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.     

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.     

Concanavalin A binding and endoglycosidase D resistance of beta1,2-xylosylated and alpha 1,3-fucosylated plant and insect oligosaccharides

The binding to concanavalin A (Con A) by pyridylaminated oligosaccharides derived from bromelain (Man alpha 1,6(Xyl beta 1, 2) Man beta 1, 4GlcNAc beta 1, 4(Fuc alpha 1, 3)GlcNAc), horseradish peroxidase (Man alpha 1,6(Man alpha 1, 3) (Xyl beta 1, 2)Man beta 1, 4GlcNAc beta 1,4(Fuc alpha 1, 3) GlcNAc), bee venom phospholipase A2 (Man alpha 1,6Man beta 1,4GlcNAc beta 1,4GlcNAc and Man alpha 1,6(Man alpha 1, 3)Man beta 1,4GlcNAc beta 1, 4 (Fuc alpha 1, 3)GlcNAc) and zucchini ascorbate oxidase (Man alpha 1,6(Man alpha 1, 3) (Xyl beta 1, 2)Man beta 1, 4 GlcNAc beta 1, 4GlcNAc) was compared to the binding by Man3GlcNAc2, Man5GlcNAc2 and the asialo-triantennary complex oligosaccharide from bovine fetuin. While the fetuin oligosaccharide did not bind, bromelain, zucchini, Man2GlcNAc2 and horseradish peroxidase were retarded (in that order). The alpha 1, 3-fucosylated phospholipase, Man3GlcNAc2 and Man5GlcNAc2 structures were eluted with 15 M alpha -methylmannoside. It is concluded that core alpha 1,3-fucosylation has little or no effect on ConA binding while xylosylation decreases affinity for ConA. In a parallel study comparing the endoglycosidase D (Endo D) sensitivities of Man3GlcNAc2, IgG-derived GlcNAc beta 1, 2Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1,4GlcNAc beta 1,4(Fuc alpha 1,6)GlcNAc, the phospholipase Man alpha 1,6(Man alpha 1, 3)Man beta 1, 4GlcNAc beta 1,4(Fuc alpha 1,3)GlcNAc, and horseradish and zucchini pyridylaminated N-linked oligosaccharides, it was found that only the Man3GlcNAc2 structure was cleaved. The IgG structure was sensitive only when beta -hexosaminidase was also present. Thus, in contrast to core alpha 1,6-fucosylated structures, such as those present in mammals, the presence of core alpha 1,3-fucose, as found in structures from plants and insects, and/or beta 1,2-xylose, as found in plants, causes resistance to Endo D.     

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Fucosebeta-1-P-Ser is a new type of glycosylation: using antibodies to identify a novel structure in Dictyostelium discoideum and study multiple types of fucosylation during growth and development

Three antibodies that recognize distinct fucose epitopes were used to study fucosylation during growth and development of Dictyostelium discoideum. mAb83.5 is known to recognize an undefined "fucose epitope" on several proteins with serine-rich domains, while mAb CAB4, and a component of anti-horse-radish peroxidase, specifically recognize Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc residues respectively in the core of N-linked oligosaccharides. We show that mAb 83.5 defines a new type of O-glycosylation. Serine-containing peptides incubated with GDPbeta[3H]Fuc and microsomes formed two fucosylated products. A neutral product accounting for 30% of the label did not react with the antibody, while the rest of the label was incorporated into a charged product which contained all the mAb83.5 reactive material. beta- Elimination of the labeled peptide or endogenous products produced [3H]Fuc-1-P, indicating phosphodiester linkage to serine. Fucbeta-1-P and GDP-betaFuc at 100 microM blocked mAb83.5 binding to endogenous and peptide products, but their alpha-linked anomers did not. Electrospray ionization mass spectra of the neutral and anionic labeled products showed major peaks of mass units corresponding to O-Fuc-Ser peptide and O-Fuc-phospho-Ser peptide, respectively. The activity of Fuc- phosphotransferase exactly paralleled the accumulation of reactive glycans during growth and development. The expressions of N-glycan core Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc and their respective fucosyl transferase activities were also synchronous, but their developmental regulation differed from one another. Fucalpha1, 6GlcNAc was expressed maximally during growth but declined during development. In contrast core Fucalpha1,3GlcNAc epitopes were expressed almost exclusively during development. These findings provide direct evidence for a novel type of O-phosphofucosylation, demonstrate the existence of an O- fucosyl transferase, and identify two different types of core fucosylation in the N-glycans of Dictyostelium.      

Identification of genes required for neural-specific glycosylation using functional genomics

Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA-binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells.     

Fucosyltransferases in Schistosoma mansoni development

Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fucalpha1,3GlcNAc, Fucalpha1,2Fuc, Fucalpha1,6GlcNAc, and perhaps Fucalpha1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophore-assisted carbohydrate electrophoresis with detection of radioactive fucose incorporation from GDP-[(14)C]-fucose into structurally defined acceptors. The total fucosyltransferase-specific activity in egg extracts was 50-fold higher than that in the other life stages tested (cercaria and adult worms). A fucosyltransferase was detected that transferred fucose to type-2 oligosaccharides (Galbeta1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by alpha2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,3Galbeta1,4Glc). A low level of fucosyltransferase that transfers fucose to no-sialylated type-1 oligosaccharides (Galbeta1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucose-type iminosugars inhibitors were tested on schistosome fucosyltransferases. A new fucose-type 1-N-iminosugar was four- to sixfold more potent as an inhibitor of schistosome fucosyltransferases in vitro than was deoxyfuconojirimycin. In vivo, this novel 1-iminosugar blocked the expression of a fucosylated epitope (mAb 128C3/3 antigen) that is associated with the pathogenesis of schistosomiasis.     

Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseadish peroxidase epitope

For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue of Drosophila melanogaster. This epitope is considered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity. Here we report that N-glycan core alpha1,3-linked fucose is, as judged by preabsorption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody. Further, we describe the identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and high performance liquid chromatography of two Drosophila N-glycans that, as already detected in other insects, carry both alpha1,3- and alpha1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding alpha1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core alpha1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core alpha1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF6; Km 11 microm) over the same structure lacking a core fucose residue (GnGn; Km 46 microm). The Drosophila core alpha1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrin in vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody.     

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.     

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.     

Characteristic structural features of schistosome cercarial N-glycans: expression of Lewis X and core xylosylation

Schistosomal egg N-glycans are the only examples in nature that have been structurally shown to contain beta2-xylosylation, alpha6-fucosylation, and alpha3-fucosylation on the N,N'-diacetyl chitobiose core. We present evidence that core difucosylated and xylosylated N-glycans are characteristics of Schistosoma japonicum eggs but not of the cercariae and adults, for which neither core xylosylation nor alpha3-fucosylation could be readily detected. In contrast, a majority of the N-glycans from Schistosoma mansoni cercariae but not the adults are core xylosylated. Tandem mass spectrometry analysis coupled with chromatographic mapping, sequential exoglycosidase digestion, and methylation analysis were employed to unambiguously define the structures of core beta2-xylosylated, alpha6-fucosylated N-glycans from S. mansoni cercariae. Unexpectedly, a majority of these N-glycans were found to carry Lewis X determinant, Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->, on the nonreducing termini of mono- and biantennary structures. The Lewis X-containing glycoproteins were found to be distinct from those carrying the complex, multifucosylated glycocalyx O-glycans reported previously. The corresponding N-glycans from S. japonicum cercariae are likewise dominated by Lewis X termini but without the core xylosylation. We concluded that the invading cercariae present an important and abundant source of Lewis X antigens, which may contribute to the induced humoral response upon infection. Following transformation and development into the adults, the N-glycans synthesized comprise a significantly larger amount of high mannose and fucosylated pauci-mannose structures in comparison with the cercarial N-glycans. A portion of the mono- and biantennary complex types were identified to carry Lewis X and fucosylated LacdiNAc termini, which could also be detected by mass spectrometry analysis on larger, complex-type structures.     

Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic alpha3-fucosylation of pure polylactosamines

We report that isomeric monofucosylhexasaccharides, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4 GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, and bifucosylhexasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 (Fucalpha1-3)GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4( Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc with GDP-fucose and alpha1,3-fucosyltransferases of human milk. The pure isomers were characterized in several ways;1H-NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Galbeta1-4(Fucalpha1-3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin. Isomeric monofucosyl derivatives of the pentasaccharides GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1- 4Gl cNAc and Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcN Ac and the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc were also obtained in pure form, implying that the methods used are widely applicable. The isomeric Lewis x glycans proved to be recognized in highly variable binding modes by polylactosamine-metabolizing enzymes, e.g., the midchain beta1,6-GlcNAc transferase (Lepp?nen et al., Biochemistry, 36, 13729-13735, 1997).     

N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system

N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then used to infect Ld652Y cells, and the recombinant protein was harvested at 120 h postinfection. N-glycans were released from the purified recombinant human serum transferrin and derivatized with 2-aminopyridine; the glycan structures were analyzed by a two-dimensional HPLC and MALDI-TOF MS. Structures of 11 glycans (88.8% of total N-glycans) were elucidated. The glycan analysis revealed that the most abundant glycans were Man1-3(+/-Fucalpha6)GlcNAc2 (75.5%) and GlcNAcMan3(+/-Fucalpha6)GlcNAc2 (7.4%). There was only approximately 6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained alpha(1,6)-fucosylation on the Asn-linked GlcNAc residue. However alpha(1,3)-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected. Inclusion of fetal bovine serum in culture media had little effect on the N-glycan structures of the recombinant human serum transferrin obtained.     

Molecular basis of the differences in binding properties of the highly related C-type lectins DC-SIGN and L-SIGN to Lewis X trisaccharide and Schistosoma mansoni egg antigens

The dendritic cell-specific C-type lectin DC-SIGN functions as a pathogen receptor that recognizes Schistosoma mansoni egg antigens through its major glycan epitope Galbeta1,4(Fucalpha1,3)GlcNAc (Lex). Here we report that L-SIGN, a highly related homologue of DC-SIGN found on liver sinusoidal endothelial cells, binds to S. mansoni egg antigens but not to the Lex epitope. L-SIGN does bind the Lewis antigens Lea, Leb, and Ley, similar as DC-SIGN. A specific mutation in the carbohydrate recognition domain of DC-SIGN (V351G) abrogates binding to all Lewis antigens. In L-SIGN Ser363 is present at the corresponding position of Val351 in DC-SIGN. Replacement of this Ser into Val resulted in a "gain of function" L-SIGN mutant that binds to Lex, and shows increased binding to the other Lewis antigens. These data indicate that Val351 is important for the fucose specificity of DC-SIGN. Molecular modeling and docking of the different Lewis antigens in the carbohydrate recognition domains of L-SIGN, DC-SIGN, and their mutant forms, demonstrate that Val351 in DC-SIGN creates a hydrophobic pocket that strongly interacts with the Fucalpha1,3/4-GlcNAc moiety of the Lewis antigens. The equivalent amino acid residue Ser363 in L-SIGN creates a hydrophilic pocket that prevents interaction with Fucalpha1,3-GlcNAc in Lex but supports interactions with the Fucalpha1,4-GlcNAc moiety in Lea and Leb antigens. These data demonstrate for the first time that DC-SIGN and L-SIGN differ in their carbohydrate binding profiles and will contribute to our understanding of the functional roles of these C-type lectin receptors, both in recognition of pathogen and self-glycan antigens.     

Fuc-TIX: a versatile alpha1,3-fucosyltransferase with a distinct acceptor- and site-specificity profile

alpha1,3-Fucosyltransferases (Fuc-Ts) convert N-acetyllactosamine (LN, Galbeta1-4GlcNAc) to Galbeta1-4(Fucalpha1-3)GlcNAc, the Lewis x (CD15, SSEA-1) epitope, which is involved in various recognition phenomena. We describe details of the acceptor specificity of alpha1,3-fucosyltransferase IX (Fuc-TIX). The unconjugated N- and O-glycan analogs LNbeta1-2Man, LNbeta1-6Manalpha1-OMe, LNbeta1-2Manalpha1-3(LNbeta1-2Manalpha1-6)Manbeta1-4GlcNAc, and Galbeta1-3(LNbeta1-6)GalNAc reacted well in vitro with Fuc-TIX present in lysates of appropriately transfected Namalwa cells. Fuc-TIX reacted well with the reducing end LN of GlcNAcbeta1-3'LN (underscored site reacted) and GlcNAcbeta1-3'LNbeta1-3'LN (both LNs reacted), but very poorly with the reducing end LN of LNbeta1-3'LN. However, Fuc-TIX reacted significantly better with the non-reducing end LN as compared to the other LN units in the glycans LNbeta1-3'LN and LNbeta1-3'LNbeta1-3'LNbeta1-3'LN, confirming our previous data on LNbeta1-3'LNbeta1-OR. In contrast, the sialylated glycan Neu5Acalpha2-3'LNbeta1-3'LNbeta1-3'LNbeta1-3'LN was fucosylated preferentially at the two most reducing end LN units. We conclude that Fuc-TIX is a versatile alpha1,3-Fuc-T, that (1) generates distal Lewis x epitopes from many different acceptors, (2) possesses inherent ability for the biosynthesis of internal Lewis x epitopes on growing polylactosamine backbones, and (3) fucosylates the remote internal LN units of alpha2,3-sialylated i-type polylactosamines.     

Formation of the immunogenic α1,3-fucose epitope: elucidation of substrate specificity and of enzyme mechanism of core fucosyltransferase A

Glycans of glycoproteins are often associated with IgE mediated allergic immune responses. Hymenoptera venoms, e.g., carry α1,3-fucosyl residues linked to the proximal GlcNAc of glycoproteins. This epitope, formed selectively by α1,3-fucosyltransferase (FucTA), is xenobiotic and as such highly immunogenic and it also shows cross-reactivity if present on different proteins. Production of post-translationally modified proteins in insect cells is however commonly used and, thus, resulting glycoproteins can carry this highly immunogenic epitope with potentially significant side effects on mammals. To analyze mechanism, specificity and reaction kinetics of the key enzyme, we chose FucTA from Apis mellifera (honeybee) and characterized it by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR) experiments. Specifically, we show here that the donor substrate, GDP-Fucose, binds mostly via its guanine and less so via pyrophosphate and fucosyl fragments and has a K_D = 37 μM. Affinity and kinetic studies with both the core α1,6-fucosylated and the unfucosylated octa- or heptasaccharides, respectively, as acceptor substrate revealed that honeybee FucTA prefers the latter structure with affinities of K_D ～ 10 mM. Establishment of progress curve analysis using an explicit solution of the integrated Michaelis–Menten equation allowed for determination of key constants of the transfer reaction of the glycosyl residue. The dominant minimum acceptor substrate is an unfucosylated heptasaccharide with K_m = 420 μM and k_cat = 6 min^?1. Time-resolved NMR spectra as well as STD NMR allow molecular insights into specificity, activity and interaction of the enzyme with substrates and acceptors.     

Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen

In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.