The N‐linked carbohydrate chains of phospholipase A₂ 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 ¹H‐NMR spectroscopy.Ten oligosaccharides consisted of mannose, N‐acetylglucosamine and fucose α1–6 and/or α1–3 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 N‐acetylgalactosamine. The structures of the 14 N‐glycans from honeybee phospholipase A₂ can be arranged into the following three series: image

The β-N-acetylglucosaminidase activity in the lepidopteran insect cell line Sf21 has been studied using pyridylaminated oligosaccharides and chromogenic synthetic glycosides as substrates. Ultracentrifugation experiments indicated that the insect cell β-N-acetylglucosaminidase exists in a soluble and a membrane-bound form. This latter form accounted for two-thirds of the total activity and was associated with vesicles of the same density as those containing GlcNAc-transferase I. Partial membrane association of the enzyme was observed with all substrates tested, i.e. 4-nitrophenyl β-N-acetylglucosaminide, tri-N-acetylchitotriose, and an N-linked biantennary agalactooligosaccharide. Inhibition studies indicated a single enzyme to be responsible for the hydrolysis of all these substrates. With the biantennary substrate, the β-N-acetylglucosaminidase exclusively removed β-N-acetylglucosamine from the α1,3-antenna. GlcNAcMan₅GlcNAc₂, the primary product of GlcNAc-transferase I, was not perceptibly hydrolyzed. β-N-Acetylglucosaminidases with the same branch specificity were also found in the lepidopteran cell lines Bm-N and Mb-0503. In contrast, β-N-acetylglucosaminidase activities from rat or frog (Xenopus laevis) liver and from mung bean seedlings were not membrane-bound, and they did not exhibit a strict branch specificity. An involvement of this unusual β-N-acetylglucosaminidase in the processing of asparagine-linked oligosaccharides in insects is suggested.

The levels of β1,2-N-acetylglucosaminyltransferase (GlcNAc-T) I and II activities in cultured cells from Bombyxmori (Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda (Sf-9 and Sf-21) were investigated. Apart from initial experiments with Manα-3(Manα1–6)-Manβ1-O(CH₂)₈COOH₃ and ³H-labelled UDP-GlcNAc as substrates, GlcNAc-T I activity was measured with a non-radioactive HPLC method using pyridylaminated Man₃-GlcNAc₂ and Man₅GlcNAc₂ as acceptor oligosaccharides. It was shown by reversed-phase HPLC, exoglycosidase digestion and methylation analysis that the product obtained with Man₃GlcNAc₂ contained a terminal GlcNAc residue linked β1,2 to the α1,3 arm of the acceptor. Compared to the enzyme from the human hepatoma cell line HepG2, insect cell GlcNAc-T I exhibited a much higher preference for the Man₅ substrate. The GlcNAc-T I from Mb-0503 cells had apparent K_m and V_max values for pyridylaminated Man₃- and Man₅GlcNAc₂ of 2.15 and 0.21 mM, and of 3.4 and 11.4 nmol/h/mg of cell protein, respectively. When Man₅GlcNAc₂ was used as the acceptor substrate, the levels of GlcNAc-T I activity in the four insect cell lines ranged between 7.5 and 14.7 nmol/h/mg of cell protein, and thus were comparable to that of HepG2 cells. Evidence is presented for the dependence of lepidopteran fucosyltransferase on the presence of terminal N-acetylglucosamine. GlcNAc-T II activity could be demonstrated by HPLC using GlcNAcβ1–2Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAc-pyridylamine as the acceptor in the presence of 6-acetamido-6-deoxycastanospermine as an inhibitor of β-N-acetylglucos-aminidase. However, the insect cells exhibited specific activities of GlcNAc-T II of only 0.02–0.11 nmol/h/mg of cell protein, much less than HepG2 cells.

Core α1,6-fucosylation is a conserved feature of animal N-linked oligosaccharides being present in both invertebrates and vertebrates. To prove that the enzymatic basis for this modification is also evolutionarily conserved, cDNAs encoding the catalytic regions of the predicted Caenorhabditis elegans and Drosophila melanogaster homologs of vertebrate α1,6-fucosyltransferases (E.C. 2.4.1.68) were engineered for expression in the yeast Pichia pastoris. Recombinant forms of both enzymes were found to display core fucosyltransferase activity as shown by a variety of methods. Unsubstituted nonreducing terminal GlcNAc residues appeared to be an obligatory feature of the substrate for the recombinant Caenorhabditis and Drosophila α1,6-fucosyltransferases, as well as for native Caenorhabditis and Schistosoma mansoni core α1,6-fucosyltransferases. On the other hand, these α1,6-fucosyltransferases could not act on N-glycopeptides already carrying core α1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core α1,3-fucosyltransferases were able to use core α1,6-fucosylated glycans as substrates. Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core α1,3-fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core α1,3-fucosyltransferase. Overall, we conclude that the strict order of fucosylation events, previously determined for fucosyltransferases in crude extracts from insect cell lines (core α1,6 before core α1,3), also applies for recombinant Drosophila core α1,3- and α1,6-fucosyltransferases as well as for core fucosyltransferases in schistosomal egg extracts.