(Received for publication, February 28, 1995)
From the
The
Mammalian cells contain in their lysosomes
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.
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.
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) .
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
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 (
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 (
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
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
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) .
We thank Barbara Swoboda for culturing insect cells
and Dr. Iain Wilson for reading the manuscript.
e 33, A-1180 Wien, Austria
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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. GlcNAcMan
GlcNAc
, 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.
-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
Man
GlcNAc
(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.
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.
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.
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
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).
-N-Acetylglucosaminidase
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) .
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.
-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).
), 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.
) 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.
-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).
1,3-arm is hydrolyzed. In addition, the inactivity
toward GlcNAcMan
GlcNAc
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.
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.
-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 GnGnF
N-glycans.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.