Abstract
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.
Highlights
Polyclonal antibodies raised against the plant glycoprotein, horseradish peroxidase (HRP),3 were used for many years in the study of invertebrate neurobiology, information as to the exact structural basis for this cross-reaction was lacking [1,2,3,4]
A Drosophila Neuronal Cell Line Is Enriched in Difucosylated N-Glycans—Due to the challenges in collecting a sufficient amount of Drosophila heads to show by direct HPLC or MS analyses that core ␣1,3-linked fucose is enriched in the neural tissue, we decided to look for suitable Drosophila cell lines to examine the expression of this epitope
We made use of two different Drosophila cell lines in order to investigate the biological role of FucTA in vivo and to show that, of the four ␣1,3-fucosyltransferase homologues, only FucTA is indispensable in synthesis of the anti-HRP neuronal epitope in these cells
Summary
Polyclonal antibodies raised against the plant glycoprotein, horseradish peroxidase (HRP), were used for many years in the study of invertebrate neurobiology, information as to the exact structural basis for this cross-reaction was lacking [1,2,3,4]. Analyses showed that the contribution of xylose in anti-HRP binding to HRP was minimal [7] The latter contrasts with more recent data demonstrating that anti-HRP binds both xylose-substituted and fucose-substituted structures to a similar extent [8]. By examining N-glycans of these cell lines, performing knock-in and knock-down experiments on ␣1,3-fucosyltransferase homologues in the respective cell lines, and determining the tissue and stage specificity of the expression of all four ␣1,3-fucosyltransferase homologues, we have gained further evidence pointing to a key role for FucTA in the biosynthesis of the anti-HRP epitope in Drosophila cells and have demonstrated that the other three fucosyltransferase homologues are not responsible for the formation of anti-HRP epitopes in the neuronal cell line
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