scholarly journals Pseudomonas aeruginosa 1244 Pilin Glycosylation: Glycan Substrate Recognition

2006 ◽  
Vol 188 (12) ◽  
pp. 4244-4252 ◽  
Author(s):  
Joseph Horzempa ◽  
Charles R. Dean ◽  
Joanna B. Goldberg ◽  
Peter Castric
Keyword(s):  
Mass Spectrometry ◽  
Pseudomonas Aeruginosa ◽  
Structural Analysis ◽  
Western Blotting ◽  
Biosynthetic Pathway ◽  
Substrate Recognition ◽  
O Antigen ◽  
Recognition Elements ◽  
Glycosylation Reaction

ABSTRACT The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics and because the sugars at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (β-N-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the wbjE gene produced an incomplete O subunit, consisting only of the monosaccharide at the reducing end (β-d-N-acetyl fucosamine), indicating that this moiety contained substrate recognition elements for WaaL. Expression of pilAO 1244 in PA103 wbjE::aacC1, followed by Western blotting of extracts of these cells, indicated that pilin produced has been modified by the addition of material consistent with a single N-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. These data suggest that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O repeat and that structures of the remaining sugars are irrelevant.

scholarly journals Structural analysis of the GPI glycan

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257435
Author(s):  
Miyako Nakano ◽  
Susana Sabido-Bozo ◽  
Kouta Okazaki ◽  
Auxiliadora Aguilera-Romero ◽  
Sofia Rodriguez-Gallardo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Structural Analysis ◽  
Cell Surface ◽  
Membrane Fraction ◽  
Biosynthetic Pathway ◽  
Cellular Membrane ◽  
Trypsin Digestion ◽  
Post Translational Modification ◽  
Structural Remodeling

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.

scholarly journals Pseudomonas fluorescens CHA0 Produces Enantio-pyochelin, the Optical Antipode of the Pseudomonas aeruginosa Siderophore Pyochelin

2007 ◽  
Vol 282 (49) ◽  
pp. 35546-35553 ◽  
Author(s):  
Zeb A. Youard ◽  
Gaëtan L. A. Mislin ◽  
Paul A. Majcherczyk ◽  
Isabelle J. Schalk ◽  
Cornelia Reimmann
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Pseudomonas Aeruginosa ◽  
Pseudomonas Fluorescens ◽  
Biosynthetic Pathway ◽  
Cysteine Residue ◽  
Iron Limitation ◽  
Chiral Hplc ◽  
Optical Antipode ◽  
Layer Chromatography

The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4 ′R, 2 ″R, 4 ″R (pyochelin I) and 4 ′R, 2 ″S, 4 ″R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.

Structural analysis of oligomeric fructans from excised leaves of Lolium temulentum

2020 ◽  
Author(s):  
Ian Sims ◽  
CJ Pollock ◽  
R Horgan
Keyword(s):  
Mass Spectrometry ◽  
Structural Analysis ◽  
Higher Plants ◽  
Water Soluble ◽  
Lolium Temulentum ◽  
Neutral Water ◽  
Alditol Acetates ◽  
Partially Methylated

Individual fructan tri-, tetra- and pentasaccharide isomers in neutral, water-soluble extracts from Lolium temulentum were purified and the linkages present in these isomeric oligosaccharides were analysed by combined GC-mass spectrometry of partially methylated alditol acetates. 1-Kestose and neokestose were the most abundant trisaccharides with 6-kestose present in much lower amounts. Analysis of isomers of DP 4 and 5 showed that multiple linkage types were present with structures based on all three trisaccharides. Oligosaccharides based on neokestose but with 2,6 linkages between adjacent fructose residues have not been previously detected in higher plants. © 1992.

scholarly journals A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAs

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 468
Author(s):  
Anthony E. Jones ◽  
Nataly J. Arias ◽  
Aracely Acevedo ◽  
Srinivasa T. Reddy ◽  
Ajit S. Divakaruni ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Biosynthetic Pathway ◽  
Solid Phase ◽  
High Sensitivity ◽  
Short Chain ◽  
Extraction Column ◽  
Chain Acyl ◽  
Precision And Accuracy ◽  
Mass Spectrometry Mass Spectrometry

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.

scholarly journals Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

2021 ◽  
Vol 22 (6) ◽  
pp. 3166
Author(s):  
Jwala Priyadarsini Sivaccumar ◽  
Antonio Leonardi ◽  
Emanuela Iaccarino ◽  
Giusy Corvino ◽  
Luca Sanguigno ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Cancer Biomarkers ◽  
Trypsin Digestion ◽  
Protein Fragments ◽  
Target Epitope ◽  
Potential Activity ◽  
Antibody Epitopes

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

Protein surface mapping by chemical oxidation: Structural analysis by mass spectrometry

2003 ◽  
Vol 313 (2) ◽  
pp. 216-225 ◽  
Author(s):  
Joshua S. Sharp ◽  
Jeffrey M. Becker ◽  
Robert L. Hettich
Keyword(s):  
Mass Spectrometry ◽  
Structural Analysis ◽  
Chemical Oxidation ◽  
Protein Surface ◽  
Surface Mapping
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