The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing.
Most processed, e.g. fucosylated, N-glycans on insect glycoproteins terminate in mannose, yet the relevant modifying enzymes require the prior action of N-acetylglucosaminyltransferase I. This led to the hypothesis that a hexosaminidase acts during the course of N-glycan maturation. To determine whether the Drosophila melanogaster genome indeed encodes such an enzyme, a cDNA corresponding to fused lobes (fdl), a putative beta-N-acetylglucosaminidase with a potential transmembrane domain, was cloned. When expressed in Pichia pastoris, the enzyme exhibited a substrate specificity similar to that previously described for a hexosaminidase activity from Sf-9 cells, i.e. it hydrolyzed exclusively the GlcNAc residue attached to the alpha1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl-beta-glucosaminide, but not chitooligosaccharides; in contrast, Drosophila HEXO1 and HEXO2 expressed in Pichia cleaved both these substrates but not N-glycans. The localization of recombinant FDL tagged with green fluorescent protein in Drosophila S2 cells by immunoelectron microscopy showed that this enzyme transits through the Golgi, is present on the plasma membrane and in multivesicular bodies, and is secreted. Finally, the N-glycans of two lines of fdl mutant flies were analyzed by mass spectrometry and reversed-phase high-performance liquid chromatography. The ratio of structures with terminal GlcNAc over those without (i.e. paucimannosidic N-glycans) was drastically increased in the fdl-deficient flies. Therefore, we conclude that the fdl gene encodes a novel hexosaminidase responsible for the occurrence of paucimannosidic N-glycans in Drosophila.
Léonard
Renaud
R
Glycobiology Group, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria. renaud.leonard@boku.ac.at
Rendic
Dubravko
D
Rabouille
Catherine
C
Wilson
Iain B H
IB
Préat
Thomas
T
Altmann
Friedrich
F
D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics* / Q000502:physiology*; D004331:Drosophila melanogaster; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D010843:Pichia / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D018076:DNA, Complementary; D029721:Drosophila Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000118:Acetylglucosaminidase; C509916:fdl protein, Drosophila