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.
Rendic
Dubravko
D
Department für Chemie and Department für Biotechnologie, Universität für Bodenkultur, Muthgasse 18, Wien A-1190, Austria. bubravko.rendic@boku.ac.at
Linder
Angela
A
Paschinger
Katharina
K
Borth
Nicole
N
Wilson
Iain B H
IB
Fabini
Gustáv
G
D000818:Animals; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002460:Cell Line; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004331:Drosophila melanogaster; D000939:Epitopes / Q000737:chemistry*; D005434:Flow Cytometry; D005647:Fucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D005822:Genetic Vectors; D006735:Horseradish Peroxidase / Q000737:chemistry; D007313:Insecta; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D016366:Open Reading Frames; D011134:Polysaccharides; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014162:Transfection
D002241:Carbohydrates; D018076:DNA, Complementary; D000939:Epitopes; D011134:Polysaccharides; D012333:RNA, Messenger; D006735:Horseradish Peroxidase; D005647:Fucosyltransferases