Endo β‐N‐acetylglucosaminidase F cleavage specificity with peptide free oligosaccharides

AbstractEndo β‐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 glycoproteins was determined. The commercial Endo F‐peptide N‐glycosidase/glycanyl amidase (PNGase)mixture readily leaved high mannose and complex oligosaccharides (neutral and sialyated) with common core α1–6 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 α1–6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides. However, it was similar to Endo H when reduced ovalbumin oligosaccharides were used as substrates, consistent with the recently isolated Endo F subfraction F1 being similar to Endo H [Trimble, R. B. and Tarentino, a. L. (1991). J. Biol. Chem. 266, 1646]. Results obtained in this study suggest that the complex oligosaccharides cleaving enzymes F2 and F3 show high specificity towards peptide free oligosaccharides with the core α1‐6 linked fucose, unlike the glycopeptide substrates. Therefore PNGase free Endo F1, F2 and F3 mixtures should be useful in the functional evaluation of the oligosaccharides in glycoproteins.