The release of oligosaccharides from glycoproteins is performed for two main reasons; first to allow further studies on the core protein, and second to elucidate the structure of the oligosaccharide moeities present. For further studies of the protein to be carried out it is essential that the amino acid peptide bonds remain intact during the release process, whereas this is not essential for further studies on the released oligosaccharides. Here we describe strategies for the release of both N-linked and O-linked oligosaccharides, which allow further characterization of both protein and oligosaccharide. N-linked oligosaccharides can be readily released enzymatically, and rational use of glycosidases, e.g., peptide N glycanase F (PNGase F), endo-β-N-acetylglucosaminidase H (Endo H), neuraminidase and endo-α-N-acetylgalactosaminidase (O-glycosidase) will reveal useful information on the type of oligosaccharide present. O-linked chains are more difficult to release as sequential glycosidase digestions (e.g., neuraminidase and O-glycosidase) will remove some but not all types of O-linked chain. For the release of all O-linked chains for further analysis a chemical method is required, which also degrades the protein.
Once the presence of monosaccharides has been established by chemical methods (see Chapter 100), the next stage of any glycoconjugate or polysaccharide analysis is to find out the amount of sugar and monosaccharide composition. The latter can give an idea as to the type of oligosaccharides present and, hence, indicate further strategies for analysis (). Analysis by high-pH anion-exchange chromatography (HPAEC) with pulsed electrochemical detection, that is described here, is the most sensitive and easiest technique (). If the laboratory does not have a biocompatible HPLC available that will withstand high salt concentrations, a sensitive labeling technique can be used with gel electrophoresis (), e.g., that marketed by Glyko Inc. (Navato, CA) or Oxford Glycosystems (Abingdon, UK), to include release of oligosaccharides/monosaccharides and labeling via reductive amination with a fluorescence label ().
Once the presence of monosaccharides has been established by chemical methods (see Chapter 128), the next stage of any glycoconjugate or polysac-charide analysis is to find out the amount of sugar and monosaccharide composition. The latter can give an idea as to the type of oligosaccharides present and, hence, indicate further strategies for analysis (1). Analysis by high-pH anion-exchange chromatography (HPAEC) with pulsed electrochemical detection, that is described here, is the most sensitive and easiest technique (2). If the laboratory does not have a biocompatible HPLC available that will withstand high salt concentrations, a sensitive labeling technique can be used with gel electrophoresis (3), e.g., that marketed by Glyko Inc. (Navato, CA) or Oxford GlycoSciences (Abingdon, UK), to include release of oligosaccharides/mon-osaccharides and labeling via reductive amination with a fluorescence label (4).
Glycoproteins were extracted from meconium samples of group O neonates of secretor type by pronase digestion followed by precipitation in 67% aqueous ethanol and separated into Ii antigen enriched and depleted fractions by affinity chromatography. The latter fraction strongly expressed the oncofoetal antigens recognised by natural antibodies in mouse sera and the hybridoma antibody FC 10.2, and this activity was enhanced after mild acid hydrolysis to remove sialic acid and fucose residues. Oligosaccharides were released from the mild‐acid‐treated fraction by base‐borohydride degradation and purified by gel permeation chromatography on Bio‐Gel P4 and high performance liquid chromatography on octadecylsilyl and aminopropylsilyl columns. The major oligosaccharides were characterised by fast atom bombardment and electron impact mass spectrometry, combined gas‐liquid chromatography/mass spectrometry and 500‐MHz proton NMR spectroscopy. Their structures, in order of abundance, were:
The vast array of possible N-linked oligosaccharides demands high-resolution HPLC columns for their purification (1, 2). Reverse-phase (C18) and normalphase (NH2) columns have been used for the separation (singly or in concert) of many N-linked oligosaccharides. The porous graphitized carbon (PGC) column described in Chapter 134 for O-linked alditol separation will give improved N-linked oligosaccharide resolution over C18 columns, and has the advantage of using salt-free buffers for preparative work (3, 4).
ABSTRACT Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogen Porphyromonas gingivalis and are encoded by rgpA and rgpB . Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the α-catalytic chain and the β-adhesin chain, the monomeric soluble Arg-gingipain comprising only the α-catalytic chain (RgpA cat ), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the α-catalytic chain (mt-RgPA cat ), are derived from rgpA . The monomeric enzymes contain between 14 and 30% carbohydrate by weight. rgpB encodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated that rgpB is involved in the glycosylation process, since inactivation of rgpB results in the loss of not only RgpB and mt-RgpB but also mt-RgpA cat . This work aims to confirm the role of RgpB in the posttranslational modification of RgpA cat and the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivated rgpB strain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream of rgpB had no effect on rgpA -derived enzymes, suggesting that the phenotype of the rgpB mutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpA cat from W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpA cat from D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived from rgpA and that this process is required for enzyme stabilization.
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