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 ␣1,3-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 ␣1,3-and ␣1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding ␣1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core ␣1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core ␣1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF 6 ; K m 11 M) over the same structure lacking a core fucose residue (GnGn; K m 46 M). The Drosophila core ␣1,3-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.Glycoproteins from plants and invertebrates are often highly immunogenic and many antibodies (both IgG and IgE) directed against them bind to core ␣1,3-fucose and/or ␤1,2-xylose residues of their N-linked oligosaccharides (1-7); since these modifications are present across plant species and one or the other modification is present in a number of invertebrates, these antibodies are highly cross-reactive. Indeed, polyclonal antibodies to horseradish peroxidase (anti-HRP) 1 have been used for nearly two decades to specifically stain neurons, and their growth pathways, in Drosophila melanogaster (8 -11); similar staining has also been described in grasshopper (11), whereas in Caenorhabditis elegans 10% of neurons are stained by this antibody (12). A number of proteins in Drosophila, such as an Na ϩ ,K ϩ ATPase christened Nervana, a receptor tyrosine phosphatase, and cell adhesion molecules (e.g. fasciclin I and II, neurotactin and neuroglian), have been found to bind anti-HRP (9 -11, 13, 14); however, no data on their glycosylation have been described. A number of Drosophila nac (neurally altered carbohydrate) mutants have been described, which are defective in anti-HRP staining of adult flies and display wing morphological defects (15) and, when under cold stress, some minor behavioral and eye developmental abnormalities (9). Neither the affected gene(s) nor the underlying biochemical defect(s) have been identified.As compared with the amount o...

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.

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Neural pathways in invertebrates are often tracked using antihorseradish peroxidase, a cross-reaction due to the presence of core ␣1,3-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 ␣1,3/␣1,6-difucosylated N-glycans, a result that correlates with anti-horseradish peroxidase binding. Of all four ␣1,3-fucosyltransferase homologues previously identified, the core ␣1 Polyclonal antibodies raised against the plant glycoprotein, horseradish peroxidase (HRP), 3 were used for many years in the study of invertebrate neurobiology, although information as to the exact structural basis for this cross-reaction was lacking (1-4). More recently, neural anti-HRP staining has been suggested to be a characteristic of Ecdysozoa (5), one of the two newly redefined clades of protostomians. Thus, not only has anti-HRP been much used to track neurons in insects (especially Drosophila melanogaster), but there is also a report that 10% of the neurons of Caenorhabditis elegans are recognized by this reagent (6). The first hints as to the basis of this staining in insects were shown by its sensitivity to reagents that destroy carbohydrate moieties and its inhibition by bromelain glycopeptides (3), which carry N-glycans decorated with ␤1,2-xylose and core ␣1,3-fucose residues. The epitopes recognized by anti-HRP were partially revealed by analysis of N-glycans attached to horseradish peroxidase (7), indicating that the ␣1,6-linked mannose of the trimannosyl core and the core ␣1,3-linked fucose contribute to the recognition the most, whereas the presence of nonreducing terminal N-acetylglucosamines greatly reduces the reactivity of the anti-HRP. In this study, analyses also showed that the contribution of xylose in anti-HRP binding to HRP was minimal (7). However, the latter contrasts with more recent data demonstrating that anti-HRP binds both xylose-substituted and fucose-substituted structures to a similar extent (8). Nonetheless, the exact molecular basis of the neuronal anti-HRP staining in insects remained unclear. Thus, we began to re-examine this problem and found that only neoglycoconjugates containing native, and not defucosylated, bromelain glycopeptides inhibit this reaction, suggesting that core ␣1,3-fucose is part of the epitope recognized in flies. Furthermore, staining fly embryos with anti-bee venom, which contains antibodies directed against core ␣1,3-fucose, and not xylose, reproduces the same pattern as using anti-HRP. In addition, we detected core ␣1,3-/␣1,6-difucosylated N-glycans in adult flies and cloned a cDNA encoding an enzymatically active core ␣1,3-fucosyltransferase (FucTA) (9). Recently, we were also able to demonstrate that anti-HRP staining in C. elegans is due to the activity of FUT-1, a core ␣1,3-fucosyltransferase (10).A number of questions, however, rem...

The hY RNAs are a group of four small cytoplasmic RNAs of unknown function that are stably associated with at least two proteins, Ro60 and La, to form Ro ribonucleoprotein complexes. Here we show that the heterogeneous nuclear ribonucleoproteins (hnRNP) I and K are able to associate with a subset of hY RNAs in vitro and demonstrate these interactions to occur also in vivo in a yeast three-hybrid system. Experiments performed in vitro and in vivo with deletion mutants of hY1 RNA revealed its pyrimidine-rich central loop to be involved in interactions with both hnRNP I and K and clearly showed their binding sites to be different from the Ro60 binding site. Both hY1 and hY3 RNAs coprecipitated with hnRNP I in immunoprecipitation experiments performed with HeLa S100 extracts and cell extracts from COS-1 cells transiently transfected with VSV-G-tagged hnRNP-I, respectively. Furthermore, both anti-Ro60 and anti-La antibodies coprecipitated hnRNP I, whereas coprecipitation of hnRNP K was not observed. Taken together, these data strongly suggest that hnRNP I is a stable component of a subpopulation of Ro RNPs, whereas hnRNP K may be transiently bound or interact only with (rare) Y RNAs that are devoid of Ro60 and La. Given that functions related to translation regulation have been assigned to both proteins and also to La, our findings may provide novel clues toward understanding the role of Y RNAs and their respective RNP complexes. Small ribonucleoprotein (RNP)1 complexes are usually composed of one molecule of a small RNA and several proteins that bind either directly to the RNA or indirectly via protein-protein interactions (1, 2). Many of these complexes exert essential functions that are often indispensable for survival, such as the small nuclear RNPs, which are major components of the spliceosomal machinery, or the signal recognition particle, which plays a key role in protein export. In contrast to these well defined complexes, the structure of the cytoplasmic Ro RNPs is still not fully resolved, and their function has remained enigmatic (3, 4). They are composed of one molecule of a small Y RNA (transcribed by RNA polymerase III) and at least two proteins, the 60-kDa protein Ro60 and the 48-kDa phosphoprotein La. However, although Ro60 and Y RNAs are present in comparable stochiometric amounts, La is ϳ50-fold more abundant, and therefore the vast majority of La molecules is not bound to Y RNAs, in contrast to Ro60 (5). Y RNAs are highly conserved in evolution (6) and have been found in all multicellular eukaryotic organisms and may also be present in some bacteria (7) but, remarkably, have so far not been detected in yeast. Interestingly, the genome of the nematode Caenorhabditis elegans contains only one functional Y RNA gene, whereas in humans and other vertebrates four closely related Y RNA species exist. Unlike other RNA polymerase III transcripts, Y RNAs retain the oligo(U) stretch at their 3Ј end that forms the binding site for La; therefore these RNAs remain permanently associated with La (5,8). On the ot...