Identification of cDNA encoding rat eosinophil cationic protein/eosinophil-associated ribonuclease

Nittoh, Takeaki; Hirakata, Mikito; Mue, Suetsugu; Ohuchi, Kazuo

doi:10.1016/s0167-4781(97)00024-9

Cited by 21 publications

(19 citation statements)

References 16 publications

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“…The calculated isoelectric points of all encoded proteins range from 8.4 to 9.2, except for rR-1 and rR-7, which are cationic ribonucleases with isoelectric points exceeding 10. Rat rR-7 differs from "rat ECP" identified by Nittoh et al (1997) by a single amino acid, and they are thus considered identical by this analysis (see Materials and Methods).…”

Section: Identification Of Novel Rat Ribonuclease Genesmentioning

confidence: 99%

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

et al. 1999

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Abstract. The two eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3), are among the most rapidly evolving coding sequences known among primates. The eight mouse genes identified as orthologs of EDN and ECP form a highly divergent, species-limited cluster. We present here the rat ribonuclease cluster, a group of eight distinct ribonuclease A superfamily genes that are more closely related to one another than they are to their murine counterparts. The existence of independent gene clusters suggests that numerous duplications and diversification events have occurred at these loci recently, sometime after the divergence of these two rodent species (∼10-15 million years ago). Nonsynonymous substitutions per site (d N ) calculated for the 64 mouse/rat gene pairs indicate that these ribonucleases are incorporating nonsilent mutations at accelerated rates, and comparisons of nonsynonymous to synonymous substitution (d N / d S ) suggest that diversity in the mouse ribonuclease cluster is promoted by positive (Darwinian) selection. Although the pressures promoting similar but clearly independent styles of rapid diversification among these primate and rodent genes remain uncertain, our recent findings regarding the function of human EDN suggest a role for these ribonucleases in antiviral host defense.

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Section: Identification Of Novel Rat Ribonuclease Genesmentioning

confidence: 99%

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

et al. 1999

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“…We first performed PCR amplification using the three degenerate primers based on the amino acid sequence of rat EAR-1, as described previously [26]. When PCR was carried out using certain primers, a 190-bp nucleotide fragment was strongly amplified (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…The first-strand cDNA was synthesized from 0.2 µg of poly(A)+ RNA by incubation at 37°C for 1 h in a 20 m M Tris-HCl buffer (pH 8.4) containing 50 m M KCl, 2.5 m M MgCl 2 , 5 µ M random hexamer oligonucleotides (Gibco-BRL, Gaithersburg, Md., USA), 200 units (U) of Moloney murine leukemia virus reverse transcriptase (Gibco-BRL), 0.5 µ M each of the deoxyribonucleoside 5′-triphosphates (dNTPs, Wako), 10 m M dithiothreitol and 100 µg/ml of bovine serum albumin. For the determination of partial sequences of the EAR-2 gene, the PCR primers were designed from the amino acid sequence of rat EAR-1 as described previously [26]. The sequences of the primers were sense (5′-TGYAARGGNATHAAYACNTT-3′), (5′-TTYGCNAAYGTNGTNGGNGT-3′) and antisense (5′-GGRTTNACNCCNACNGTRTA-3′) (N = G, A, T or C; R = A or G; Y = T or C; H = A, T or C).…”

Section: Methodsmentioning

confidence: 99%

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Expression and Purification of Recombinant Rat Eosinophil-Associated Ribonucleases, Homologues of Human Eosinophil Cationic Protein and Eosinophil-Derived Neurotoxin, and Their Characterization

Nakajima

Hirakata²,

Nittoh³

et al. 2001

Int Arch Allergy Immunol

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Background: Human eosinophils contain two eosinophil ribonucleases, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). In rats, 8 homologues of human ECP and EDN have been identified. To clarify the biological activity of rat eosinophil ribonucleases, we cloned rat eosinophil-associated ribonuclease (EAR)-1/rat ribonuclease 7 and rat EAR-2/rat ribonuclease 4, and produced recombinant rat pre-EAR-1 and pre-EAR-2 in a bacterial expression system. Methods: As we have already cloned the complete nucleotide sequence for rat EAR-1, we determined that for rat EAR-2 cDNA by the rapid amplification of cDNA ends procedure. Recombinant rat pre-EAR-1 and pre-EAR-2 were expressed in Escherichia coli as N-terminal 6 × histidine-tagged proteins, isolated from the insoluble fraction of the cell lysate and purified by a single-step method using an Ni-NTA resin column after solubilization with a 6 M guanidine solution. Results: The deduced amino acid sequence revealed that the molecular weight of EAR-2 containing the signal peptide is 17.3 kD and the isoelectric point is 8.59. The homology in amino acid sequence between rat pre-EAR-2, and human pre-ECP and human pre-EDN is 51 and 53%, respectively. The purified and refolded recombinant rat pre-EAR-1 and pre-EAR-2 showed bactericidal activity against E. coli and Staphylococcus aureus. Conclusions: These findings suggest that rat EAR-1 and EAR-2 act as host defense factors against bacterial infection in rats.

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“…8 Larson et al 9 isolated the first two of these B15 ribonucleases from Mus musculus, and coined the term mouse eosinophil-associated ribonucleases, or mEars. [9][10][11][12][13] While all 15 are potential ribonucleases (ie, all 15 have appropriate structural and catalytic elements), ribonuclease and antiviral activity have thus far been documented only for mEar 2.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the divergent eosinophil ribonuclease, mEar 6, and its expression in response to Schistosoma mansoni infection in vivo

et al. 2004

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The eosinophil-associated ribonucleases (Ears) are rapidly evolving proteins found in multigene clusters that are unique to each rodent species. Of the 15 independent genes in the Mus musculus cluster, only mEars 1 and 2 are expressed at significant levels at homeostasis. Here we characterize the expression of mEar 6 in the liver and spleen in mice in response to infection with the helminthic parasite, Schistosoma mansoni. Interestingly, expression of mEar 6 is not directly related to the elevated levels of serum IL-5 or tissue eosinophilia characteristic of this disease, as no mEar 6 transcripts were detected in the liver or the spleen from uninfected IL-5-transgenic mice. The coding sequence of mEar 6 has diverged under positive selection pressure (K a /K s 41.0) and has a unique unpaired cysteine near the carboxy-terminus of the protein. The high catalytic efficiency of recombinant mEar 6 (k cat /K m ¼ 0.9 Â 10 6 /M/s) is similar to that of the cluster's closest human ortholog, eosinophilderived neurotoxin (EDN/RNase 2). In summary, we have identified mEar 6 as one of only two RNase A superfamily ribonucleases known to be expressed specifically in response to pathophysiologic stress in vivo.

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Identification of cDNA encoding rat eosinophil cationic protein/eosinophil-associated ribonuclease

Cited by 21 publications

References 16 publications

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

Expression and Purification of Recombinant Rat Eosinophil-Associated Ribonucleases, Homologues of Human Eosinophil Cationic Protein and Eosinophil-Derived Neurotoxin, and Their Characterization

Characterization of the divergent eosinophil ribonuclease, mEar 6, and its expression in response to Schistosoma mansoni infection in vivo

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