©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insect Cells Contain an Unusual, Membrane-bound -N-Acetylglucosaminidase Probably Involved in the Processing of Protein N-Glycans (*)

(Received for publication, February 28, 1995)

Friedrich Altmann (§) , Herwig Schwihla (1), Erika Staudacher , Josef Glössl (1), Leopold März

From the Institut für Chemie der Universität für Bodenkultur Zentrum für Angewandte Genetik der Universität für Bodenkultur, Gregor Mendelstrae 33, A-1180 Wien, Austria

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The -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 -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 -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 -N-acetylglucosaminidase exclusively removed -N-acetylglucosamine from the 1,3-antenna. GlcNAcManGlcNAc, the primary product of GlcNAc-transferase I, was not perceptibly hydrolyzed.

-N-Acetylglucosaminidases with the same branch specificity were also found in the lepidopteran cell lines Bm-N and Mb-0503. In contrast, -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 -N-acetylglucosaminidase in the processing of asparagine-linked oligosaccharides in insects is suggested.


INTRODUCTION

Mammalian cells contain in their lysosomes -N-acetylglucosaminidase (GlcNAcase)()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(1, 2) . 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 1,3-linked as known for plant glycoproteins. Such structures are not consistent with the classical N-glycan processing pathway established for mammalian cells (3) since in mammals both -mannosidase II and fucosyltransferase require the presence of GlcNAc-1, which is transferred by GlcNAc-transferase I (Fig. 1)(4, 5) . In principle, the fucosylated paucimannose structures could be explained by a different specificity of both the -mannosidase removing the two terminal mannosyl residues from the 1,6-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(6, 7) . (ii) The fucosyltransferase(s) of insect cells do not act upon ManGlcNAc(6) . (iii) -Mannosidase II from insect cells is also strictly dependent on the presence of GlcNAc-1 (8) . (iv) GlcNAc-2 is transferred to the 1,6-antenna only after the action of GlcNAc-transferase I(6) . 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'' -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(9) . In mammals, most processing enzymes such as GlcNAc-transferase I, -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(10, 11) , whereas catabolic enzymes exist in the lysosomes as soluble proteins.


Figure 1: Part of N-glycan processing in mammalian and insect cells. The bold arrows depict the well established pathway in mammalian cells(3) . The fine arrows represent hypothetical routes for the formation of fucosylated paucimannose structures in insect cells.



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.


MATERIALS AND METHODS

Preparation of Microsomes from Insect Cells

Cells of the strains IPLB-Sf21AE, Bm-N, and Mb-0503 as well as human HepG2 cells were grown and harvested as described(6) . Typically, insect cells (10 10/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 10 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.

Other Enzyme Sources

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.

Enzyme Substrates and Inhibitors

4-Nitrophenyl N-acetyl--glucopyranoside (pNP-GlcNAc), other 4-nitrophenyl glycosides, and 4-methylumbelliferyl N-acetyl--glucosaminide (MU-GlcNAc) were obtained from Sigma. The oligosaccharide substrate GnGn-PA (see Table 1for 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(12) . The product was degraded with Aspergillus oryzae -galactosidase (6) and finally purified by reverse-phase HPLC(12) , lyophilized, and redissolved in water. By the same procedure, GnGnF-PA from human IgG was prepared(13) . GnM-PA and MGn-PA were isolated by reverse-phase HPLC from a partial digest of GnGn-PA with jack bean -N-acetylhexosaminidase(6, 12) . M5Gn-PA was synthesized from M5-PA by the use of rabbit GlcNAc-transferase I as described(6, 8) . GlcNAc-PA was prepared from tri-N-acetylchitotriose (Sigma).



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).

Assay of -N-Acetylglucosaminidase

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(6, 12) . Kinetic data were estimated by a self-written arithmetic version of the direct linear plot method(14) .

Determination of Other Enzyme Activities

-Mannosidase II activity was determined as described(8) . Insect GlcNAc-transferase I was measured using M5-PA as the substrate, whereas for mammalian GlcNAc-transferase I, MM-PA was used(6) . The GlcNAc-transferase I substrate M5-PA also constitutes a substrate for an insect endo--N-acetylglucosaminidase (Endo L)(6) . Therefore, GlcNAc-transferase I assays simultaneously yielded values of Endo L activity(6) .

Analytical Techniques

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(15) .


RESULTS

Effect of GlcNAcase from Insect Cells on Complex Glycans

The biantennary oligosaccharide GnGn-PA (see Table 1for 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 1,3-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 GnGnF-PA, Sf21 cells exhibited strict specificity toward the 1,3-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 -mannosidase II(8) . Indeed, probing with jack bean -GlcNAcase revealed the M5Gn-PA digestion product to be M4Gn-PA or MGn-PA rather than M5-PA. In the presence of the -mannosidase II inhibitor swainsonine, M5Gn-PA was not processed by GlcNAcase ( Fig. 3and Table 2). Since swainsonine did not directly inhibit Sf21 GlcNAcase (see Table 4), this result indicates that the terminal mannosyl residues in M5Gn-PA inhibited the action of GlcNAcase. Jack bean GlcNAcase did not exhibit such a restriction of its substrate specificity (data not shown). As the Sf21 cell homogenate also exhibited activity toward pNP-GlcNAc, pNP-GalNAc, and tri-N-acetylchitotriose (GlcNAc-PA) (Table 2), it proved necessary to determine whether a single enzyme was responsible for the hydrolysis of all these substrates.


Figure 2: Hydrolysis of GnGn-PA by various cell or tissue homogenates. Incubations were performed at pH 6.0 for 20 h, and the digests were analyzed by reverse-phase chromatography. A and B, Sf21 cells (5 and 50 µg of protein, respectively); C, Xenopus liver (26 µg); D, HepG2 cells (30 µg); E, mung bean seedlings (18 µg). The elution positions of MGn-, MM-, GnGn-, and GnM-PA are designated by the lines1-4, respectively. Peak5 is GlcNAc-PA, which stems from the action of Endo L.






Figure 3: Effect of Sf21 cell homogenate on different pyridylaminated oligosaccharides. A, 5 µg of Sf21 cell protein with the substrate MGn-PA; B, 50 µg with the nonsubstrate GnM-PA; C, 50 µg with M5Gn-PA incubated in the presence of swainsonine (4 µg/ml; the possible products M5-, M4Gn-, and MGn-PA are not separable by reverse-phase HPLC and would elute at peak6); D, GlcNAc-PA digested with 0.8 µg of protein (peaks 7-9 represent GlcNAc-, GlcNAc-, and GlcNAc-PA, respectively). Other details are described in the legend to Fig. 2.





Membrane Association of Insect Cell GlcNAcase

Upon ultracentrifugation of homogenates, about two-thirds of the Sf21 cell GlcNAcase activity was found in the pellet, regardless of the disrupture procedure (Table 3), whereas nearly all of the activity of the processing enzymes GlcNAc-transferase I and mannosidase II was found in the pellet. This proportion remains unaffected by pretreatment with salt, which is thought to solubilize proteins that are only peripherally attached to vesicles (Table 3). Treatment of the cell homogenate with Triton X-100 reduced the proportion of both GlcNAcase and mannosidase II sedimented to 35% (Table 3). The same distribution between pellet and supernatant was observed when activity was measured with the substrates GnGn-PA and GlcNAc-PA (data not shown). Both the soluble and the sedimented forms of the enzyme hydrolyzed the GlcNAc residue only from the 1,3-arm of GnGn-PA. It is noteworthy that also in Bm-N cells, about two-thirds of GlcNAcase appeared to be membrane-bound (data not shown).



Acidic phosphatase, which serves as a lysosomal marker in mammalian cells, and Endo L, which probably likewise resides in insect cell vacuoles, do not sediment ( Fig. 4and Table 3). The influence of Triton X-100 on the apparent activity of GlcNAcase and mannosidase II was investigated with microsomes obtained with the Potter-Elvehjem procedure. Both enzymes exhibited a small but definite activation by Triton (Table 4).


Figure 4: Sucrose density gradient centrifugation of insect cell organelles. Sf21 cells were disrupted using a Potter-Elvehjem homogenizer as described under ``Materials and Methods.'' After centrifugation at 47,000 rpm for 90 min in a Ti-50 rotor, the gradient was fractionated, and fractions were analyzed for GlcNAcase (), GlcNAc-transferase I (), Endo L (), and acidic phosphatase (). Results are given as relative activities. The density at 20 °C was calculated from the refractive index.



Centrifugation of the Sf21 homogenate in a density gradient resulted in two GlcNAcase peaks (Fig. 4). One part of the activity remained in the zone of sample application. The larger part, however, gave a broad band with maximal activity at 1.12 g/ml. Endo L and acidic phosphatase both did not leave the zone of sample application.

Enzymological Characterization

GlcNAcase appears to be a fairly stable enzyme as reactions proceeded linearly for up to 20 h (Fig. 5). Table 2gives the relative rates of hydrolysis as well as kinetic parameters for the different substrates of GlcNAcase from the whole cell homogenate of Bm-N cells. This cell line contains only minute amounts of Endo L and was therefore preferred to Sf21 cells, which, however, exhibited essentially similar GlcNAcase specificities. The pH optima for the substrates GnGn-PA and pNP-GlcNAc differed substantially (Fig. 6). At first sight, this suggests the existence of two different enzymes. However, as will be shown, several other observations argue against this interpretation.


Figure 5: Time course of product formation by Sf21 GlcNAcase. Lines A-D were obtained with 103, 34, 8.6, and 0 µg of Sf21 cell protein, respectively, as the enzyme source and with pNP-GlcNAc as the substrate.




Figure 6: pH dependence of GlcNAcase from Sf21 cells. These experiments were performed with microsomes to eliminate any buffering capacity of the cell content. The rates of hydrolysis of GnGn-PA () and pNP-GlcNAc () were determined in citrate/phosphate buffers at varying pH. GlcNAc-PA gives a graph comparable to that of GnGn-PA.



The activities against both the complex and the synthetic substrates were inhibited by NACS and GlcNAc (Table 4). For practical reasons, the concentrations of the two substrates differed. Consequently, the inhibitor concentrations giving 50% inhibition likewise differed. The hydrolysis of GnGn-PA was inhibited by pNP-GlcNAc and also by pNP-GalNAc at a concentration at which GlcNAc itself was ineffective (Table 4). On the other hand, 4-nitrophenyl -glucopyranoside or 4-nitrophenyl -xylopyranoside exhibited only a marginal effect on activity.

Comparison with GlcNAcases from Other Species

Incubation of a homogenate of vertebrate tissue such as Xenopus liver or cultured HepG2 cells with GnGn-PA gave three products when analyzed by reverse-phase chromatography (Fig. 2). Two of these peaks, intermediate products arising from the removal of one GlcNAc residue from either the 1,3- or 1,6-arm, were formed in approximately equal yield, and were readily converted to the final product, MM-PA. Similar results were obtained with both rat liver and plant material such as, in this case, mung bean seedlings (Fig. 2) and also with jack bean and bovine kidney GlcNAcases (data not shown). Ultracentrifugation of mung bean or rat liver homogenates led to the sedimentation of only 4% of the GlcNAcase activity. Obviously, these enzymes are not membrane-bound.

There are even more differences making insect cell GlcNAcase unique. For most GlcNAcases, the rate of hydrolysis of the N-glycan GnGn-PA and of the chitooligosaccharide GlcNAc-PA was substantially lower than that observed with pNP-GlcNAc (Table 5). This might explain why jack bean GlcNAcase has erroneously been reported to be inactive toward chitooligosaccharides (16) . Insect cell GlcNAcase, however, displays an unusually high activity toward both natural substrates compared with pNP-GlcNAc ( Table 3and Table 6). It is noteworthy that the activity of Sf21 GlcNAcase toward natural substrates was highest at near neutral pH (Fig. 5), whereas all other GlcNAcases were more effective at pH 4.5 regardless of the substrate (Table 5), a feature consistent with their localization in lysosomes. While GlcNAcases usually are inhibited by acetate buffers, Sf21 GlcNAcase was unaffected by acetate (Table 4). In contrast, the activities of all GlcNAcases were reduced to 20% in 0.2 M sodium acetate at pH 4.5 (data not shown).






DISCUSSION

Insect cells contain a GlcNAcase with several remarkable features. One is the unique mode of action on N-linked biantennary agalactooligosaccharide (GnGn), where only the GlcNAc residue on the 1,3-arm is hydrolyzed. In addition, the inactivity toward GlcNAcManGlcNAc and the unusually high activity toward chitooligosaccharides should be noted. The other most distinctive property of this enzyme is its membrane association. One-third of GlcNAcase activity exists as a freely soluble protein. The indistinguishable substrate specificities and the results of the inhibition experiments (Table 2) suggest (i) that a single enzyme was responsible for the hydrolysis of the different substrates and (ii) that soluble and membrane-bound forms essentially possess the same catalytic domain. There is a regrettable lack of knowledge of potential marker enzymes for insect cell organelles; and thus, the localization of these two enzymes in insect cells has not been definitely established. However, the data on Endo L and acidic phosphatase ( Fig. 4and Table 3) suggest that both homogenization procedures led to the complete release of the soluble contents of the vacuoles. Thus, the two-thirds of GlcNAcase found in the pellet cannot be attributed to soluble enzyme entrapped in vesicles derived from the large and therefore fragile vacuole. Moreover, sonication is assumed to release the soluble content of every membrane compartment. Thus, it appears improbable that the sedimented form of GlcNAcase existed as a soluble protein inside any kind of endomembrane compartment. Overall, this indicates that about two-thirds of the GlcNAcase activity in Sf21 cells exists in a membrane-bound form.

The unique membrane association of insect cell GlcNAcase points to a special biological function of this enzyme. As noted above, a processing GlcNAcase would explain the occurrence of fucosylated, paucimannosidic N-glycans on insect glycoproteins (Fig. 1). All known N-glycan-processing enzymes are membrane proteins residing either in the endoplasmic reticulum or in the Golgi apparatus. Little is known about the effect of various disruption procedures on insect cell organelles, their buoyant density, and appropriate marker enzymes. Therefore, an unambiguous intracellular localization of GlcNAcase by density gradient centrifugation is impossible. Although the cosedimentation of GlcNAcase with GlcNAc-transferase I indicates that GlcNAcase should be located in the Golgi apparatus, this cannot be proven without further study.

However, the unusual branch specificity of insect cell GlcNAcase offers credence that it serves as a processing enzyme in the maturation of insect N-glycans. A recent paper has reported the structures of the N-glycans of membrane glycoproteins from Sf21, Bm-N, and Mb-0503 cells(2) . 40% of the N-glycans of membrane glycoproteins from these insect cells were of the paucimannosidic type, most of them fucosylated (Table 6). According to the current knowledge of the specificity of insect cell -mannosidase II and fucosyltransferase(s), these glycans must have experienced the action of GlcNAc-transferase I(6, 8) . However, only a tiny fraction of the structures contained a GlcNAc residue on the 1,3-arm (Table 6). On the other hand, a comparable fraction exhibited a single terminal GlcNAc residue linked to the 1,6-arm, although GlcNAc-transferase II activity was lower by 2 orders of magnitude than GlcNAc-transferase I activity in these cells (Table 6). It is therefore highly implausible that a similar number of structures originally carried GlcNAc linked to the 3- and 6-arms, which was then removed by a nonspecific GlcNAcase. The data can, however, be explained by a nonrandom removal of only the 3-arm-linked GlcNAc residue by a branch-specific GlcNAcase. The GlcNAcase described herein provides exactly this branch specificity. The formation of paucimannosidic structures by a variety of insect species (for a review, see (1) ) points to the widespread significance of this processing GlcNAcase throughout the insect phylum.


FOOTNOTES

*
This work was supported by a grant from the Austrian Bundesministerium für Wissenschaft und Forschung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 43-1-47654-6062; Fax: 43-1-3105176.

The abbreviations used are: GlcNAcase, N-acetyl--glucosaminidase; pNP-GlcNAc, 4-nitrophenyl N-acetyl--glucopyranoside; MU-GlcNAc, 4-methylumbelliferyl N-acetyl--glucopyranoside; HPLC, high pressure liquid chromatography; PA, pyridylamino; Gn, N-acetylglucosamine; M, mannose; F, fucose; GlcNAc, tri-N-acetylchitotriose; NACS, 6-acetamido-6-deoxycastanospermine; Endo L, endo-N-acetylglucosaminidase from lepidopteran cells. See Table I for structures of GnGn, MGn, GnM, MM, M5Gn, and GnGnFN-glycans.


ACKNOWLEDGEMENTS

We thank Barbara Swoboda for culturing insect cells and Dr. Iain Wilson for reading the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.