Revision of the structure for an endo-beta-N-acetylglucosaminidase H substrate using a novel modification of the Smith degradation.
(Man)5(GlcNAc)2Asn was shown in a previous study (Trimnble, R. B., Tarentino, A. L., Plummer, T. H., Jr., and Maley, F. (1978) J. Biol. Chem. 253, 4508-4511) to be hydrolyzed by alpha-mannosidase to Man alpha 1 leads to 6Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 2 leads to 4GlcNAc-Asn. The latter is the most effective substrate for endo-beta-N-acetylglucosaminidase H tested to date. By employing a new and highly sensitive modification of the Smith degradation, it is shown that this compound is in reality Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc beta 1eads to 4GlcNAc-Asn. The method entails the conversion of a glycosyl asparagine derivative to its corresponding dimethylaminonaphthyl sulfonyl analogue, which after periodate oxidation is treated directly with Dowex 50-H+ to eliminate the modified carbohydrate residues. The dansylated products, which are eluted from the resin with ammonium hydroxide, can be identified rapidly by thin layer chromatography.
D000118:Acetylglucosaminidase / Q000378:metabolism*; D001216:Asparagine / Q000031:analogs & derivatives*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006020:Glycopeptides; D006596:Hexosaminidases / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008722:Methods; D009844:Oligosaccharides; D013379:Substrate Specificity
D006020:Glycopeptides; D009844:Oligosaccharides; C028361:(Man)5(GlcNAc)2Asn; D001216:Asparagine; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase