The ability of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N‐glycopeptides and N‐glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase‐C glycoprotein, which contain xylose linked β1 → 2 to β‐mannose and fucose linked α1 → 3 to the innermost N‐acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100‐fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of α1 → 3‐linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that α1 → 3 fucosylation of the inner N‐acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.

The N-linked carbohydrate chains of phospholipase A, from honeybee (Apis mellifera) were released from glycopeptides with peptide-N-glycanase A and reductively aminated with 2-aminopyridine. The fluorescent derivatives were separated by size-fractionation and reverse-phase HPLC, yielding 14 fractions. Structural analysis was accomplished by compositional and methylation analyses, by comparison of the HPLC elution patterns with reference oligosaccharides, by stepwise exoglycosidase digestions which were monitored by HPLC, and, where necessary, by 500-MHz 'H-NMR spectroscopy.Ten oligosaccharides consisted of mannose, N-acetylglucosamine and fucose al-6 and/or al-3 linked to the innermost N-acetylglucosamine. Four compounds, which comprised 10% of the oligosaccharide pool from phospholipase A2, contained a rarely found terminal element with Nacetylgalactosamine. The structures of the 14 N-glycans from honeybee phospholipase A2 can be arranged into the following three series :Manal -6 \ / /

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The reactivity of sera from honeybee venom allergic patients with the N-glycan of phospholipase A₂ was investigated using neoglycoproteins with an enzyme-linked immunosorbent assay. Of 122 sera with appreciable levels of IgE antibodies directed against bee venom as measured by radioallergosorbent test, 34 sera exhibited significant amounts of glycan-reactive IgE. These sera cross-reacted with the N-glycan from the plant glycoprotein bromelain. The interaction of IgE with the N-glycan from phospholipase could be inhibited with glycopeptides from bromelain which shares the α1,3-fucosylation of the asparagine-bound N-acetylglucosamine with bee venom phospholipase. Since defucosylated bromelain glycopeptides or glycopeptides containing a Man₃GlcNAc₂ oligosaccharide were not recognized by most of these sera, we conclude that α1,3-fucosylation of the innermost N-acetylglucosamine residue of N-glycoproteins forms an IgE-reactive determinant. This structural element is frequent in glycoproteins from plants, and it occurs also in insects. It is suspected to be one of the major causes of the broad allergenic cross-reactivity among various allergens from insects and plants.