurable anti-staphylococcal activity, suggesting functional as well as structural similarity to EDN. However, with yeast tRNA as substrate, mEDN had significantly less ribonuclease activity than EDN; Michaelis constants were nearly identical (Km (mEDN) = 0.67 microM; Km (EDN) = 0.70 microM), while turnover numbers differed by a factor of 100 (kcat (mEDN) = 0.91 s-1; kcat (EDN) = 0.64 x 10(-2) s-1). Thus, evolutionary constraints appear to have promoted two novel functions: increased cationicity/toxicity (ECP) and enhanced ribonuclease activity (EDN). The latter result is particularly intriguing, as it suggests a crucial role for ribonuclease activity in the (as yet to be determined) physiologic function of EDN.
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PMID:Eosinophil cationic protein and eosinophil-derived neurotoxin. Evolution of novel function in a primate ribonuclease gene family. 853 Apr 35

The biochemistry, the molecular biology and the biological activity of the eosinophil granule proteins, major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPO) are reviewed. MBP is present in the core of the eosinophil granule and is toxic to parasite and host cells. ECP and EDN are proteins in the matrix of the granule and share sequence similarity and ribonuclease activity. These two proteins can provoke the Gordon phenomenon in rabbits and are toxic to parasites. EPO consists of two polypeptide and is a toxin for parasite and host cells with or without H2O2. The common characteristics of these proteins are their high isoelectric points and cytotoxic activities.
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PMID:[Biochemistry and biological activities of eosinophil granule proteins]. 768 96

Eosinophil cationic protein (ECP) is a toxin secreted by activated human eosinophils that has anti-parasitic, antibacterial, and neurotoxic activities; ECP also has ribonuclease activity and structural homology to other mammalian ribonucleases. To determine the relationship between the ribonuclease activity and cytotoxicity of ECP, a method for producing recombinant ECP (rECP) in a prokaryotic expression system was devised. Periplasmic isolates from induced bacterial transfectants contained enzymatically active rECP; micromolar concentrations of rECP were shown to be toxic for Staphylococcus aureus (strain 502A). In contrast, recombinant eosinophil-derived neurotoxin, with 67% amino acid sequence identity to ECP, had little to no toxicity for S. aureus; these findings are analogous to those obtained with purified, granule-derived ECP and eosinophil-derived neurotoxin. Two single base pair mutations were introduced into the coding sequence of rECP (Lys38 to Arg and His128 to Asp) to convert ribonuclease active-site residues into non-functional counterparts. These mutations eliminated the ribonuclease activity of rECP but had no discernible effect on the antibacterial activity of this protein, demonstrating that ribonuclease activity and cytotoxicity are, in this case, independent functions of ECP.
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PMID:Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity. 771 81

The gene for the human recombinant eosinophil-derived neurotoxin (rEDN) was synthesized and fused to the gene encoding a single chain antibody (sFv) to the human transferrin receptor (EDNsFv). Both rEDN and EDNsFv were expressed as insoluble proteins in inclusion bodies in Escherichia coli BL21(DE3). Following denaturation and renaturation, EDN and EDNsFv were partially purified by chromatography on heparin-Sepharose. Final purification of EDN was achieved by Sephadex G-100, whereas EDNsFv which contained a 6-histidyl residue carboxyl terminus was highly purified using the metal chelate resin, Ni(2+)-nitriloacetic acid. Whereas the recombinant EDN had ribonuclease activity that was similar to the native protein, the fusion protein had enzymatic activity that was 6-13% that of native EDN. The fusion protein was able to bind to the human transferrin receptor. In contrast to rEDN that had no inherent cytotoxicity to human tumor cells, the EDNsFv fusion protein was cytotoxic to human leukemia cells that express the human transferrin receptor with an IC50, 0.2-1 nM. At 1.3 nM EDNsFv, no cytotoxicity was observed on cells that lack the human transferrin receptor. Free antibody to the human transferrin receptor, E6, inhibited the cytotoxicity of the EDNsFv. Human enzymes may be engineered to acquire cytotoxic properties by fusing them to antibodies. Thus, they may be candidates for the construction of immunofusion proteins that may be less immunogenic than immunotoxins containing bacterial- or plant-derived toxin moieties.
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PMID:Expression and characterization of recombinant human eosinophil-derived neurotoxin and eosinophil-derived neurotoxin-anti-transferrin receptor sFv. 792 8

Although ribonucleases fold into correct tertiary conformation in vitro guided solely by information contained in the primary amino acid sequence (Sela, M., White, F. H., and Anfinsen, C. B. (1957) Science 124, 691-693), it is not clear whether folding of these proteins proceeds unassisted in a complex intracellular environment. We describe here the specific and high affinity binding of groEL, the prokaryotic homolog of the heat shock protein 60 family of molecular chaperones, to recombinant eosinophil cationic protein and eosinophil-derived neurotoxin, two members of the human ribonuclease gene family. We have determined that groEL binds to a unique peptide sequence near the amino terminus of nascent eosinophil cationic protein that includes the first of eight cysteine residues. This binding site functions independently and can confer groEL binding activity on an unrelated carrier protein. GroEL dissociates from the binding site upon addition of ATP and Mg2+; no other cations or cofactors are necessary. These findings suggest the possibility that interaction with a groEL-like molecular chaperone may be a requirement for correct folding and/or translocation of eukaryotic ribonucleases in vivo.
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PMID:Characterization of a distinct binding site for the prokaryotic chaperone, GroEL, on a human granulocyte ribonuclease. 809 49

Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Toxicity of an antitumor ribonuclease to Purkinje neurons. 830 53

Urinary gonadotrophin peptide (UGP) was originally identified by immunoassay in the urine of patients with various types of cancer and by immunohistochemistry in human cancers of various histological types. Extracts of normal adult male urine also contained UGP by immunoassay. Purified UGP from different starting material was subjected to high pressure liquid chromatography (HPLC) prior to defining amino acid sequences. Chromatographed UGP after HPLC showed three distinct fractions. The N-terminal sequence of peptide 2 was completely homologous with the beta-core fragment of human chorionic gonadotrophin (hCG) and this was found associated with two smaller peptides. The N-terminal sequence of peptide 1 has not been described previously whilst the N-terminus of peptide 3 that was sequenced showed complete homology with the N-terminal sequence of eosinophil derived neurotoxin and non-secretory ribonuclease. The monoclonal antibodies 2C2 and 6D3 only bind beta core-fragment (peptide 2) whilst the polyclonal (rabbit) antibody AK12 could bind all three peptides. The radioimmunoassay system using AK12 could be inhibited by all three peptides and the immunoradiometric assay although based on a capture antibody (2C2) that only bound peptide 2, had the potential to measure all three peptides (when bound together as UGP) at the second step when 125I-AK12 was introduced as the detector. A specific radioimmunoassay for peptide 3 was generated using 125I-peptide 3 and the AK12 antibody. Beta core-fragment on iso-electric focusing was found to have a pI > 9.5, peptide 3 showed two bands at pI = 3.5 and 3.8 whilst insufficient purified peptide 1 was available to determine its iso-electric point. Bioassay studies on UGP showed that any biological activity could be attributed to trace contamination with hCG.
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PMID:Characterisation of UGP and its relationship with beta-core fragment. 847 26

An antibody that recognizes human nonpancreatic-type ribonuclease was obtained by immunizing a rabbit with a 14-residue synthetic peptide corresponding to the N-terminal sequence of eosinophil-derived neurotoxin which is identical to human liver ribonuclease. This amino acid sequence is unique to this protein. The anti N-peptide antibody was purified by protein A-Sepharose and by using ELISA and SDS-PAGE immunoblot techniques, the antibody reactivity against EDN and partially purified nonpancreatic-type ribonucleases from human plasma and urine was observed. Cross-reactivity with bovine pancreatic ribonuclease A and other proteins was not detected. In addition, the activity of the nonpancreatic-type ribonuclease was not affected by the antibody. The immune response was elicited without the need for a carrier protein showing that the N-terminal sequence of nonpancreatic ribonuclease contains a specific epitope. This antibody can be used for the immunological identification of both the native and denatured forms of this type of enzyme.
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PMID:Identification of human nonpancreatic-type ribonuclease by antibodies obtained against a synthetic peptide. 856 14

The eosinophil-derived neurotoxin (EDN/RNS2) is a member of the mammalian ribonuclease gene family and is one of four proteins found in the large specific granules of human eosinophilic leukocytes. The gene encoding EDN consists of two exons, including a noncoding exon 1, separated by a single intron from the coding sequence in exon 2. We have identified a functional promoter of the EDN gene and shown that optimal expression depends on interaction between the promoter and one or more sequence elements found in the single intron. Cells of the clone 15 eosinophilic variant of the human promyelocytic HL-60 cell line were transfected with constructs that included the promoter region of the EDN gene alone, promoter with exon 1, and promoter with both exon 1 and the intron positioned 5' to the chloramphenicol acetyltransferase (CAT) reporter gene (constructs referred to as PrCAT, PrExCAT, and PrExIn CAT, respectively). Although reporter gene activity from either PrCAT or PrExCAT was only 2-3 fold higher than baseline (CAT alone), inclusion of the single intron (PrExInCAT) resulted in a 28-fold increase in reporter gene activity in uninduced clone 15 cells, and an 80-fold in activity when clone 15 cells were induced to differentiate toward eosinophils with butyric acid. The intron-mediated enhancer activity was reproduced in other human hematopoietic cell lines (K562, Jurkat, U937, and HL-60), but was not found in human 293 kidney cells, suggesting that the function of the enhancer element(s) may be tissue-specific. A significant portion of the observed enhancer activity resides in the first 60 base pairs the the intron, which includes consensus binding sites for both AP-1, and NF-ATp transcription factors, and a 15-base pair segment that is identical to a sequence found in the promoter of the gene encoding the neutrophil granule protein, lactoferrin. The noncoding exon 1/single intron/coding exon 2 genomic structure is a common feature among the mammalian ribonucleases; this finding suggests the possibility of a conserved mechanism of regulation in this gene family.
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PMID:Enhanced expression of the eosinophil-derived neurotoxin ribonuclease (RNS2) gene requires interaction between the promoter and intron. 864 42


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