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

Abstract

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 β-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 α1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl-β-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. 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 β-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 α1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl-β-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. Insect cells are considered to be an interesting alternative to mammalian, yeast, or bacterial cells for the expression of recombinant proteins (1Altmann F. Staudacher E. Wilson I.B.H. März L. Glycoconj. J. 1999; 16: 109-123Crossref PubMed Scopus (283) Google Scholar, 2Morton C.L. Potter P.M. Mol. Biotechnol. 2000; 16: 193-202Crossref PubMed Scopus (89) Google Scholar), because the potentially high levels of expression are associated with the ability to perform many eukaryotic post-translational modifications. Nevertheless, glycoproteins produced in insects and insect cells have been mainly found to carry paucimannosidic N-glycans, i.e. glycans consisting of the pentasaccharide core with or without α1,6- and/or α1,3-linked fucose (1Altmann F. Staudacher E. Wilson I.B.H. März L. Glycoconj. J. 1999; 16: 109-123Crossref PubMed Scopus (283) Google Scholar, 3Tretter V. Altmann F. Kubelka V. März L. Becker W.M. Int. Arch. Allergy Immunol. 1993; 102: 259-266Crossref PubMed Scopus (195) Google Scholar, 4Marchal I. Jarvis D.L. Cacan R. Verbert A. Biol. Chem. 2001; 382: 151-159Crossref PubMed Scopus (153) Google Scholar, 5Fabini G. Freilinger A. Altmann F. Wilson I.B.H. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). In addition to the possible presence of the immunogenic core α1,3-linked fucose, the lack of complex type N-glycans with complete, sialylated antennae as found on mammalian glycoproteins makes insect cells unsuitable for the production of many therapeutic glycoproteins (6Tomiya N. Betenbaugh M.J. Lee Y.C. Acc. Chem. Res. 2003; 36: 613-620Crossref PubMed Scopus (49) Google Scholar). Only in a few cases have substitutions of the terminal mannose residues of insect N-glycans been described. The most common substitution is the presence of terminal GlcNAc on the α1,3-linked mannosyl residue (GlcNAc-1) 3The abbreviations used are: GlcNAc-1, the non-reducing terminal GlcNAc on the α1,3-linked mannosyl residue of the N-glycans core; MALDI, matrix-assisted laser desorption-ionization; TOF, time-of-flight; RT, reverse transcription; GlcNAc3-PA, GlcNAcβ1,4GlcNAcβ1,4GlcNAc-PA; HPLC, high-performance liquid chromatography; MS, mass spectrometry; GFP, green fluorescent protein; pNP, p-nitrophenyl; NACS, mannosamine, 6-acetamido-6-deoxycastanospermine. added by GlcNAc-transferase I, although in a few cases further modifications such as galactose, N-acetylgalactosamine, fucose, and aminoethylphosphonate (i.e. phosphorylethanolamine) have been found linked to this GlcNAc residue (7Wagner R. Geyer H. Geyer R. Klenk H.D. J. Virol. 1996; 70: 4103-4109Crossref PubMed Google Scholar, 8Hsu T.A. Takahashi N. Tsukamoto Y. Kato K. Shimada I. Masuda K. Whiteley E.M. Fan J.Q. Lee Y.C. Betenbaugh M.J. J. Biol. Chem. 1997; 272: 9062-9070Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Kubelka V. Altmann F. Staudacher E. Tretter V. März L. Hård K. Kamerling J.P. Vliegenthart J.F. Eur. J. Biochem. 1993; 213: 1193-1204Crossref PubMed Scopus (221) Google Scholar, 10Ogonah O.W. Freedman R.B. Jenkins N. Rooney B.C. Biochem. Soc. Trans. 1995; 23: 100SCrossref PubMed Google Scholar, 11Watanabe S. Kokuho T. Takahashi H. Takahashi M. Kubota T. Inumaru S. J. Biol. Chem. 2002; 277: 5090-5093Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The occurrence of larger and sialylated N-glycans has been reviewed elsewhere (1Altmann F. Staudacher E. Wilson I.B.H. März L. Glycoconj. J. 1999; 16: 109-123Crossref PubMed Scopus (283) Google Scholar, 4Marchal I. Jarvis D.L. Cacan R. Verbert A. Biol. Chem. 2001; 382: 151-159Crossref PubMed Scopus (153) Google Scholar). In most instances, however, the α1,3-linked mannosyl residue was found as terminal residue, even though the enzymes involved in the biosynthesis of other features of the glycan structures found in insects require the presence of GlcNAc-1. Indeed in Sf-9 cells, in the same way as previously found in mammals (12Schachter H. Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (492) Google Scholar), all the activities of relevant enzymes such as α-mannosidase II (13Altmann F. März L. Glycoconj. J. 1995; 12: 150-155Crossref PubMed Scopus (47) Google Scholar), α1,3-fucosyltransferase (14Staudacher E. März L. Glycoconj. J. 1998; 15: 355-360Crossref PubMed Scopus (38) Google Scholar), α1,6-fucosyltransferase (14Staudacher E. März L. Glycoconj. J. 1998; 15: 355-360Crossref PubMed Scopus (38) Google Scholar, 15Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar), and GlcNAc-transferase II (15Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar) were found to depend upon this GO signal; similar results have been obtained with recombinant forms of the Drosophila core α1,3- and α1,6-fucosyltransferases FucTA and FucT6 (5Fabini G. Freilinger A. Altmann F. Wilson I.B.H. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 16Paschinger K. Staudacher E. Stemmer U. Fabini G. Wilson I.B.H. Glycobiology. 2005; 15: 463-474Crossref PubMed Scopus (101) Google Scholar). Furthermore, the presence of significant levels of the GlcNAc-transferase I has been demonstrated in several insect cell lines (15Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar), whereas the Drosophila GlcNAc-transferase I cDNA has been cloned and shown to encode an active enzyme (17Sarkar M. Schachter H. Biol. Chem. 2001; 382: 209-217Crossref PubMed Scopus (48) Google Scholar). It was therefore hypothesized that in insect cells, as in plant cells, the GlcNAc-1 is cleaved off In the of it that of GlcNAc residues the of the protein in the of the plant cell A. M.J. J. Cell Biol. PubMed Scopus Google Scholar). it that in proteins in the lack the GlcNAc-1 residue, whereas in insects this residue is from the of those that are N. Lee Y.C. Betenbaugh M.J. Biotechnol. 2005; PubMed Scopus Google Scholar). hexosaminidase a and during the insect In in Sf-9 cells as and were to a in the F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar). It is that this enzyme not of N-glycan structures are in but further on and it GlcNAc-1 F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar). cell in the presence of hexosaminidase (7Wagner R. Geyer H. Geyer R. Klenk H.D. J. Virol. 1996; 70: 4103-4109Crossref PubMed Google Scholar, 11Watanabe S. Kokuho T. Takahashi H. Takahashi M. Kubota T. Inumaru S. J. Biol. Chem. 2002; 277: 5090-5093Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, I. Verbert A. Cacan R. Glycobiology. 1999; PubMed Scopus Google Scholar) or of recombinant E. Takahashi N. Tsukamoto Y. Masuda K. Jarvis D.L. Lee Y.C. Betenbaugh M.J. Glycobiology. 2000; PubMed Scopus Google Scholar, Jarvis D.L. Glycobiology. 1998; PubMed Scopus Google Scholar) the putative of a It is that this enzyme in to glycoproteins with in insect have been to this the of mammalian GlcNAc-transferase I was because of the with the most of the N-glycans not carry GlcNAc-1 R. S. E. Geyer H. Geyer R. Klenk H.D. Glycobiology. 1996; PubMed Scopus (68) Google Scholar). is to J. E. M. H. Jarvis D.L. 2002; PubMed Scopus Google Scholar), has the of the substrate for the or to the M. Biotechnol. 1999; 15: PubMed Scopus Google Scholar). the most be to or the hexosaminidase To perform of the it is to the relevant To this we have and expressed the of in the Furthermore, we have the substrate and localization of of we conclude to be the melanogaster flies used in this were of the The fdl to the of a was a for I. R. M. M. T. J. 2000; PubMed Scopus Google Scholar). The fdl S. J. A. H. 2003; PubMed Scopus Google Scholar), was a from A. H. and of from the putative Drosophila were found by for of hexosaminidase gene of the Drosophila genome The of the and transmembrane were and K. Biol. Chem. 1993; Scholar). was from Drosophila S2 cells and was the of FDL the and were for HEXO1 and the and or and were The were with the relevant enzymes and cloned the expression The and of the as as and of were by an and were analyzed and protein were and analyzed F. Res. 16: PubMed Scopus Google Scholar). The hexosaminidase were expressed in Pichia as previously described for M. Fabini G. E.M. Altmann F. Wilson I.B.H. 2003; PubMed Scopus Google Scholar). of of forms of the were with the substrates for with the substrate was in a of of The were by the addition of of and was with a were used a of in a of of was by the addition of of of were analyzed by as described previously (15Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar, F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar). of of and fdl mutant or were and with addition of of and for The of N-glycans was as previously described F. Fabini G. H. Wilson I.B.H. 2001; PubMed Scopus Google Scholar) of The glycans were and in prior to of the N-glycans by or was to S. S. S. T. J. Biochem. PubMed Scopus Google Scholar). In the of fdl were by as previously described F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar). were in and analyzed by both and N-glycans, was a as fdl cDNA was cloned with a the a of the a Drosophila S2 cells were the for cells with modifications. The cells cells were with of of and of was added to and cells were for by a was and the cells were in in and for immunoelectron microscopy as described Cell Biol. 1996; PubMed Scopus Google Scholar). to were on an and the by protein to and of Drosophila hypothesis was that the putative insect hexosaminidase be to we of the Drosophila genome and found and of the and these have been previously to the in the A. Biochem. J. 1993; PubMed Scopus Google Scholar). The two have been previously HEXO1 and by on and and to be most to a β-N-acetylglucosaminidase from Y. I. M. E. Biotechnol. Biochem. 1995; PubMed Scopus Google Scholar) and to two proteins with both β-N-acetylglucosaminidase and activity from Drosophila F. M. M. Insect Biochem. Mol. Biol. 2002; PubMed Scopus Google Scholar). The is by the fdl on because of the fused lobes of the corresponding Drosophila mutant I. R. M. M. T. J. 2000; PubMed Scopus Google Scholar). all were by from from Drosophila S2 The were of the and the and the The fdl cDNA a a protein of The of the cDNA with that of the corresponding genome that the fdl the other and a common with and both encode proteins of the the Drosophila FDL is to be a type II transmembrane and with the HEXO1 and and with the HEXO2 The by that both HEXO1 and FDL are plasma membrane whereas HEXO2 is most to be secreted. that insect are two The is of enzymes to the β-N-acetylglucosaminidase from Y. I. M. E. Biotechnol. Biochem. 1995; PubMed Scopus Google Scholar) and as as The the of for we from and the all the putative insect FDL is most similar to the and over a of insect hexosaminidase are from the mammalian J. Biol. Chem. Full Text PDF PubMed Google Scholar) and even from the of previously in I. S. Y. K. H. K. Biotechnol. Biochem. 2003; PubMed Scopus Google of the hexosaminidase The protein were the and to a putative hexosaminidase putative melanogaster melanogaster hexosaminidase melanogaster hexosaminidase from and H. hexosaminidase H. hexosaminidase hexosaminidase M. hexosaminidase and T. hexosaminidase of of the Drosophila the residues (i.e. the putative and transmembrane was by and the was cloned the prior to the two other Drosophila HEXO1 and were also expressed as forms in the and In an the of were for hexosaminidase activity and indeed all this of FDL was also active expressed in insect cells, as by a in the of and in to cells not To determine the the activities of and FDL were by reversed-phase with N-glycans, such as for on N. Lee Y.C. T. Y. J. M. Takahashi N. Biochem. PubMed Scopus Google Scholar), the of the GlcNAc residue from the to is to in a to whereas of the residue from the to a in as with of of FDL with this a of be that with a the was the other the of was not the of was used in the that the is to the expressed Furthermore, is by FDL of or from specificity and ability to exclusively the GlcNAc linked to the residue of the core mass was used to that a GlcNAc residue was by a as a in of is to GlcNAc residue, was not these results that the recombinant Drosophila FDL the GlcNAc residue linked to the from the This is with the on the insect hexosaminidase F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar). in to on the Sf-9 hexosaminidase FDL not as by an as This be to the presence of a hexosaminidase activity in the present therefore that FDL is not involved in the other both HEXO1 and HEXO2 that N-glycans are not substrates for HEXO1 or HEXO2 not This was to the hexosaminidase Y. I. M. E. Biotechnol. Biochem. 1995; PubMed Scopus Google Scholar). a of the that FDL the activity most of the hexosaminidase from cells, we on this The recombinant FDL for ability to an of substrates such as and the was found to be the most by whereas was not that FDL not that FDL the prior action of whereas a was a substrate not This that core the presence of GlcNAc-1, not the action of When an substrate i.e. was the was that a N-glycan this is also with the on the insect hexosaminidase F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar) and is to the of the FDL not on not and therefore to have it not to to the previously from Drosophila cells or Drosophila both and activities F. M. M. Insect Biochem. Mol. Biol. 2002; PubMed Scopus Google Scholar, U. Arch. Insect Biochem. PubMed Scopus Google Scholar, U. Arch. Insect Biochem. PubMed Scopus Google Scholar). the and of FDL were with as the substrate not by be whereas FDL was by and hexosaminidase and were also for on the recombinant The obtained with and are and is a of This is with the of the hexosaminidase from Sf-9 cells by F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar). FDL is also by mannosamine, and of results and that of is obtained for a of This is the previously used to glycans with antennae on glycoproteins M. Biotechnol. 1999; 15: PubMed Scopus Google Scholar). of FDL by all in the we to determine the localization of FDL by a of the protein in Drosophila S2 cells of an results with microscopy showed a but of the with the the localization of the protein was by immunoelectron The cells were and processed, and were an by protein to The protein is found in all of the the and the but a on the plasma membrane and in multivesicular and In the high of that the protein is to a the This is with the that FDL transits through the to the plasma membrane and is not a enzyme of this in cells a high of the not an and this with on such as The FDL localization in the from to the the through the or from the of FDL to both the plasma membrane and the of from and whether FDL was responsible for the of Drosophila N-glycans to the paucimannosidic type in N-glycan were on flies with a or in the fdl The N-glycans by from obtained of the were to mass spectrometry and in the of to not of and fdl mutant flies The of glycans from the of the N-glycan were by that was by to the and were on from The glycan structures are in other structures be not on for on previously for on for on previously for on for on previously for on for on previously for on for on previously for on for on previously for on for on previously for and complex on for on for fdl and on previously for not on for on previously for (5Fabini G. Freilinger A. Altmann F. Wilson I.B.H. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) in a The N-glycan obtained with these were with the of the flies previously described (5Fabini G. Freilinger A. Altmann F. Wilson I.B.H. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, PubMed Scopus Google Scholar), the structures present in flies are of paucimannosidic or type and indeed for of the N-glycans. the other in the fdl the occurrence of paucimannosidic N-glycans from to although is in the of the same the of N-glycans in the fdl from to of the the is by not Furthermore, in this complex glycans two GlcNAc were not in of the of N-glycans are in the the fdl gene as as and of are to have a in N-glycan In addition to a of the the of paucimannosidic structures in this mutant is as with flies the glycan that FDL has an in the biosynthesis of the N-glycans in Drosophila. The a of from and associated with of the two most are the mammalian by the and of are associated with the and and in 1999; PubMed Scopus Google Scholar). In are of this in of the corresponding have been previously demonstrated I. R. M. M. T. J. 2000; PubMed Scopus Google Scholar) to of the the the were fused in mutant for the same gene has been M. Biotechnol. 1999; 15: PubMed Scopus Google Scholar). to the the for this mutant be mainly to gene and be from this mutant the of an fdl Nevertheless, as shown in the present FDL is a hexosaminidase with a on as by the of structures present in fdl we it has a during the the of N-glycans. N-glycan also has a in is shown by the of in and in a of and J. J. J. Schachter H. J. 1996; Google Scholar, Y. Schachter H. 2002; PubMed Scopus Google Scholar, M. V. N. F. L. M. L. J. 2001; PubMed Scopus Google Scholar). The of the fdl as as the of a II mutant G. L. J. 2001; PubMed Google Scholar), to that N-glycan is also for in the fdl mutant was i.e. activity I. R. M. M. T. J. 2000; PubMed Scopus Google Scholar), and it was to whether a mutant a of the for fdl that mutant was also to have a in this a in the N-glycan be the flies and the an of paucimannosidic structures flies. Nevertheless, even in this paucimannosidic structures in this mutant the of the fdl the of structures from a or plasma hexosaminidase activity in or all The also be the of an alternative the enzyme previously described in insect K. Jarvis D.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). from the previously described Drosophila α-mannosidase II A. R. T. M. J. Cell 1999; PubMed Google Scholar), Drosophila enzyme, α-mannosidase has been but not further by and is a for the of The that by is is not with the previously obtained with recombinant forms of the two Drosophila core FucTA and FucT6 (5Fabini G. Freilinger A. Altmann F. Wilson I.B.H. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 16Paschinger K. Staudacher E. Stemmer U. Fabini G. Wilson I.B.H. Glycobiology. 2005; 15: 463-474Crossref PubMed Scopus (101) Google Scholar) but be in because such a core α1,3-fucosyltransferase in the K. G. V. Wilson I.B.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the and insect is the presence of the paucimannosidic N-glycans structures in the This type of is also present in A. 2003; PubMed Scopus Google Scholar), and indeed a has been in S. S. Staudacher E. Schachter H. Biochem. J. 2003; PubMed Google Scholar). structures are a common of plant N-glycans, but in the occurrence of complex structures the a is common V. M. M. L. J. 1997; 12: PubMed Scopus Google Scholar). In it that the of plant N-glycans to the paucimannosidic type is a glycoproteins to the A. M.J. J. Cell Biol. PubMed Scopus Google Scholar, M. V. L. Mol. Biol. 1998; PubMed Google Scholar). Therefore, it is that the plant involved in this is in the or in a from the the in the hexosaminidase activity previously F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar), we have as is not in the The localization of FDL was by of and this an in such N. F. R. G. T. J. Cell 1995; PubMed Google Scholar, K. L. H. S. 2000; PubMed Scopus Google Scholar, S. M. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Jarvis D.L. Glycobiology. 2002; 12: PubMed Scopus (38) Google Scholar). we both proteins showed the same i.e. that FDL mainly to the plasma the and the and to a in the these from the from the F. H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (195) Google Scholar), we other the i.e. the of glycoproteins through the is to the action of This is of for because recombinant therapeutic glycoproteins produced in insect cells are the cell and therefore with FDL and This be an as to the of recombinant proteins expressed in insect cells paucimannosidic structures (1Altmann F. Staudacher E. Wilson I.B.H. März L. Glycoconj. J. 1999; 16: 109-123Crossref PubMed Scopus (283) Google Scholar, N. Lee Y.C. Betenbaugh M.J. Biotechnol. 2005; PubMed Scopus Google Scholar, R. H. K. I. T. T. Biochem. Res. 2003; PubMed Scopus Google Scholar, H. M. V. 2003; PubMed Scopus Google Scholar). The of insect cells to complex N-glycans has been a of that the genome of Drosophila encodes two N. Glycobiology. 2005; 15: PubMed Scopus Google Scholar) and a K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google whereas has a that in in the of complex N-glycans N. Jarvis D.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to is for such structures in flies complex structures such as are found on glycoproteins V. Altmann F. Staudacher E. Tretter V. März L. Hård K. Kamerling J.P. Vliegenthart J.F. Eur. J. Biochem. 1993; 213: 1193-1204Crossref PubMed Scopus (221) Google to the in of recombinant glycoproteins produced in insect cells and those from mammalian several to insect cell for the production of therapeutic glycoproteins have been of the in this has been to the of structures a GlcNAc residue linked to the of the N-glycan in further of this To this several have been from the of S. Kokuho T. Takahashi H. Takahashi M. Kubota T. Inumaru S. J. Biol. Chem. 2002; 277: 5090-5093Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) to the expression of mammalian of of and to the antennae to of GlcNAc-1 by (6Tomiya N. Betenbaugh M.J. Lee Y.C. Acc. Chem. Res. 2003; 36: 613-620Crossref PubMed Scopus (49) Google Scholar, J. E. M. H. Jarvis D.L. 2002; PubMed Scopus Google Scholar). the N-glycan of the Drosophila fdl mutant in the of the of the enzyme mainly responsible for the occurrence of paucimannosidic structures not to the of N-glycan structures with the in structures GlcNAc-1 that the of FDL expression to be an way the of N-glycans in insect The of and in fdl mutant flies that it is not to GlcNAc-transferase I insect the of FDL is also to the of GlcNAc-transferase II and enzymes in insect Therefore, we conclude that of the for the of GlcNAc-1 in insect cell lines with N-glycans. A. H. for with the and for on cell and the activity with and and for in with the N-glycan and for flies. The Drosophila S2 cells were by from and for with the and of for and for on the of S2