Isolation of null alleles of the Caenorhabditis elegans gly-12, gly-13 and gly-14 genes,all of which encode UDP-GlcNAc: alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I activity

UDP-GlcNAc: alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT I) is a Golgi-resident enzyme which transfers a GlcNAc residue in beta1,2 linkage to the Manalpha1,3Manbeta-terminus of (Manalpha1,6(Manalpha1,3)Manalpha1,6)(Manalpha1,3)Manbeta1,4GlcNAcbeta1,4GlcNAc-Asn-protein, thereby initiating the synthesis of hybrid N-glycans. Three Caenorhabditis elegans genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) have been cloned. All three cDNAs encode proteins with GnT I enzyme activity. We report in this paper the preparation by ultra-violet (UV) light irradiation in the presence of trimethylpsoralen, of mutants lacking either gly-12, gly-13 or gly-14. A double null mutation in the gly-12 and gly-14 genes (gly-14; gly-12) has also been prepared. These mutations are intragene deletions, removing large portions of the GnT I catalytic domain, and are therefore, all molecular nulls. The gly-12 and gly-14 mutants as well as the gly-14; gly-12 double mutant all displayed wild-type phenotypes, indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. In contrast, about 60% of the mutants lacking the gly-13 gene arrested as L1 larvae at 20 degrees C and the remaining 40% homozygous worms grew to adulthood but displayed severe morphological and behavioral defects despite the presence of the other two GnT I genes, gly-12 and gly-14. Attempts to rescue the gly-13 null phenotype with the wild type transgene were not successful. However, lethality co-segregated with the gly-13 deletion within 0.02 map units (mu) in genetic mapping experiments, suggesting that the gly-13 mutation is responsible for the phenotype.     

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I gene.

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.     

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II) are key enzymes in the synthesis of Asn-linked hybrid and complex glycans. We have cloned cDNAs from Caenorhabditis elegans for three genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) and one gene homologous to mammalian GnT II. All four cDNAs encode proteins which have the domain structure typical of previously cloned Golgi-type glycosyltransferases and show enzymatic activity (GnT I and GnT II, respectively) on expression in transgenic worms. We have isolated worm mutants lacking the three GnT I genes by the method of ultraviolet irradiation in the presence of trimethylpsoralen (TMP); null mutants for GnT II have not yet been obtained. The gly-12 and gly-14 mutants as well as the gly-14;gly-12 double mutant displayed wild-type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that the GLY-13 protein is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behavioral defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Our data indicate that complex and hybrid N-glycans may play critical roles in the morphogenesis of C. elegans, as they have been shown to do in mice and men.     

Native gibberellins and the metabolism of [14C]gibberellin A53 and of [17-13C, 17-3H2]gibberellin A20 in tassels of Zea mays

The native hormones from tassels of maize (Zea mays) were re-investigated. The previous identification by GC/SIM of GA₁, GA₈ and GA₂₉ in normal tassels was confirmed by full GC/MS scans at the correct Kovats retention indices. In tassels of dwarf-1 mutants, GA₄₄,^?GA₁₉, GA₁₇, GA₂₀ and the 16,17-dihydro, 7β,16α,17-trihydroxy derivative of ent-kaurenoic acid were identified by GC/MS. Gibberellin A₁ was not found in the mutant tassels. [¹⁴C]Gibberellin A₅₃ was fed to tassels of the dwarf-5 mutant. In the ethyl acetate-soluble acidic fraction from the feeds, [¹⁴C]GA₄₄ was identified by GC/MS; [¹⁴C]GA₁₉ and [¹⁴C]GA₂₉ were identified by GC/SIM. The GA₂₉ is probably a metabolite of the feeds because the dwarf-5 mutant is known to control the step copalyl pyrophosphate to ent-kaurene in the maize GA-biosynthetic pathway and because GA₂₉ was not identified in a control experiment. The n-butanol fractions obtained from the feeds were shown, by GC/MS, to contain [¹⁴C]GA₅₃ after hydrolysis, suggesting that conjugated [¹⁴C]GA₅₃ is a major metabolite from GA₅₃ feeds. [17-¹³C, 17-³H₂]Gibberellin A₂₀ was fed to normal, dwarf-1 and dwarf-5 tassels. In each case, analysis of the purified ethyl acetate-soluble acidic extracts by GC/MS led to the identification of [¹³C]GA₂₉ and unmetabolized [¹³C]GA₂₀ in which no ¹³C-isotope dilution was observed.     

Rapid high yield production of different glycoforms of Ebola virus monoclonal antibody

Background

Fc-glycosylation of monoclonal antibodies (mAbs) has profound implications on the Fc-mediated effector functions. Alteration of this glycosylation may affect the efficiency of an antibody. However, difficulties in the production of mAbs with homogeneous N-glycosylation profiles in sufficient amounts hamper investigations of the potential biological impact of different glycan residues.

Methodology/Principal Findings

Here we set out to evaluate a transient plant viral based production system for the rapid generation of different glycoforms of a monoclonal antibody. Ebola virus mAb h-13F6 was generated using magnICON expression system in Nicotiana benthamiana, a plant species developed for commercial scale production of therapeutic proteins. h-13F6 was co-expressed with a series of modified mammalian enzymes involved in the processing of complex N-glycans. Using wild type (WT) plants and the glycosylation mutant ΔXTFT that synthesizes human like biantennary N-glycans with terminal N-acetylglucosamine on each branch (GnGn structures) as expression hosts we demonstrate the generation of h-13F6 complex N-glycans with (i) bisected structures, (ii) core α1,6 fucosylation and (iii) β1,4 galactosylated oligosaccharides. In addition we emphasize the significance of precise sub Golgi localization of enzymes for engineering of IgG Fc-glycosylation.

Conclusion

The method described here allows the efficient generation of a series of different human-like glycoforms at large homogeneity of virtually any antibody within one week after cDNA delivery to plants. This accelerates follow up functional studies and thus may contribute to study the biological role of N-glycan residues on Fcs and maximizing the clinical efficacy of therapeutic antibodies.     

Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N - acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.      

Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom

BackgroundPrevious glycophylogenetic comparisons of dipteran and lepidopteran species revealed variations in the anionic and zwitterionic modifications of their N-glycans; therefore, we wished to explore whether species- and order-specific glycomic variations would extend to the hymenoptera, which include the honeybee Apis mellifera, an agriculturally- and allergologically-significant social species.MethodsIn this study, we employed an off-line liquid chromatography/mass spectrometry approach, in combination with enzymatic and chemical treatments, to analyse the N-glycans of male honeybee larvae and honeybee venom in order to facilitate definition of isomeric structures.ResultsThe neutral larval N-glycome was dominated by oligomannosidic and paucimannosidic structures, while the neutral venom N-glycome displayed more processed hybrid and complex forms with antennal N-acetylgalactosamine, galactose and fucose residues including Lewis-like epitopes; the anionic pools from both larvae and venom contained a wide variety of glucuronylated, sulphated and phosphoethanolamine-modified N-glycans with up to three antennae. In comparison to honeybee royal jelly, there were more fucosylated and fewer Man_4/5-based hybrid glycans in the larvae and venom samples as well as contrasting antennal lengths.ConclusionsCombining the current data on venom and larvae with that we previously published on royal jelly, a total honeybee N-glycomic repertoire of some 150 compositions can be proposed in addition to the 20 previously identified on specific venom glycoproteins.SignificanceOur data are indicative of tissue-specific modification of the core and antennal regions of N-glycans in Apis mellifera and reinforce the concept that insects are capable of extensive processing to result in rather complex anionic oligosaccharide structures.     

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I gene

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.     

Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum

BackgroundThe porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible.MethodsDifferent enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC.ResultsWhereas the L3 N-glycome was simpler and more dominated by phosphorylcholine-modified structures, the male and female worms express a wide range of core fucosylated N-glycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered ‘male’, while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc_2–4PC_2–4 motifs not observed in our previous studies on other nematodes.ConclusionThe interspecies tendency of class V nematodes to share most, but not all, N-glycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation.General significanceUnusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.     

Complex N-glycans: the story of the “yellow brick road”

The synthesis of complex asparagine-linked glycans (N-glycans) involves a multi-step process that starts with a five mannose N-glycan structure: [Manα1-6(Manα1-3)Manα1-6][Manα1-3]-R where R?=?Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-protein. N-acetylglucosaminyltransferase I (GlcNAc-TI) first catalyzes addition of GlcNAc in β1-2 linkage to the Manα1-3-R terminus of the five-mannose structure. Mannosidase II then removes two Man residues exposing the Manα1-6 terminus that serves as a substrate for GlcNAc-T II and addition of a second GlcNAcβ1-2 residue. The resulting structure is the complex N-glycan: GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)-R. This structure is the precursor to a large assortment of branched complex N-glycans involving four more N-acetylglucosaminyltransferases. This short review describes the experiments (done in the early 1970s) that led to the discovery of GlcNAc-TI and II.     

GC/MS analysis of the plant hormones in seeds of Cucurbita maxima

The GC/MS detection is reported of over 30 compounds, in extracts of the endosperm and embryos from seeds of Cucurbita maxima. The compounds which were identified from reference spectra include: cis,trans-ABA; trans,trans-ABA; dihydrophaseic acid; IAA; GA₄; GA₁₂; GA₁₃; GA₂₅; GA₃₉; GA₄₃; GA₄₉; ent-13-hydroxy-, ent-6α,7α-and ent-7α,13-dihydroxy-, and ent-6α,7α,13-trihydroxykaur-16-en-19-oic acids; ent-7α,16,17-trihydroxy- and ent-6α,7α,16,17-tetrahydroxy-kauran-19-oic acids, ent-6,7-seco-7-oxokauren-6,19-dioic acid and/or ent-6,7-secokauren-6,7,19-trioic acid, and 7β,12α-dihydroxykaurenolide. New compounds, the structures of which were deduced from GC/MS data, include: the 12α-hydroxy-derivatives of GA₁₂, GA₁₄, GA₃₇ and GA₄, and the 12β-hydroxy-derivatives of ent-7α-hydroxy- and ent-6α,7α-dihydroxykaurenoic acids.     

Intramolecular triple helix as a model for regular polyribonucleotide (CAA)n

The regular (CAA)_n polyribonucleotide, as well as the omega leader sequence containing (CAA)-rich core, have recently been shown to form cooperatively melted and compact structures. In this report, we propose a structural model for the (CAA)_n sequence in which the polyribonucleotide chain is folded upon itself, so that it forms an intramolecular triple helix. The triple helix is stabilized by hydrogen bonding between bases thus forming coplanar triads, and by stacking interactions between the base triads. A distinctive feature of the proposed triple helix is that it does not contain the canonical double-helix elements. The difference from the known triple helices is that Watson-Crick hydrogen bond pairings do not take place in the interactions between the bases within the base triads.     

The non-classical N-glycan processing pathway of bovine brain ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is brain specific and not due to mannose-6-phosphorylation

Ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is a glycosylphosphatidylinositol (GPI)-anchored alkaline lysophospholipase C which is predominantly expressed in brain myelin and kidney. Due to shedding of the GPI-anchor eNPP6 occurs also as a soluble isoform (s-eNPP6). eNPP 6 consists of two identical monomers of 55 kDa joined by a disulfide bridge, and possesses four N-glycans in each monomer. In brain s-eNPP6 the N-glycans are mainly hybrid and high mannose type structures, reminiscent of processed mannose-6-phosphorylated glycans. Here we completed characterization of the site-specific glycan structures of bovine brain s-eNPP6, and determined the endo H-sensitivity glycan profiles of s-eNPP6 from bovine liver and kidney. Whereas in brain s-eNPP6 all of the N-glycans were endo H-sensitive, in liver and kidney only one of the glycosylation sites was occupied by an endo H-sensitive glycan, likely N406, which is located within the cleft formed by the dimer interface. Thus, the non-classical glycan processing pathway of brain eNPP 6 is not due to mannose-6-phosphorylation, suggesting that there is an alternative Golgi glycan-processing pathway of eNPP6 in brain. The resulting brain-specific expression of accessible hybrid and oligomannosidic glycans may be physiologically important within the cell–cell communication system of the brain.     

Characterization of the single-chain Fv-Fc antibody MBP10 produced in <Emphasis Type="Italic">Arabidopsis alg3</Emphasis> mutant seeds

ER resident glycoproteins, including ectopically expressed recombinant glycoproteins, carry so-called high-mannose type N-glycans, which can be at different stages of processing. The presence of heterogeneous high-mannose type glycans on ER-retained therapeutic proteins is undesirable for specific therapeutic applications. Previously, we described an Arabidopsis alg3-2 glycosylation mutant in which aberrant Man₅GlcNAc₂ mannose type N-glycans are transferred to proteins. Here we show that the alg3-2 mutation reduces the N-glycan heterogeneity on ER resident glycoproteins in seeds. We compared the properties of a scFv-Fc, with a KDEL ER retention tag (MBP10) that was expressed in seeds of wild type and alg3-2 plants. N-glycans on these antibodies from mutant seeds were predominantly of the intermediate Man₅GlcNAc₂ compared to Man₈GlcNAc₂ and Man₇GlcNAc₂ isoforms on MBP10 from wild-type seeds. The presence of aberrant N-glycans on MBP10 did not seem to affect MBP10 dimerisation nor binding of MBP10 to its antigen. In alg3-2 the fraction of underglycosylated MBP10 protein forms was higher than in wild type. Interestingly, the expression of MBP10 resulted also in underglycosylation of other, endogenous glycoproteins.     

Formation of UDP-Xylose and Xyloglucan in Soybean Golgi Membranes

Soybean (Glycine max) membranes co-equilibrating with Golgi vesicles in linear sucrose gradients contained UDP-glucuronate carboxy-lyase and xyloglucan synthase activities. Digitonin solubilized and increased the activity of the membrane-bound UDP-glucuronate carboxy-lyase. UDP-xylose did not inhibit the transport of UDP-glucuronate into the lumen of Golgi vesicles but repressed the decarboxylation of the translocated UDP-glucuronate. The results suggest that UDP-glucuronate is transported into the vesicles by a specific carrier and decarboxylated to UDP-xylose within the lumen. On incubation of UDP-[¹⁴C]glucuronate with Golgi membranes in the presence of UDP-glucose, [¹⁴C]xylose-labeled xyloglucan was formed. Although the K_m value of UDP-glucuronate for the decarboxylation was 240 micromolar, the affinity of UDP-glucuronate for xyloglucan formation (31 micromolar) was similar to that of UDP-xylose (28 micromolar), suggesting a high turnover of UDP-xylose. The biosynthesis of UDP-xylose from UDP-glucuronate probably occurs in Golgi membranes, where xyloglucan subsequently forms from UDP-xylose and UDP-glucose.     

Stage-dependent localization of LHCP II apoprotein in the Golgi of synchronized cells of Euglena gracilis by immunogold electron microscopy

We have localized LHCP II apoprotein in the Golgi and thylakoids of Euglena gracilis Klebs var. bacillaris Cori and strain Z Pringsheim by electron microscopy using a specific antibody and protein A-gold. Using synchronized cells (light, 14 h:dark, 10 h) we show that thylakoids are always immunoreactive. There is no reaction in the Golgi at 0 h (the beginning of the light period) but immunoreaction appears in the Golgi soon thereafter, rises to a peak at 8 h and declines to zero by 16 h (2 h into the dark period). The peak in immunoreaction in the Golgi immediately precedes the peak in cellular ¹⁴C-labeling of thylakoid LHCP II apoprotein seen by Brandt and von Kessel (Plant Physiol. (1983) 72, 616), supporting our suggestion that processing in the Golgi precedes deposition of LHCP apoprotein in the thylakoids. Substitution of preimmune serum for antiserum eliminates the immunoreaction in the Golgi, and thylakoids of synchronized cells of mutant Gr₁BSL which lacks LHCP II apoprotein show no immunoreaction in the Golgi or thylakoids at any stage. Random observations indicate that the compartmentalized osmiophilic structure (COS) shows an immunoreaction with anti-LHCP II apoprotein antibody at 1 h into the light period (when the Golgi is not immunoreactive) and at 10 h into the light period (when the Golgi is fully reactive), suggesting that the COS remains immunoreactive throughout the cell cycle.     

More Than Just Oligomannose: An N-glycomic Comparison of Penicillium Species

N-glycosylation is an essential set of post-translational modifications of proteins; in the case of filamentous fungi, N-glycans are present on a range of secreted and cell wall proteins. In this study, we have compared the glycans released by peptide/N-glycosidase F from proteolysed cell pellets of three Penicillium species (P. dierckxii, P. nordicum and P. verrucosum that all belong to the Eurotiomycetes). Although the major structures are all within the range Hex_5–11HexNAc₂ as shown by mass spectrometry, variations in reversed-phase chromatograms and MS/MS fragmentation patterns are indicative of differences in the actual structure. Hydrofluoric acid and mannosidase treatments revealed that the oligomannosidic glycans were not only in part modified with phosphoethanolamine residues and outer chain och1-dependent mannosylation, but that bisecting galactofuranose was present in a species-dependent manner. These data are the first to specifically show the modification of N-glycans in fungi with zwitterionic moieties. Furthermore, our results indicate that mere mass spectrometric screening is insufficient to reveal the subtly complex nature of N-glycosylation even within a single fungal genus.Fungi and yeast have been used for hundreds of years as modifiers of foodstuffs, e.g. in fermentation, baking or cheese production. Ecologically significant in degradation of biological materials or as symbionts, they have within the last century also become biotechnologically important as sources of antibiotics, enzymes and recombinant proteins. On the other hand, their harmful potential is reflected by their ability to produce mycotoxins, their association with allergy or their pathogenicity toward plants and animals. For the fungal kingdom (1) currently five divisions are defined, with the Ascomycota and the Basidiomycota being the most familiar. Within the Ascomycota, the yeasts form a separate clade with unicellular species, whereas filamentous fungi including various molds are examples of multicellular mycelium-forming species. Among the most familiar of the latter is the class of Eurotiomycetes including Penicillium species (2), which are of general interest because of their production of antibiotics such as penicillin and griseofulvin as well as their role in food spoilage. Some species cause opportunistic infections as exemplified by Penicillium (Talaromyces) marneffei, which result in severe symptoms in immunocompromised humans and other animals (3). In terms of allergens, IgE reactivity toward Pen c 30 from P. citrinum is apparently reduced by periodate oxidation, suggesting IgE binding to the carbohydrate moieties of this protein (4).The fungi and yeasts may be morphologically diverse, but share fundamental characteristics such as possessing a polysaccharide-rich cell wall. Furthermore, basic scientific research on the model ascomycete Saccharomyces cerevisiae has revealed conserved aspects of genetics and metabolism also in the field of glycoscience, e.g. by the study of the alg genes involved in the formation of N-glycans (5). Regarding the observed N- and O-glycans of fungi and yeasts hypermannosylation seems to be a general feature; here there is a key role for the Och1p α1,6-mannosyltransferase which initiates the formation of ''outer chains'' of variable lengths on the α1,3-antennae. Additional modifications dependent on the species, with phosphodiesters, galactopyranose, N-acetylglucosamine or pyruvate residues, xylose and fucose have also been reported (6–8). In the case of some of the Eurotiomycetes, galactofuranose residues are also known to be present and glycan analyses of secreted glycoproteins have indicated that often one or more such residues can be present on the dominant Hex_8–10HexNAc₂ structures (9). Nevertheless, there are major gaps in our knowledge regarding the glycans and glycosylation pathways in most fungi. This is due, in part, to the fungal N-glycans containing primarily hexose residues, which presents a methodological challenge as compared with mammalian complex N-glycans with their variety of monosaccharide components. The Penicillium genus is exemplary for the lack of in-depth reports regarding the N-glycan structures of filamentous fungi in general.As part of our general efforts directed at defining the fine structure of N-glycans from diverse species, we have compared the N-glycomes of three members of the Penicillium genus, including two mycotoxin producers. In addition to the typical high mannose glycans, in part featuring the precursors for ''outer chain'' formation, we detected structures containing phosphoethanolamine and galactofuranose. The former modification is also known on glycoconjugates of, e.g. bacteria as well as of some eukaryotes such as trichomonads and insects (10–12), whereas the latter is present not just in other fungi, but also in, e.g. mycobacteria and kinetoplasts (13, 14). Certainly, interactions of these glycan modifications with proteins of the human immune system are possible and may be relevant in terms of allergy or when considering these members of the Eurotiomycetes for manufacturing biotechnological products.     

Myrosinases TGG1 and TGG2 from Arabidopsis thaliana contain exclusively oligomannosidic N-glycans

In all eukaryotes N-glycosylation is the most prevalent protein modification of secretory and membrane proteins. Although the N-glycosylation capacity and the individual steps of the N-glycan processing pathway have been well studied in the model plant Arabidopsis thaliana, little attention has been paid to the characterization of the glycosylation status of individual proteins. We report here the structural analysis of all N-glycans present on the endogenous thioglucoside glucohydrolases (myrosinases) TGG1 and TGG2 from A. thaliana. All nine glycosylation sites of TGG1 and all four glycosylation sites of TGG2 are occupied by oligomannosidic structures with Man₅GlcNAc₂ as the major glycoform. Analysis of the oligomannosidic isomers from wild-type plants and mannose trimming deficient mutants by liquid chromatography with porous graphitic carbon and mass spectrometry revealed that the N-glycans from both myrosinases are processed by Golgi-located α-mannosidases.     

Molecular cloning and characterization of cDNA coding for beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) from Nicotiana tabacum.

In plants as well as in animals beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from oligomannosidic N-glycans to complex or hybrid type N-linked oligosaccharides. Employing degenerated primers deduced from known GlcNAc-TI genes from animals, we were able to identify the cDNA coding for GlcNAc-TI from a Nicotiana tabacum cDNA library. The complete nucleotide sequence revealed a 1338 base pair open reading frame that codes for a polypeptide of 446 amino acids. Comparison of the deduced amino acid sequence with that of already known GlcNAc-TI polypeptides revealed no similarity of the tobacco clone within the putative cytoplasmatic, transmembrane, and stem regions. However, 40% sequence similarity was found within the putative C-terminal catalytic domain containing conserved single amino acids and peptide motifs. The predicted domain structure of the tobacco polypeptide is typical for type II transmembrane proteins and comparable to known GlcNAc-TI from animal species. In order to confirm enzyme activity a truncated form of the protein containing the putative catalytic domain was expressed using a baculovirus/insect cell system. Using pyridylaminated Man(5)- or Man(3)GlcNAc(2)as acceptor substrates and HPLC analysis of the products GlcNAc-TI activity was shown. This demonstrates that the C-terminal region of the protein comprises the catalytic domain. Expression of GlcNAc-TI mRNA in tobacco leaves was detected using RT-PCR. Southern blot analysis gave two hybridization signals of the gene in the amphidiploid genomes of the two investigated species N. tabacum and N.benthamiana.