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We have previously reported three Caenorhabditis elegans genes (gly-12, gly-13 and gly-14) encoding UDP-N-acetyl-d-glucosamine:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid and complex N-glycan synthesis. GLY-13 was shown to be the major GnT I in worms and to be the only GnT I cloned to date which can act on [Manα1,6(Manα1,3)Manα1,6](Manα1,3)Manβ1, 4GlcNAcβ1,4GlcNAc-R, but not on Manα1,6(Manα1,3)Manβ1-O-R substrates. We now report the kinetic constants, bivalent-metal-ion requirements, and optimal pH, temperature and Mn2+ concentration for this unusual enzyme. C. elegans glycoproteins are rich in oligomannose (Man6–9GlcNAc2) and ‘paucimannose’ Man3–5GlcNAc2(±Fuc) N-glycans, but contain only small amounts of complex and hybrid N-glycans. We show that the synthesis of paucimannose Man3GlcNAc2 requires the prior actions of GnT I, α3,6-mannosidase II and a membrane-bound β-N-acetylglucosaminidase similar to an enzyme previously reported in insects. The β-N-acetylglucosaminidase removes terminal N-acetyl-d-glucosamine from the GlcNAcβ1, 2Manα1,3Manβ- arm of Manα1,6(GlcNAcβ1,2Manα1,3) Manβ1,4GlcNAcβ1,4GlcNAc-R to produce paucimannose Man3GlcNAc2 N-glycan. N-acetyl-d-glucosamine removal was inhibited by two N-acetylglucosaminidase inhibitors. Terminal GlcNAc was not released from [Manα1,6(Manα1,3)Manα1,6] (GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-R nor from the GlcNAcβ1,2Manα1,6Manβ- arm. These findings indicate that GLY-13 plays an important role in the synthesis of N-glycans by C. elegans and that therefore the worm should prove to be a suitable model for the study of the role of GnT I in nematode development.

The occurrence of N‐acetylneuraminic acid and N‐glycolylneuraminic acid residues in preparations of the slug Arion lusitanicus(Gastropoda) was determined by sodium dodecyl sulphate electrophoresis of the proteins followed by lectin blots stained with the sialic acid specific lectin from Maackia amurensis, by the sensitivity of this binding to sialidase from Clostridium perfringens, by specific fluorescent labelling of sialic acids with 1,2‐diamino‐4,5‐methylenedioxybenzene, by the determination of the sensitivity to sialate‐pyruvate‐lyase, by co‐migration with standards on high performance anion exchange chromatography with pulsed amperometric detection and by identification of the typical masses in the fragmentation patterns of the trimethylsilyl derivatives after gas chromatography. It is the first time sialic acids are identified in gastropods.

The occurrence of N-acetylneuraminic acid and N-glycolylneuraminic acid residues in preparations of the slug Arion lusitanicus(Gastropoda) was determined by sodium dodecyl sulphate electrophoresis of the proteins followed by lectin blots stained with the sialic acid specific lectin from Maackia amurensis, by the sensitivity of this binding to sialidase from Clostridium perfringens, by specific fluorescent labelling of sialic acids with 1,2-diamino-4,5-methylenedioxybenzene, by the determination of the sensitivity to sialate-pyruvate-lyase, by co-migration with standards on high performance anion exchange chromatography with pulsed amperometric detection and by identification of the typical masses in the fragmentation patterns of the trimethylsilyl derivatives after gas chromatography. It is the first time sialic acids are identified in gastropods.

Substitution of the asparagine-linked GlcNAc by α1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-l-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase (core α1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6.8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core α1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial α1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn, GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4(Fucα1–6)GlcNAcβ1-Asn, and GlcNAcβ1–2Manα1–3[Manα1–3(Manα1–6)Manα1–6]Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core α1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Chymotryptic glycopeptides were prepared from a honeybee (Apis mellifica) venom phospholipase A2(E.C. 3.1.1.4) fraction, with high affinity towards lentil (Lens culinaris) lectin. Treatment of the glycopeptide mixture with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase A, followed by HPLC fractionation, yielded two oligosaccharides, which were analysed by 500 MHz1H-NMR spectroscopy to give the following structures $$\begin{array}{*{20}c} {Man\alpha 1} & \backslash & {Fuc\alpha 1} & | \\ {} & 6 & {Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc} & 6 \\ {} & 3 & {} & {} \\ {Man\alpha 1} & / & {} & {} \\ \end{array} \begin{array}{*{20}c} {Man\alpha 1} & \backslash & {Fuc\alpha 1} & | \\ {} & 6 & {Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc} & 6 \\ {} & 3 & 3 & | \\ {Man\alpha 1} & / & {Fuc\alpha 1} & {} \\ \end{array}$$ This is the first report on a naturally occurring glycoprotein N-glycan with two fucose residues linked to the asparagine-boundN-acetylglucosamine.

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.

Analysis of the coenzyme Q system and the monosaccharide pattern of purified cell walls were used for species characterization in the genus Kluyveromyces. All the type strains of the genus possess coenzyme Q-6 and the mannose-glucose (‘Saccharomyces type’) cell wall sugar pattern. With the help of Random Amplified Polymorphic DNA-Polymerase Chain Reaction analysis 17 species were separated: K. aestuarii, K. africanus, K. bacillisporus, K. blattae, K. delphensis, K. dobzhanski, K. lactis (anamorph Candida sphaerica), K. lodderae, K. marxianus (syn. K. fragilis, K. bulgaricus, K. cicerisporus anamorphs Candida macedoniensis, C. pseudotropicalis, C. kefyr), K. phaffii, K. piceae, K. polysporus, K. sinensis, K. thermotolerans (syn. K. veronae, anamorph Candida dattila), K. waltii, K. wickerhamii, K. yarrowii (anamorph Candida tannotolerans). A strain of K. drosophilarum showed with the type strain of K. lactis only 63% similarity. The strain originally described as the type strain of K. cellobiovorus nom. nud. was excluded from the genus (Q-9), and found to be conspecific with the type strain of Candida intermedia.

An α1,3-fucosyltransferase was purified 3000-fold from mung bean seedlings by chromatography on DE 52 cellulose and Affigel Blue, by chromatofocusing, gelfiltration and affinity chromatography resulting in an apparently homogenous protein of about 65 kDa on SDS-PAGE. The enzyme transferred fucose from GDP-fucose to the Asn-linkedN-acetylglucosaminyl residue of an N-glycan, forming an α1,3-linkage. The enzyme acted upon N-glycopeptides and related oligosaccharides with the glycan structure GlcNAc2Man3GlcNAc2. Fucose in α1,6-linkage to the asparagine-linked GlcNAc had no effect on the activity. No transfer to N-glycans was observed when the terminal GlcNAc residues were either absent or substituted with galactose.N-acetyllactosamine, lacto-N-biose andN-acetylchito-oligosaccharides did not function as acceptors for the α1,3-fucosyltransferase. The transferase exhibited maximal activity at pH 7.0 and a strict requirement for Mn2+or Zn2+ions. The enzyme's activity was moderately increased in the presence of Triton X-100. It was not affected byN-ethylmaleimide.

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.

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 Km and Vmax 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.