Endo beta-N-acetylglucosaminidase F cleavage specificity with peptide free oligosaccharides.
Endo beta-N-acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N-acetylglucosamines to the oligosaccharides with a single N-acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl-N,N'-diacetylchitobiose, N,N'-diacetylchitobiose and N-acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the endo beta-N-acetylglucosaminidase F (Endo F) activity on various free oligosaccharides obtained from the standard glycoproteins was determined. The commercial Endo F-peptide N-glycosidase/glycanyl amidase (PNGase) mixture readily cleaved high mannose and complex oligosaccharides (neutral and sialyated) with common core alpha 1-6 linked fucose found in porcine thyroglobulin including the trimannosyl-chitobiose core structure. However, the same Endo F mixture did not cleave the non-fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core alpha 1-6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)
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Macromolecular Sciences and Analytical Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005643:Fucose / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D009961:Orosomucoid / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009844:Oligosaccharides; D009961:Orosomucoid; D005643:Fucose; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008358:Mannose