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J. Biol. Chem., Vol. 281, Issue 8, 4867-4875, February 24, 2006

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The Drosophila fused lobes Gene Encodes an N-Acetylglucosaminidase Involved in N-Glycan Processing^*

Renaud Léonard¹², Dubravko Rendi¹, Catherine Rabouille, Iain B. H. Wilson, Thomas Préat^¶, and Friedrich Altmann

From the Glycobiology Group, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria, the Department of Cell Biology and Institute of Biomembranes, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands, and the ^¶Développement, Evolution, Plasticité du Système Nerveux, CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France

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 -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 1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl--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.

Received for publication, October 11, 2005 , and in revised form, November 28, 2005.

^* This work was supported by the Austrian Science Fund (Grants S8803 to F. A. and P17681 [GenBank] to I. B. H. W.) and by a Glycoscience Research Award from Neose Technologies (to I. B. H. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors have contributed equally to this work.

² To whom correspondence should be addressed: Tel.: 43-1-36006-6065; Fax: 43-1-36006-6059; E-mail: renaud.leonard{at}boku.ac.at.

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