Insect Cells Contain an Unusual, Membrane-bound β-N-Acetylglucosaminidase Probably Involved in the Processing of Protein N-Glycans

Abstract

The β-N-acetylglucosaminidase activity in the lepidopteran insect cell line Sf21 has been studied using pyridylaminated oligosaccharides and chromogenic synthetic glycosides as substrates. Ultracentrifugation experiments indicated that the insect cell β-N-acetylglucosaminidase exists in a soluble and a membrane-bound form. This latter form accounted for two-thirds of the total activity and was associated with vesicles of the same density as those containing GlcNAc-transferase I. Partial membrane association of the enzyme was observed with all substrates tested, i.e. 4-nitrophenyl β-N-acetylglucosaminide, tri-N-acetylchitotriose, and an N-linked biantennary agalactooligosaccharide. Inhibition studies indicated a single enzyme to be responsible for the hydrolysis of all these substrates. With the biantennary substrate, the β-N-acetylglucosaminidase exclusively removed β-N-acetylglucosamine from the α1,3-antenna. GlcNAcMan5GlcNAc2, the primary product of GlcNAc-transferase I, was not perceptibly hydrolyzed.β-N-Acetylglucosaminidases with the same branch specificity were also found in the lepidopteran cell lines Bm-N and Mb-0503. In contrast, β-N-acetylglucosaminidase activities from rat or frog (Xenopus laevis) liver and from mung bean seedlings were not membrane-bound, and they did not exhibit a strict branch specificity. An involvement of this unusual β-N-acetylglucosaminidase in the processing of asparagine-linked oligosaccharides in insects is suggested. The β-N-acetylglucosaminidase activity in the lepidopteran insect cell line Sf21 has been studied using pyridylaminated oligosaccharides and chromogenic synthetic glycosides as substrates. Ultracentrifugation experiments indicated that the insect cell β-N-acetylglucosaminidase exists in a soluble and a membrane-bound form. This latter form accounted for two-thirds of the total activity and was associated with vesicles of the same density as those containing GlcNAc-transferase I. Partial membrane association of the enzyme was observed with all substrates tested, i.e. 4-nitrophenyl β-N-acetylglucosaminide, tri-N-acetylchitotriose, and an N-linked biantennary agalactooligosaccharide. Inhibition studies indicated a single enzyme to be responsible for the hydrolysis of all these substrates. With the biantennary substrate, the β-N-acetylglucosaminidase exclusively removed β-N-acetylglucosamine from the α1,3-antenna. GlcNAcMan5GlcNAc2, the primary product of GlcNAc-transferase I, was not perceptibly hydrolyzed. β-N-Acetylglucosaminidases with the same branch specificity were also found in the lepidopteran cell lines Bm-N and Mb-0503. In contrast, β-N-acetylglucosaminidase activities from rat or frog (Xenopus laevis) liver and from mung bean seedlings were not membrane-bound, and they did not exhibit a strict branch specificity. An involvement of this unusual β-N-acetylglucosaminidase in the processing of asparagine-linked oligosaccharides in insects is suggested. Mammalian cells contain in their lysosomes β-N-acetylglucosaminidase (GlcNAcase)1 1The abbreviations used are: GlcNAcaseN-acetyl-β-glucosaminidasepNP-GlcNAc4-nitrophenyl N-acetyl-β-glucopyranosideMU-GlcNAc4-methylumbelliferyl N-acetyl-β-glucopyranosideHPLChigh pressure liquid chromatographyPApyridylaminoGnN-acetylglucosamineMmannoseFfucoseGlcNAc3tri-N-acetylchitotrioseNACS6-acetamido-6-deoxycastanospermineEndo Lendo-N-acetylglucosaminidase from lepidopteran cells. See Table I for structures of GnGn, MGn, GnM, MM, M5Gn, and GnGnF6N-glycans. activity, which serves a vital role in the degradation of various glycoconjugates. GlcNAcases can also been found in a variety of plant seeds, where they may serve a similar task. Insect cells have so far not been analyzed for GlcNAcase activity, although the current knowledge of the structures and biosynthesis of asparagine-linked oligosaccharides in insects or insect cells creates a strong impetus for such an investigation. Glycoproteins produced in insects or cultured insect cells mostly contain N-glycans of either the oligomannosidic or the “paucimannosidic” type(1März L. Altmann F. Staudacher E. Kubelka V. Montreuil J. Schachter H. Vliegenthart J.F.G. Protein Glycosylation in Insects. Elsevier Science Publishers B. V., Amsterdam1995Google Scholar, 2Kubelka V. Altmann F. Kornfeld G. März L. Arch. Biochem. Biophys. 1994; 308: 148-157Crossref PubMed Scopus (128) Google Scholar). The latter type contains three (or even only two) mannosyl residues and frequently fucose attached to the asparagine-linked GlcNAc, whereby this fucose may also be α1,3-linked as known for plant glycoproteins. Such structures are not consistent with the classical N-glycan processing pathway established for mammalian cells (3Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar) since in mammals both α-mannosidase II and fucosyltransferase require the presence of GlcNAc-1, which is transferred by GlcNAc-transferase I (Fig. 1)(4Longmore G.D. Schachter H. Carbohydr. Res. 1982; 100: 365-392Crossref PubMed Scopus (156) Google Scholar, 5Tulsiani D.R.P. Hubbard S.C. Robbins P.W. Touster O. J. Biol. Chem. 1982; 257: 3660-3668Abstract Full Text PDF PubMed Google Scholar). In principle, the fucosylated paucimannose structures could be explained by a different specificity of both the α-mannosidase removing the two terminal mannosyl residues from the α1,6-branch and the fucosyltransferase(s) acting on the innermost GlcNAc. However, four recent observations make this hypothesis unlikely. (i) Insect cells do contain appreciable GlcNAc-transferase I activity(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar, 7Velardo M.A. Bretthauer R.H. Boutaud A. Reinhold B. Reinhold V.N. Castellino F.J. J. Biol. Chem. 1993; 268: 17902-17907Abstract Full Text PDF PubMed Google Scholar). (ii) The fucosyltransferase(s) of insect cells do not act upon Man3GlcNAc2(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar). (iii) α-Mannosidase II from insect cells is also strictly dependent on the presence of GlcNAc-1 (8Altmann F. März L. Glycoconjugate J. 1995; 12: 150-155Crossref PubMed Scopus (47) Google Scholar). (iv) GlcNAc-2 is transferred to the α1,6-antenna only after the action of GlcNAc-transferase I(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar). Therefore, it may be speculated that the biosynthesis in insects largely follows the “classical” pathway, but instead of galactosylation and further chain elongations, GlcNAc-1 is removed by a dedicated enzyme: a “processing”β-N-acetylglucosaminidase. Most of the insect glycoproteins investigated were secretory or plasma membrane proteins. Therefore, an enzyme involved in the processing of these glycoproteins must act at some point along the normal transport route of such proteins. This appears to be a situation different from that encountered by legume storage proteins, which were shown to experience removal of a “transient” GlcNAc residue in protein bodies(9Vitale A. Chrispeels M.J. J. Cell Biol. 1984; 99: 133-140Crossref PubMed Scopus (112) Google Scholar). In mammals, most processing enzymes such as GlcNAc-transferase I, α-mannosidase II, galactosyltransferase, sialyltransferase, and others are confined to one or more compartments of the Golgi apparatus, where they are anchored by a transmembrane domain(10Joziasse D.H. Glycobiology. 1992; 2: 271-277Crossref PubMed Scopus (117) Google Scholar, 11Moreman K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (324) Google Scholar), whereas catabolic enzymes exist in the lysosomes as soluble proteins. N-acetyl-β-glucosaminidase 4-nitrophenyl N-acetyl-β-glucopyranoside 4-methylumbelliferyl N-acetyl-β-glucopyranoside high pressure liquid chromatography pyridylamino N-acetylglucosamine mannose fucose tri-N-acetylchitotriose 6-acetamido-6-deoxycastanospermine endo-N-acetylglucosaminidase from lepidopteran cells. See Table I for structures of GnGn, MGn, GnM, MM, M5Gn, and GnGnF6N-glycans. Here we characterize a membrane-bound GlcNAcase from Sf21 cells and other insect cell lines. The enzyme is compared with GlcNAcase activities in plant and vertebrate tissues. Cells of the strains IPLB-Sf21AE, Bm-N, and Mb-0503 as well as human HepG2 cells were grown and harvested as described(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar). Typically, insect cells (10 × 106/ml) were homogenized in the cold by sonication (3 × 10 s at 30 watts) in isotonic buffer (5 mM imidazole HCl buffer at pH 7.3 containing 250 mM sucrose). All experiments were done with this whole cell homogenate if not otherwise stated. For ultracentrifugation experiments, this homogenate was centrifuged at 2000 × g for 10 min. When centrifugation experiments were performed with additional agents such as NaCl or Triton X-100, these were included before sonication. In an alternative, more gentle procedure, 50 × 106 Sf21 cells were suspended in 2 ml of hypotonic buffer (1 volume isotonic buffer and 1 volume of water). After 1 h at 4°C, the cells were disrupted using a Potter-Elvehjem homogenizer equipped with a tight fitting pestle for a total of 5 min. Only about one-half of the cells were broken up by this treatment as judged by light microscopy. The pellet obtained upon centrifugation at 2000 × g was therefore again subjected to this procedure. The 2000 × g supernatants were combined and subjected to ultracentrifugation. Microsomes were obtained by centrifugation for 40 min in a Beckman Ti-50 rotor at 40,000 rpm (∼110,000 × g). The pellet was gently suspended in isotonic buffer by a few strokes of a Dounce homogenizer. Part of this microsome preparation was subjected to density gradient centrifugation using the Ti-50 rotor at 47,000 rpm for 90 min with a stepwise gradient from 10 to 45% (w/w) sucrose in 5 mM imidazole HCl buffer at pH 7.3. The density of the fractions obtained was assessed by refractometry. Mung beans were soaked in tap water overnight and germinated at 37°C using a dedicated ceramic apparatus. Seedlings as well as Xenopus laevis and rat livers were homogenized in isotonic buffer by three 10-s bursts in an IKA-Ultra Turrax homogenizer containing 0.5 mM 2-mercaptoethanol in the case of the bean seedlings. Separation of membrane-bound and soluble proteins was performed as described above. Purified GlcNAcases from beef kidney and jack beans were obtained from Sigma. 4-Nitrophenyl N-acetyl-β-glucopyranoside (pNP-GlcNAc), other 4-nitrophenyl glycosides, and 4-methylumbelliferyl N-acetyl-β-glucosaminide (MU-GlcNAc) were obtained from Sigma. The oligosaccharide substrate GnGn-PA (see Table 1 for structures of glycans) was prepared from bovine fibrin by digestion with pepsin, chemical deglycosylation, and digestion of the glycopeptides by N-glycosidase A (Boehringer Mannheim) as described(12Kubelka V. Altmann F. Staudacher E. Tretter V. März L.H.K. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1993; 213: 1193-1204Crossref PubMed Scopus (220) Google Scholar). The product was degraded with Aspergillus oryzae β-galactosidase (6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar) and finally purified by reverse-phase HPLC(12Kubelka V. Altmann F. Staudacher E. Tretter V. März L.H.K. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1993; 213: 1193-1204Crossref PubMed Scopus (220) Google Scholar), lyophilized, and redissolved in water. By the same procedure, GnGnF6-PA from human IgG was prepared(13Staudacher E. Kubelka V. März L. Eur. J. Biochem. 1992; 207: 987-993Crossref PubMed Scopus (72) Google Scholar). GnM-PA and MGn-PA were isolated by reverse-phase HPLC from a partial digest of GnGn-PA with jack bean β-N-acetylhexosaminidase(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar, 12Kubelka V. Altmann F. Staudacher E. Tretter V. März L.H.K. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1993; 213: 1193-1204Crossref PubMed Scopus (220) Google Scholar). M5Gn-PA was synthesized from M5-PA by the use of rabbit GlcNAc-transferase I as described(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar, 8Altmann F. März L. Glycoconjugate J. 1995; 12: 150-155Crossref PubMed Scopus (47) Google Scholar). GlcNAc3-PA was prepared from tri-N-acetylchitotriose (Sigma).TABLE I Open table in a new tab Swainsonine was purchased from Sigma. 6-Acetamido-6-deoxycastanospermine (NACS) (compound MDL 102.373) was kindly provided by Drs. E. H. W. Bohme and M. S. Kang (Marion Merell Dow Research Institute, Cincinnati, OH). Enzyme incubations were conducted at 37°C for 20 h in the presence of 0.02% sodium azide and 0.5% Triton X-100 if not otherwise stated. For experiments with pNP-GlcNAc or other 4-nitrophenyl substrates, the substrate concentration was 5 mM in a total volume of 0.04 ml of 0.1 M citrate/phosphate buffer at pH 4.5. The reactions were terminated by the addition of 0.26 ml of 0.4 M glycine/NaOH buffer at pH 10.4, and absorbance at 405 nm was measured with a microtiter plate reader. In the case of MU-GlcNAc, various substrate concentrations were used in a total volume of 0.2 ml. For measurement of fluorescence at excitation and emission wavelengths of 362 and 451 nm, respectively, the volume was adjusted to 1 ml with 0.1 M sodium carbonate. All enzyme activities are expressed in international units (micromoles/minute). Pyridylaminated oligosaccharides were used at a final concentration of 0.1 mM in a total volume of 0.02 ml of 0.1 M citrate/phosphate buffer at pH 6.0 or, where indicated, 4.5. Incubation was terminated by the addition of 0.18 ml of 20 mM ice-cold sodium borate. Aliquots of 0.05 ml were routinely submitted to reverse-phase chromatography, which was carried out as described(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar, 12Kubelka V. Altmann F. Staudacher E. Tretter V. März L.H.K. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1993; 213: 1193-1204Crossref PubMed Scopus (220) Google Scholar). Kinetic data were estimated by a self-written arithmetic version of the direct linear plot method(14Eisenthal R. Cornish-Bowden A. Biochem. J. 1974; 139: 715-720Crossref PubMed Scopus (1238) Google Scholar). α-Mannosidase II activity was determined as described(8Altmann F. März L. Glycoconjugate J. 1995; 12: 150-155Crossref PubMed Scopus (47) Google Scholar). Insect GlcNAc-transferase I was measured using M5-PA as the substrate, whereas for mammalian GlcNAc-transferase I, MM-PA was used(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar). The GlcNAc-transferase I substrate M5-PA also constitutes a substrate for an insect endo-β-N-acetylglucosaminidase (Endo L)(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar). Therefore, GlcNAc-transferase I assays simultaneously yielded values of Endo L activity(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar). Protein concentrations were determined with the micro-bicinchoninic acid protein assay (Pierce) after treatment of homogenates with 10% (w/v) NaOH at 95°C for 3 min and subsequent neutralization with acetic acid. Oligosaccharide concentrations were determined by amino sugar analysis(15Altmann F. Anal. Biochem. 1992; 204: 215-219Crossref PubMed Scopus (93) Google Scholar). The biantennary oligosaccharide GnGn-PA (see Table 1 for oligosaccharide structures) contains two terminal GlcNAc residues. Incubation with homogenates of X. laevis liver, HepG2 cells, or mung bean seedlings led to the isomers GnM-PA and MGn-PA and finally to MM-PA (Fig. 2). However, when a homogenate of insect cells of the lepidopteran cell line Sf21 was incubated with GnGn-PA, only the isomer GnM-PA was obtained as the product (Fig. 2). Thus, only the GlcNAc residue linked to the α1,3-arm of the core pentasaccharide had been removed by the insect cell GlcNAcase. The same observation was made with homogenates of Bm-N and Mb-0503 cells (data not shown). Also in the case of the fucosylated substrate GnGnF6-PA, Sf21 cells exhibited strict specificity toward the α1,3-arm (data not shown). While MGn-PA was a substrate for Sf21 GlcNAcase, the isomer GnM-PA was not detectably hydrolyzed (Table 2). M5Gn-PA was rapidly degraded by the Sf21 cell homogenate. However, M5-PA, the product of GlcNAcase action, has an almost identical elution position to M4Gn-PA and MGn-PA, the products of α-mannosidase II(8Altmann F. März L. Glycoconjugate J. 1995; 12: 150-155Crossref PubMed Scopus (47) Google Scholar). Indeed, with jack bean the M5Gn-PA digestion product to be M4Gn-PA or MGn-PA In the presence of the α-mannosidase II M5Gn-PA was not by GlcNAcase (Fig. 3 and Table 2). did not Sf21 GlcNAcase (see Table this that the terminal mannosyl residues in M5Gn-PA the action of GlcNAcase. bean GlcNAcase did not exhibit such a of substrate specificity (data not shown). the Sf21 cell homogenate also exhibited activity toward and tri-N-acetylchitotriose (Table it to a single enzyme was responsible for the hydrolysis of all these II Open table in a new tab of Sf21 cell homogenate on different pyridylaminated 5 of Sf21 cell protein with the substrate 50 with the 50 with M5Gn-PA incubated in the presence of the products and MGn-PA are not by reverse-phase HPLC and at GlcNAc3-PA with of protein and are described in the to Open table in a new tab ultracentrifugation of about two-thirds of the Sf21 cell GlcNAcase activity was found in the of the (Table whereas all of the activity of the processing enzymes GlcNAc-transferase I and II was found in the This by with which is to proteins that are only attached to vesicles (Table of the cell homogenate with Triton X-100 the of both GlcNAcase and II to (Table The same pellet and was observed when activity was measured with the substrates GnGn-PA and GlcNAc3-PA (data not shown). the soluble and the of the enzyme hydrolyzed the GlcNAc residue only from the α1,3-arm of is that also in Bm-N cells, about two-thirds of GlcNAcase to be membrane-bound (data not Open table in a new tab which serves as a in mammalian cells, and Endo which in insect cell do not (Fig. and Table The of Triton X-100 on the activity of GlcNAcase and II was investigated with obtained with the Potter-Elvehjem procedure. enzymes exhibited a but by Triton (Table of the Sf21 homogenate in a density gradient in two GlcNAcase (Fig. of the activity in the of The a with activity at Endo L and both did not the of GlcNAcase appears to be a enzyme as reactions for up to 20 h (Fig. Table 2 the of hydrolysis as well as for the different substrates of GlcNAcase from the whole cell homogenate of Bm-N cells. This cell line contains only of Endo L and was therefore to Sf21 cells, exhibited similar GlcNAcase The pH for the substrates GnGn-PA and pNP-GlcNAc (Fig. this the of two different However, as be other observations this of GlcNAcase from Sf21 cells. experiments were performed with to of the cell The of hydrolysis of GnGn-PA and pNP-GlcNAc were determined in citrate/phosphate at GlcNAc3-PA a to that of The activities both the and the synthetic substrates were by and GlcNAc (Table For the concentrations of the two substrates the concentrations The hydrolysis of GnGn-PA was by pNP-GlcNAc and also by at a concentration at which GlcNAc was (Table the other 4-nitrophenyl or 4-nitrophenyl exhibited only a on Incubation of a homogenate of vertebrate such as Xenopus liver or cultured HepG2 cells with GnGn-PA three products when analyzed by reverse-phase chromatography (Fig. 2). of these products from the removal of one GlcNAc residue from either the or were in and were to the final were obtained with both rat liver and plant such in this mung bean seedlings (Fig. and also with jack bean and bovine kidney GlcNAcases (data not shown). Ultracentrifugation of mung bean or rat liver homogenates led to the of only of the GlcNAcase these enzymes are not are even more insect cell GlcNAcase For most the of hydrolysis of the N-glycan GnGn-PA and of the GlcNAc3-PA was that observed with pNP-GlcNAc (Table This jack bean GlcNAcase has been to be toward T. A. A. S. Biochem. 1993; Scopus Google Scholar). Insect cell GlcNAcase, an high activity toward both substrates compared with pNP-GlcNAc is that the activity of Sf21 GlcNAcase toward substrates was at pH (Fig. whereas all other GlcNAcases were more at pH of the substrate (Table a consistent with their in While GlcNAcases are by Sf21 GlcNAcase was by (Table In contrast, the activities of all GlcNAcases were to in 0.2 M sodium at pH (data not Open table in a new tab Insect cells contain a GlcNAcase with is the of action on N-linked biantennary where only the GlcNAc residue on the α1,3-arm is hydrolyzed. In the toward and the high activity toward be The other most of this enzyme is membrane of GlcNAcase activity exists as a soluble The substrate and the of the experiments (Table (i) that a single enzyme was responsible for the hydrolysis of the different substrates and (ii) that soluble and membrane-bound the same is a of knowledge of enzymes for insect cell and the of these two enzymes in insect cells has not been However, the data on Endo L and (Fig. and Table that both led to the of the soluble of the Thus, the two-thirds of GlcNAcase found in the pellet be to soluble enzyme in vesicles from the and therefore sonication is to the soluble of membrane Thus, it appears that the form of GlcNAcase as a soluble protein of this that about two-thirds of the GlcNAcase activity in Sf21 cells exists in a membrane-bound form. The membrane association of insect cell GlcNAcase to a of this a processing GlcNAcase the of N-glycans on insect glycoproteins (Fig. All known enzymes are membrane proteins either in the or in the Golgi apparatus. is known about the of various on insect cell their and Therefore, an of GlcNAcase by density gradient centrifugation is the of GlcNAcase with GlcNAc-transferase I that GlcNAcase be in the Golgi apparatus, this be further However, the unusual branch specificity of insect cell GlcNAcase that it serves as a processing enzyme in the of insect A recent has the structures of the N-glycans of membrane glycoproteins from Bm-N, and Mb-0503 V. Altmann F. Kornfeld G. März L. Arch. Biochem. Biophys. 1994; 308: 148-157Crossref PubMed Scopus (128) Google Scholar). of the N-glycans of membrane glycoproteins from these insect cells were of the most of fucosylated (Table to the current knowledge of the specificity of insect cell α-mannosidase II and these must have the action of GlcNAc-transferase I(6Altmann F. Kornfeld G. Dalik T. Staudacher E. Glössl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar, 8Altmann F. März L. Glycoconjugate J. 1995; 12: 150-155Crossref PubMed Scopus (47) Google Scholar). However, only a of the structures a GlcNAc residue on the α1,3-arm (Table the other a exhibited a single terminal GlcNAc residue linked to the although GlcNAc-transferase II activity was by of GlcNAc-transferase I activity in these cells (Table is therefore that a similar of structures carried GlcNAc linked to the and which was removed by a GlcNAcase. The data be explained by a removal of only the GlcNAc residue by a GlcNAcase. The GlcNAcase described this branch specificity. The of structures by a variety of insect a L. Altmann F. Staudacher E. Kubelka V. Montreuil J. Schachter H. Vliegenthart J.F.G. Protein Glycosylation in Insects. Elsevier Science Publishers B. V., Amsterdam1995Google to the of this processing GlcNAcase the insect Open table in a new tab for insect cells and for the