Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells

Neural pathways in invertebrates are often tracked using anti-horseradish peroxidase, a cross-reaction due to the presence of core alpha1,3-fucosylated N-glycans. In order to investigate the molecular basis of this epitope in a cellular context, we compared two Drosophila melanogaster cell lines: the S2 and the neuronal-like BG2-c6 cell lines. As shown by mass spectrometric and chromatographic analyses, only the BG2-c6 cell line expresses alpha1,3/alpha1,6-difucosylated N-glycans, a result that correlates with anti-horseradish peroxidase binding. Of all four alpha1,3-fucosyltransferase homologues previously identified, the core alpha1,3-fucosyltransferase (FucTA; EC 2.4.1.214) is expressed in the neuronal cell line as well as throughout fly development and in heads and bodies of flies of both sexes. This pattern is distinctive in comparison with the expression of the other three alpha1,3-fucosyltransferase homologues (FucTB, FucTC, and FucTD). Furthermore, only transfection of FucTA cDNA into S2 cells resulted in expression of the anti-horseradish peroxidase epitope, a result compatible with its substrate specificity in vitro. Finally, silencing of FucTA by RNAi in the neuronal cell line led to a significant reduction of anti-horseradish peroxidase binding. The present study, in conjunction with our previous in vitro data, thereby shows that FucTA is indispensable for expression of the neural carbohydrate epitope in Drosophila cells.     

An endogenous Drosophila receptor for glycans bearing alpha 1,3-linked core fucose residues

The genome of Drosophila melanogaster encodes several proteins that are predicted to contain Ca(2+)-dependent, C-type carbohydrate-recognition domains. The CG2958 gene encodes a protein containing 359 amino acid residues. Analysis of the CG2958 sequence suggests that it consists of an N-terminal domain found in other Drosophila proteins, a middle segment that is unique, and a C-terminal C-type carbohydrate-recognition domain. Expression studies show that the full-length protein is a tetramer formed by noncovalent association of disulfide-linked dimers that are linked through cysteine residues in the N-terminal domain. The expressed protein binds to immobilized yeast invertase through the C-terminal carbohydrate-recognition domain. Competition binding studies using monosaccharides demonstrate that CG2958 interacts specifically with fucose and mannose. Fucose binds approximately 5-fold better than mannose. Blotting studies reveal that the best glycoprotein ligands are those that contain N-linked glycans bearing alpha1,3-linked fucose residues. Binding is enhanced by the additional presence of alpha1,6-linked fucose. It has previously been proposed that labeling of the Drosophila neural system by anti-horseradish peroxidase antibodies is a result of the presence of difucosylated N-linked glycans. CG2958 is a potential endogenous receptor for such neural-specific carbohydrate epitopes.     

Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates

Core alpha1,6-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 alpha1,6-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 alpha1,6-fucosyltransferases, as well as for native Caenorhabditis and Schistosoma mansoni core alpha1,6-fucosyltransferases. On the other hand, these alpha1,6-fucosyltransferases could not act on N-glycopeptides already carrying core alpha1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core alpha1,3-fucosyltransferases were able to use core alpha1,6-fucosylated glycans as substrates. Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core alpha1,3-fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core alpha1,3-fucosyltransferase. Overall, we conclude that the strict order of fucosylation events, previously determined for fucosyltransferases in crude extracts from insect cell lines (core alpha1,6 before core alpha1,3), also applies for recombinant Drosophila core alpha1,3- and alpha1,6-fucosyltransferases as well as for core fucosyltransferases in schistosomal egg extracts.     

Molecular basis of anti-horseradish peroxidase staining in Caenorhabditis elegans

Cross-reactivity with anti-horseradish peroxidase antiserum is a feature of many glycoproteins from plants and invertebrates; indeed staining with this reagent has been used to track neurons in Drosophila melanogaster and Caenorhabditis elegans. Although in insects the evidence indicates that the cross-reaction results from the presence of core alpha1,3-fucosylated N-glycans, the molecular basis for anti-horseradish peroxidase staining in nematodes has been unresolved to date. By using Western blots of wild-type and mutant C. elegans extracts in conjunction with specific inhibitors, we show that the cross-reaction is due to core alpha1,3-fucosylation. Of the various mutants examined, one with a deletion of the fut-1 (K08F8.3) gene showed no reaction to anti-horseradish peroxidase; the molecular phenotype was rescued by injection of either the K08F8 cosmid or the fut-1 open reading frame under control of the let-858 promoter. Furthermore, expression of fut-1 cDNA in Pichia and insect cells in conjunction with antibody staining, high pressure liquid chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses showed that FUT-1 is a core alpha1,3-fucosyltransferase with an unusual substrate specificity. It is the only core fucosyltransferase in plants and animals described to date that does not require the prior action of N-acetylglucosaminyltransferase I.     

Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin

Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing beta1,2-xylose and core alpha1,3-fucose, the latter also found in insect glycoproteins. To determine the relative contributions of these two sugar residues to antibody binding, we prepared an array of glycomodified forms of human apo-transferrin. Using core-alpha1, 3-fucosyltransferase (EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF), or neither (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the alpha1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in western blot, ELISA, and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.     

The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases

Fucoconjugates are key mediators of protein-glycan interactions in prokaryotes and eukaryotes. As examples, N-glycans modified with the non-mammalian core α1,3-linked fucose have been detected in various organisms ranging from plants to insects and are immunogenic in mammals. The rabbit polyclonal antibody raised against plant horseradish peroxidase (anti-HRP) is able to recognize the α1,3-linked fucose epitope and is also known to specifically stain neural tissues in the fruit fly Drosophila melanogaster. In this study, we have detected and localized the anti-HRP cross-reactivity in another insect species, the malaria mosquito vector Anopheles gambiae. We were able to identify and structurally elucidate fucosylated N-glycans including core mono- and difucosylated structures (responsible for anti-HRP cross reactivity) as well as a Lewis-type antennal modification on mosquito anionic N-glycans by applying enzymatic and chemical treatments. The three mosquito fucosyltransferase open reading frames (FucT6, FucTA and FucTC) required for the in vivo biosynthesis of the fucosylated N-glycan epitopes were identified in the Anopheles gambiae genome, cloned and recombinantly expressed in Pichia pastoris. Using a robust MALDI-TOF MS approach, we characterised the activity of the three recombinant fucosyltransferases in vitro and demonstrate that they share similar enzymatic properties as compared to their homologues from D. melanogaster and Apis mellifera. Thus, not only do we confirm the neural reactivity of anti-HRP in a mosquito species, but also demonstrate enzymatic activity for all its α1,3- and α1,6-fucosyltransferase homologues, whose specificity matches the results of glycomic analyses.     

Identification of an Arabidopsis gene encoding a GH95 alpha1,2-fucosidase active on xyloglucan oligo- and polysaccharides

alpha1,2-linked fucose can be found on xyloglucans which are the main hemicellulose compounds of dicotyledons. The fucosylated nonasaccharide XXFG derived from xyloglucans plays a role in cell signaling and is active at nanomolar concentrations. The plant enzyme acting on this alpha1,2-linked fucose residues has been previously called fucosidase II; here we report on the molecular identification of a gene from Arabidopsis thaliana (At4g34260 hereby designed AtFuc95A) encoding this enzyme. Analysis of the predicted protein composed of 843 amino acids shows that the enzyme belongs to the glycoside hydrolase family 95 and has homologous sequences in different monocotyledons and dicotyledons. The enzyme was expressed recombinantly in Nicotiana bentamiana, a band was visible by Coomassie blue staining and its identity with the alpha1,2-fucosidase was assessed by an antibody raised against a peptide from this enzyme as well as by peptide-mass mapping. The recombinant AtFuc95A is active towards 2-fucosyllactose with a Km of 0.65 mM, a specific activity of 110 mU/mg and a pH optimum of 5 but does not cleave alpha1,3, alpha1,4 or alpha1,6-fucose containing oligosaccharides and p-nitrophenyl-fucose. The recombinant enzyme is able to convert the xyloglucan fragment XXFG to XXLG, and is also active against xyloglucan polymers with a Km value for fucose residues of 1.5mM and a specific activity of 36 mU/mg. It is proposed that the AtFuc95A gene has a role in xyloglucan metabolism.     

The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter

Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac(1) flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac(1) mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac(1) Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac(1) mutant. These results validate the Drosophila nac(1) mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency.     

Drosophila sperm surface alpha-l-fucosidase interacts with the egg coats through its core fucose residues

Sperm–oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein–carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α-l-fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α-l-fucosidases have fundamental function(s) during gamete interactions. An α-l-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila. Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster. In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-l-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α-l-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. The ability of sperm α-l-fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α-l-fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α-l-fucosidase was pre-incubated with fucoidan, a polymer of α-l-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-l-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm–egg interactions in Drosophila.     

Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose

The plant glycosyltransferases, beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT), are responsible for the transfer of beta1,2-linked xylose and core alpha1,3-linked fucose residues to glycoprotein N-glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modification of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete deficiency of XylT and FucT. These plants lack antigenic protein-bound N-glycans and instead synthesise predominantly structures with two terminal betaN-acetylglucosamine residues (GlcNAc(2)Man(3)GlcNAc(2)).     

Strict order of (Fuc to Asn-linked GlcNAc) fucosyltransferases forming core-difucosylated structures

In insect cells fucose can be either α1,6- or α1,3-linked to the asparagine-bound GlcNAc residue of N-glycans. Difucosylated glycans have also been found. Kinetic studies and acceptor competition experiments demonstrate that two different enzymes are responsible for this α1,6- and α1,3-linkage of fucose. Using dansylated acceptor substrates a strict order of these enzymes can be established for the formation of difucosylated structures. First, the α1,6-fucosyltransferase catalyses the transfer of fucose into α1,6-linkage to the non-fucosylated acceptor and then the α1,3-fucosyltransferase completes the difucosylation. © 1998 Rapid Science Ltd     

Definition of immunogenic carbohydrate epitopes

Carbohydrates are known as sources of immunological cross-reactivity of allergenic significance. In celery and in cypress pollen, the major allergens Api g 5 and Cup a 1 are recognised by antisera raised against anti-horseradish peroxidase and by patients' IgE which apparently bind carbohydrate epitopes; mass spectrometric analysis of the tryptic peptides and of their N-glycans showed the presence of oligosaccharides carrying both xylose and core alpha1,3-fucose residues. Core alpha1,3-fucose residues are also a feature of invertebrates: genetic and biochemical studies on the fruitfly Drosophila melanogaster, the parasitic trematode Schistosoma mansoni and the nematode worm Caenorhabditis elegans indicate that these organisms possess core alpha1,3-fucosyltransferases. Various experiments have shown that fucosyltransferases from both fly and worm are responsible in vivo and in vitro for the synthesis of N-glycans which cross-react with anti-horseradish peroxidase; thus, we can consider these enzymes as useful tools in generating standard compounds for testing cross-reactive carbohydrate epitopes of allergenic interest.     

Galactosylated Fucose Epitopes in Nematodes: INCREASED EXPRESSION IN A CAENORHABDITIS MUTANT ASSOCIATED WITH ALTERED LECTIN SENSITIVITY AND OCCURRENCE IN PARASITIC SPECIES

The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.     

Structure of Golgi alpha-mannosidase II: a target for inhibition of growth and metastasis of cancer cells.

Golgi alpha-mannosidase II, a key enzyme in N-glycan processing, is a target in the development of anti- cancer therapies. The crystal structure of Drosophila Golgi alpha-mannosidase II in the absence and presence of the anti-cancer agent swainsonine and the inhibitor deoxymannojirimycin reveals a novel protein fold with an active site zinc intricately involved both in the substrate specificity of the enzyme and directly in the catalytic mechanism. Identification of a putative GlcNAc binding pocket in the vicinity of the active site cavity provides a model for the binding of the GlcNAcMan(5)GlcNAc(2) substrate and the consecutive hydrolysis of the alpha1,6- and alpha1,3-linked mannose residues. The enzyme-inhibitor interactions observed provide insight into the catalytic mechanism, opening the door to the design of novel inhibitors of alpha-mannosidase II.     

Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose

The alpha1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the alpha1,6-fucosylated chain among alpha1,2-, alpha1,3-, alpha1,4-, and alpha1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the alpha1,2-fucosylated PA-sugar chain (H antigen) relative to the alpha1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with alpha1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the alpha1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from alpha1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA.     

A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.     

A unique beta1,3-galactosyltransferase is indispensable for the biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuc alpha1-4(Gal beta1-3)GlcNAc-R] structures. This process involves the sequential attachment of beta1,3-galactose and alpha1,4-fucose residues by beta1,3-galactosyltransferase and alpha1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, beta1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana beta1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring beta1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with alpha1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of beta1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.     

Molecular cloning and heterologous expression of beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from maize

Maize is considered a promising alternative production system for pharmaceutically relevant proteins. However, like in all other plant species asparagine-linked oligosaccharides of maize glycoproteins are modified with beta1,2-xylose and core alpha1,3-fucose sugar residues, which are considered to be immunogenic in mammals. This altered N-glycosylation when compared to mammalian cells may reduce the potential of maize as a production system for heterologous glycoproteins. Here we report the cloning and characterization of the cDNA sequences coding for the maize enzymes beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT). The cloned XylT and FucT cDNAs were shown to encode enzymatically active proteins, which were independently able to convert a mammalian acceptor glycoprotein into an antigen binding anti-plant N-glycan antibodies. The complete sequence of the XylT gene was determined. Evidence for the presence of at least three XylT and FucT gene loci in the maize genome was obtained. The identification of the two enzymes and their genes will allow the targeted downregulation or even elimination of beta1,2-xylose and core alpha1,3-fucose addition to recombinant glycoproteins produced in maize.     

Biosynthesis of truncated N-linked oligosaccharides results from non-orthologous hexosaminidase-mediated mechanisms in nematodes, plants, and insects

In many invertebrates and plants, the N-glycosylation profile is dominated by truncated paucimannosidic N-glycans, i.e. glycans consisting of a simple trimannosylchitobiosyl core often modified by core fucose residues. Even though they lack antennal N-acetylglucosamine residues, the biosynthesis of these glycans requires the sequential action of GlcNAc transferase I, Golgi mannosidase II, and, finally, beta-N-acetylglucosaminidases. In Drosophila, the recently characterized enzyme encoded by the fused lobes (fdl) gene specifically removes the non-reducing N-acetylglucosamine residue from the alpha1,3-antenna of N-glycans. In the present study, we examined the products of five beta-N-acetylhexosaminidase genes from Caenorhabditis elegans (hex-1 to hex-5, corresponding to reading frames T14F9.3, C14C11.3, Y39A1C.4, Y51F10.5, and Y70D2A.2) in addition to three from Arabidopsis thaliana (AtHEX1, AtHEX2, and AtHEX3, corresponding to reading frames At1g65590, At3g55260, and At1g05590). Based on homology, the Caenorhabditis HEX-1 and all three Arabidopsis enzymes are members of the same sub-family as the aforementioned Drosophila fused lobes enzyme but either act as chitotriosidases or non-specifically remove N-acetylglucosamine from both N-glycan antennae. The other four Caenorhabditis enzymes are members of a distinct sub-family; nevertheless, two of these enzymes displayed the same alpha1,3-antennal specificity as the fused lobes enzyme. Furthermore, a deletion of part of the Caenorhabditis hex-2 gene drastically reduces the native N-glycan-specific hexosaminidase activity in mutant worm extracts and results in a shift in the N-glycan profile, which is a demonstration of its in vivo enzymatic relevance. Based on these data, it is hypothesized that the genetic origin of paucimannosidic glycans in nematodes, plants, and insects involves highly divergent members of the same hexosaminidase gene family.