Escherichia coli ribosome is inactivated by Mirabilis antiviral protein which cleaves the N-glycosidic bond at A2660 of 23 S ribosomal RNA

Habuka, Noriyuki; Miyano, Masashi; Kataoka, Jiro; Noma, Masana

doi:10.1016/0022-2836(91)80168-t

Cited by 27 publications

(30 citation statements)

References 20 publications

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“…This site of action is a highly conserved loop-and-stem structure, and the conditions necessary for its identification by ricin have been determined by Endo and co-workers [7]. This is the only known site of action of RIPs on eukaryotic ribosomes, although ricin [7] and Mirabilis antiviral protein [3] also depurinated naked 16 S Escherichia coli rRNA at another site, A-1014. We report here that saporins (RIPs from seeds, leaves and roots of Saponaria officinalis), pokeweed antiviral protein from Phytolacca americana roots (PAP-R), and trichokirin from Trichosanthes kirilowii seeds depurinate rat liver ribosomes more extensively than do other known RIPs.…”

Section: Introduci Onmentioning

confidence: 95%

Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites

Barbieri

Ferreras

Barraco

et al. 1992

Biochemical Journal

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Saporin-S6, a ribosome-inactivating protein (RIP) from Saponaria officinalis released more than 1 mol of adenine/mol of ribosomes from house fly (Musca domestica) larvae and from rat liver. The release of adenine from rat liver ribosomes by several RIPs (plant enzymes with RNA N-glycosidase activity) was examined. Saporins, pokeweed antiviral protein from roots of Phytolacca americana (PAP-R), and trichokirin from Trichosanthes kirilowii seeds depurinated rat liver ribosomes at more than one site. Up to 33 mol of adenine were released from 1 mol of ribosomes. This property is not common to all RIPS. INTRODUCI ONRibosome-inactivating proteins (RIPs) from plants (reviewed in [11), of either type I (single-chain proteins) or type 2 (twochain proteins, ricin and related toxins), damage ribosomes from eukaryotes and, at higher concentrations, ribosomes from prokaryotes 12-4J. They are RNA N-glycosidases which depurinate the major ribosomal RNA at a position corresponding to adenine-4324 of rat liver 28 S rRNA [5,6]. This site of action is a highly conserved loop-and-stem structure, and the conditions necessary for its identification by ricin have been determined by Endo and co-workers [7]. This is the only known site of action of RIPs on eukaryotic ribosomes, although ricin [7] and Mirabilis antiviral protein [3] also depurinated naked 16 S Escherichia coli rRNA at another site, A-1014. We report here that saporins (RIPs from seeds, leaves and roots of Saponaria officinalis), pokeweed antiviral protein from Phytolacca americana roots (PAP-R), and trichokirin from Trichosanthes kirilowii seeds depurinate rat liver ribosomes more extensively than do other known RIPs.

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Section: Introduci Onmentioning

confidence: 95%

Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites

Barbieri

Ferreras

Barraco

et al. 1992

Biochemical Journal

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“…The NH2-terminal sequence was determined using a 447A protein sequencer (Applied Biosystems Incorporated) using automatic Edman degradation. The inhibitory activity of the protein synthesis was measured using E. coli and rabbit reticulocyte systems as described by Habuka et al (1991). Quantification of total Y72F was determined by enzyme-linked immunosorbent assay using the antisera against wild-type MAP.…”

Section: Methodsmentioning

confidence: 99%

“…The specific N-glycosidase © 1994 International Union of Crystallography Printed in Great Britain -all rights reserved activity of MAP proteins inactivates the ribosomes (Habuka, Miyano, Kataoka & Noma, 1991). This class of ribosome-inactivating proteins (RIPs) are found in a variety of plants (Barbieri & Stirpe, 1982).…”

Section: Introductionmentioning

confidence: 99%

Improved crystals of the toxic protein MAP by protein engineering towards the host specificity

Ago¹,

Habuka²,

Kataoka³

et al. 1994

Acta Cryst D

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Mirabilis anti-viral protein (MAP) is a ribosomeinactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F which exhibits a lower (1/100) activity against E. coli ribosomes while retaining almost full activity against mammalian cells . J. Biol. Chem. 267,[7758][7759][7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F MAP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the native MAP crystals . J. Mol. Biol. 226,[281][282][283]: 50% ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3~21 (or P3221) with a = b = 104.1 and c = 134.3 A. The crystals are isomorphous with the wild-type crystals but diffract to higher resolution. Imaging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.

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“…We confirmed that under these conditions hydrolysis of the N-glycosidic bond in the ribosomes was 100%, as has been reported. 16 ) The RNA extracted from the ribosomes was treated with acid/aniline to cleave the phosphoribose on the 3'-side of the modified nucleotide. 17) The sample was diluted systematically with the RNA of S. Y OSHINARI et al In A, acid/aniline-treated rRNA prepared from MAP-treated rat liver ribosomes was mixed with the rRNA from untreated flbosomes at the indicated molar ratios and separated on the gel.…”

Section: Densitometric Determination Of the Extent Of Depurination Ofmentioning

confidence: 99%

Gypsophilin, a New Type 1 Ribosome-inactivating Protein fromGypsophila elegans: Purification, Enzymatic Characterization, and Subcellular Localization

Yoshinari

Koresawa

Yokota

et al. 1997

Bioscience, Biotechnology, and Biochemistry

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Gypsophila elegans contains a new type 1 ribosome-inactivating protein, which we named gypsophilin.The protein was purified to apparent homogeneity by (NH 4 }z80 4 fractionatioJr), ion-exchange chromatography, and adsorption chromatography. The protein was found to have a moleeular mass of 28.0 kDa and a pI of about 10.1. It does not contain glycosidic linkages. The sequence of the N-terminal 22 amino acids of the protein shows a close relationship to other RIPs. The enzyme strongly inhibits protein synthesis in a rabbit reticulocyte lysate and depurinates 288 rRNA in rat liver ribosomes in a manner identical to that of ricin A-chain and other RIPs. Using a direct method for the measurement of the RNA N-glycosidas~ activity, the substrate spcificity of gypsophilin was identified. EC so of the protein for ribosomes of rat liver l wheat germ, and E. coli was 39.8 PM, 0.24 nM, and 0.82 pM, respectively. Gypsophilin may be one of th~' most active RNA jV-glycosidases among the RIPs known to date. Immunoelectron microscopic localization of gypsophilin in the leaves shows that the protein is accumulated densely in the intercellular spaces and is: also distributed within vacuoles in the cytoplasm.Key words: ribosome-inactivating protein; RNA N-glycosidase; characterization; subcellular localization; Gypsophila elegansRibosome-inactivating proteins [RIPs, Ee 3.2.2.22, see ref. 1 for a review] constitute a diverse family of proteins that inhibit polypeptide chain elongation by inactivating ribosomes. They are present in many higher plants but their biological role is unknown. The molecular basis of the inhibition of protein synthesis is the hydrolysis of the N-glycosidic bond betwe(~n the base and the ribose at position A 4324 in 28S rRNA of rat.2) The cleavage site is embedded in a purine-rich single-stranded segment of 14 nucleotides that is nearly universal. RIPs are classified into two groups: type 1, single-chain polypeptides; and type 2, two-chain proteins consisting of an A chain with an enzymatic activity equivalent to the type 1 RIPs and a B chain with lectin properties. In spite of their apparently identical enzymatic activity, RIPs from different plants seem to have different potencies in their activities on animal, plant, or bacterial ribosomes. 1) There are three methods to assay the N-glycosidase activity of RIPs: 1) measurement of the inhibition of protein synthesis in vitro, 2) chromatographic measurement of adenine released from ribosomes, and 3) acid/aniline induced chain scission at the apurinic site in rRNA and gel electrophoretic identification of the 3' fragment. For measurement of enzyme activity the first method has been used widely because of its simplicity. There is, however, a limitation to this indirect method: it is not known whether Rl[Ps act on functioning and nonfunctioning ribosomes with equal efficiency, and the population of ribosomes actively engaged in protein synthesis is usually unknown in a given cell-free system. This method therefore does not reflect accurately the extent of the d...

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Escherichia coli ribosome is inactivated by Mirabilis antiviral protein which cleaves the N-glycosidic bond at A2660 of 23 S ribosomal RNA

Cited by 27 publications

References 20 publications

Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites

Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites

Improved crystals of the toxic protein MAP by protein engineering towards the host specificity

Gypsophilin, a New Type 1 Ribosome-inactivating Protein fromGypsophila elegans: Purification, Enzymatic Characterization, and Subcellular Localization

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