Abstract
In the preceding paper, preliminary analysis revealed a new type of O-linked oligosaccharide of 1244 Da at each of two proposed glycosylation sites on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum (Plummer, T. H., Jr., Tarentino, A. L., and Hauer, C. R. (1995) J. Biol. Chem. 270, 13192-13196). In this report we detail the linkage, sequence, and branching of this unusual heptasaccharide by electrospray (ES) ionization mass spectrometry (MS), and collision-induced dissociation (CID). The proposed structure was supported by a combination of isotopic labeling, composition and methylation analysis, and the preparation of several chemical analogs and derivatives with each product evaluated by MS and CID. The singly branched structure contained seven residues, including three different uronyl analogs: a methylated rhamnose and mannose, a glucose, and a reducing terminal mannose. Only pyranose ring forms were detected ((2-OMe)Man1-4GlcNAcU1-4GlcU1-4Glc1-4(2-OMe)G lcU-4 [(2-OMe)Rham1-2]Man).
Highlights
C;;:o to be modified with carbohydrate during secretion
Because of its unusual sugar composition, which included three different uronic acid derivatives, initial attempts at structural analysis of the Flavobacterium oligosaccharide by high resolution 3H1 NMR spectroscopy were unsuccessful in spite of the relative homogeneity (>80%) of the oligosaccharide chain
In the course of studies on the hydrolases produced by Flavobacterium meningosepticum, eight major proteins have been purified, two of which were cloned and expressed in Escherichia coli,' When compared on SDS-polyacrylamide gel electrophoresis, the native enzymes were slightly larger than their cloned counterparts and mass spectrometry
Summary
13197-13203, 1995 Printed in U.S.A. Detailed Structural Analysis of a Novel, Specific O-Linked Glycan from the Prokaryote Flavobacterium meningosepticum*. (Received for publication, December 27, 1994, and in revised form, March 23, 1995). Preliminary analysis revealed a new type ofO-linked oligosaccharide of1244 Da at each of two proposed glycosylation sites on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum The proposed structure was supported by a combination of isotopic labeling, composition and methylation analysis, and the preparation of several chemical analogs and derivatives with each product evaluated by MS and cm. The singly branched structure contained seven residues, including three different uronyl analogs: a methylated rhamnose and mannose, a glucose, and a reducing terminal mannose.
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