RNase B (ribonucleaseB)

The binding properties of low- and high-adhesive forms of FimH adhesins from Salmonella enterica serovars Enteritidis and Typhimurium (S. Enteritidis and S. Typhimurium) were studied using chimeric proteins containing an additional peptide that represents an N-terminal extension of the FimF protein. This modification, by taking advantage of a donor strand exchange mechanism, closes the hydrophobic groove in the fimbrial domain of the FimH adhesin. Such self-complemented adhesins (scFimH) did not form aggregates and were more stable (resistant to proteolytic cleavage) than native FimH. High-adhesive variants of scFimH proteins, with alanine at position 61 and serine at position 118, were obtained by site-directed mutagenesis of fimH genes from low-adhesive variants of S. Enteritidis and S. Typhimurium, with glycine at position 61 and phenylalanine at position 118. Direct kinetic analysis using surface plasmon resonance (SPR) and glycoproteins carrying high-mannose carbohydrate chains (RNase B, horseradish peroxidase and mannan-BSA) revealed the existence of high- and low-adhesive allelic variants, not only in S. Typhimurium but also in S. Enteritidis. Using two additional mutants of low-adhesive FimH protein from S. Enteritidis (Gly61Ala and Phe118Ser), SPR analysis pointed to Ser118 as the major determinant of the high-adhesive phenotype of type 1 fimbriae from S. Enteritidis. These studies demonstrated for the first time that the functional differences observed with whole fimbriated bacteria could be reproduced at the level of purified adhesin. They strongly suggest that the adhesive properties of type 1 fimbriae are determined only by structural differences in the FimH proteins and are not influenced by the fimbrial shaft on which the adhesin is located.

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Ultrastructural characterization of an extracellular fibrillar sheath on cells of Ascocalyx abietina, the scleroderris canker agent of conifers

Canadian Journal of Botany ◽

10.1139/b87-022 ◽

1987 ◽

Vol 65 (1) ◽

pp. 154-167 ◽

Cited By ~ 12

Author(s):

Nicole Benhamou ◽

G. B. Ouellete

Keyword(s):

Rnase A ◽

Host Cells ◽

Biological Functions ◽

Intact Cells ◽

Physiological Conditions ◽

Physical Conditions ◽

Ultrastructural Characterization ◽

Cationic Compounds ◽

Rnase B

Morphology, ultrastructure, and some aspects of the chemical composition of a fibrillar sheath surrounding cells of the fungus Ascocalyx abietina (Lagerberg.) Schlaepfer-Bernhard were studied using electron microscopy and gold-labeled ligands. Although consistently present around all cells, the fibrillar matrix was found to vary greatly in morphology within the same isolate, depending apparently on age and (or) physiological conditions of the cells. Around cells considered younger, the sheath appeared always to be constituted of fibrillar masses that varied in size and shape but were delineated by a well-defined border. In contrast, cells expected to be older were generally bordered by a regular and uniform matrix composed of numerous intertwined fine fibrils, some being associated with small osmiophilic knobs. The presence of RNA in the denser layers of the sheath was revealed through gold complexes with either RNase A or RNase B. Continuity of portions of the sheath with similar material surrounding endocells or intact cells through gaps in the wall was frequently observed. This observation was considered as one of the possible explanations for the presence of RNA in the sheath. Association of sugars such as β-glucopyranosides and especially sialic acid with the extracellular matrix is, most probably, relevant to specific biological functions such as attachment to host cells, protection against unfavourable physical conditions and transport of cationic compounds. Peculiarities of this sheath produced by A. abietina contribute, therefore, to distinguish it from those described in other fungi.

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Novel Mutation and Structural RNA Analysis of the Noncoding RNase MRP Gene in Cartilage-Hair Hypoplasia

Molecular Syndromology ◽

10.1159/000430970 ◽

2015 ◽

Vol 6 (2) ◽

pp. 77-82 ◽

Cited By ~ 3

Author(s):

Imane Cherkaoui Jaouad ◽

Fatima Z. Laarabi ◽

Siham Chafai Elalaoui ◽

Stanislas Lyonnet ◽

Alexandra Henrion-Caude ◽

...

Keyword(s):

Novel Mutation ◽

Rna Analysis ◽

Cartilage Hair Hypoplasia ◽

Rnase B

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Production of an Endo-β-N-Acetylglucosaminidase Activity Mediates Growth ofEnterococcus faecalis on a High-Mannose-Type Glycoprotein

Journal of Bacteriology ◽

10.1128/jb.182.4.882-890.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 882-890 ◽

Cited By ~ 18

Author(s):

Gretta Roberts ◽

Edward Tarelli ◽

Karen A. Homer ◽

John Philpott-Howard ◽

David Beighton

Keyword(s):

Bacterial Growth ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Anion Exchange Chromatography ◽

Glycosylation Site ◽

Exchange Chromatography ◽

Maldi Tof ◽

Rnase B ◽

High Mannose

ABSTRACT Enterococcus faecalis is associated with a high proportion of nosocomial infections; however, little is known of the ability of this organism to proliferate in vivo. The ability of RNase B, a model glycoprotein with a single N-glycosylation site occupied by a family of high-mannose-type glycans (Man5- to Man9-GlcNAc2), to support growth of E. faecalis was investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of RNase B demonstrated a reduction in the molecular mass of this glycoprotein during bacterial growth. Further analysis by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry revealed that this mass shift was due to the degradation of all high-mannose-type glycoforms to a single N-linked N-acetylglucosamine residue. High-pH anion-exchange chromatography analysis during exponential growth demonstrated the presence of RNase B-derived glycans in the culture supernatant, indicating the presence of an endoglycosidase activity. The free glycans were eluted with the same retention times as those generated by the action of Streptomyces plicatusendo-β-N-acetylglucosaminidase H on RNase B. The cleavage specificity was confirmed by MALDI-TOF analysis of the free glycans, which showed glycan species containing only oneN-acetylglucosamine residue. No free glycans were detectable after 5 h of bacterial growth, and we have subsequently demonstrated the presence of mannosidase activity in E. faecalis, which releases free mannose from RNase B-derived glycans. We propose that this deglycosylation of glycoproteins containing high-mannose-type glycans and the subsequent degradation of the released glycans by E. faecalis may play a role in the survival and persistence of this nosocomial pathogen in vivo.

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Functional characterization of the FimH adhesin from Salmonella enterica serovar Enteritidis

Microbiology ◽

10.1099/mic.0.28588-0 ◽

2006 ◽

Vol 152 (5) ◽

pp. 1337-1346 ◽

Cited By ~ 37

Author(s):

Dagmara Kisiela ◽

Anna Laskowska ◽

Anna Sapeta ◽

Maciej Kuczkowski ◽

Alina Wieliczko ◽

...

Keyword(s):

Horseradish Peroxidase ◽

Salmonella Enterica ◽

Functional Characterization ◽

Infectious Agent ◽

Binding Specificity ◽

Human Colon ◽

Binding Properties ◽

Intact Cells ◽

Serovar Typhimurium ◽

Rnase B

Salmonella enterica serovar Enteritidis has emerged during the last 20 years as the major causative agent of food-borne gastroenteritis in humans and as the major infectious agent on poultry farms, replacing Salmonella enterica serovar Typhimurium as the dominant pathogenic serovar. Because adhesion to gut tissues and colonization of the alimentary tract, mediated in large part by the FimH adhesins located on type 1 fimbriae, is an important stage in the pathogenesis of both serovars, the binding properties of the FimH adhesins from these two enteropathogens were compared. Salmonella Enteritidis FimH protein and the Salmonella Typhimurium low-adhesive variant of this adhesin were expressed in Escherichia coli and the recombinant proteins were analysed for their ability to bind glycoproteins carrying different oligomannosidic structures and different types of eukaryotic cells. In static binding assays (ELISA and Western blotting) both FimH proteins bound equally well to all three tested glycoproteins (RNase B, horseradish peroxidase and mannan-BSA). In addition, no differences were found in the binding specificity of the FimH proteins and intact cells of Salmonella Enteritidis and Salmonella Typhimurium to human colon carcinoma or bladder cancer cells. The presence of the same amino acid residues at positions 61 (glycine) and 118 (phenylalanine) and the similar binding properties of these two adhesins suggest that the newly described FimH protein of Salmonella Enteritidis represents the low-adhesive variant found in Salmonella Typhimurium. To study the binding specificity of Salmonella Enteritidis FimH protein further, direct kinetic analysis using surface plasmon resonance was performed. With this method it was found that Salmonella Enteritidis FimH adhesin bound with the highest K d value to high-mannose type N-glycans carried by RNase B; about 100 times lower K d values were obtained in the interactions with mannan-BSA and horseradish peroxidase.

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The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

Journal of Protein Chemistry ◽

10.1007/bf00248053 ◽

1987 ◽

Vol 6 (4) ◽

Author(s):

Roger Williams ◽

Herbert Axelrod ◽

Marie Greene ◽

Alexander McPherson

Keyword(s):

Crystalline State ◽

Rnase A ◽

Rnase B

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Studying the Glycan Moiety of RNase B by Means of Raman and Raman Optical Activity

ChemPhysChem ◽

10.1002/cphc.201402029 ◽

2014 ◽

Vol 15 (11) ◽

pp. 2252-2254 ◽

Cited By ~ 11

Author(s):

Carl Mensch ◽

Robert Pendrill ◽

Göran Widmalm ◽

Christian Johannessen

Keyword(s):

Optical Activity ◽

Raman Optical Activity ◽

Rnase B

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Glycosylation States on Intact Proteins Determined by NMR Spectroscopy

Molecules ◽

10.3390/molecules26144308 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4308

Author(s):

Audra A. Hargett ◽

Aaron M. Marcella ◽

Huifeng Yu ◽

Chao Li ◽

Jared Orwenyo ◽

...

Keyword(s):

Protein Interactions ◽

Protein Glycosylation ◽

Protein Digestion ◽

Liquid Chromatography Mass Spectrometry ◽

Protein Protein Interactions ◽

Adhesion Protein ◽

Intact Proteins ◽

Rnase B ◽

Non Destructive ◽

Nmr Signals

Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.

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Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

10.1101/2021.06.04.447131 ◽

2021 ◽

Author(s):

Simon Ngao Mule ◽

Livia Rosa-Fernandes ◽

Joao V. P. Coutinho ◽

Vinicius De Morais Gomes ◽

Janaina Macedo-da-Silva ◽

...

Keyword(s):

Conformational Changes ◽

Protein Misfolding ◽

Structural Changes ◽

3D Structure ◽

Differential Rate ◽

Solvent Exposure ◽

Experimental Conditions ◽

Rnase B ◽

Wide Scale ◽

Folded Proteins

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion under denaturing conditions, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of the formerly N-glycosylated peptides. LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases.

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