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INTRODUCTION |
Glycoproteins constitute a large and diverse group of
macromolecules with central roles in many biological processes. Among them are the Asn-linked glycoproteins, which consist of a
polypeptide backbone and an amide-linked oligosaccharide side chain
or N-glycan. The N-glycan precursor,
Glc3Man9GlcNAc2, is initially
transferred to newly synthesized proteins in the lumen of the
endoplasmic reticulum (ER)1
and is converted to various mature forms by processing enzymes distributed along the secretory pathway. Processing begins with cleavage of the three glucose residues by ER
-glucosidases, which produce Man9GlcNAc2, generally known as a
"high mannose" N-glycan. In higher eukaryotes, high
mannose N-glycans may be further processed by ER and Golgi
-mannosidases, which cleave up to six mannose residues. Cleavage of
these mannose residues is required before N-glycans can be
converted to "hybrid" or "complex" N-glycans by
various glycosyltransferases (1).
Three types of
-mannosidases are involved in
N-glycan processing: endo-
-mannosidase, class I
-mannosidases, and class II
-mannosidases (2-5).
Endo-
-mannosidase cleaves the reducing terminus of the mannose
residue to which the glucose residues are attached if a glycoprotein
containing
Glc(1-3)Man9GlcNAc2 exits
the ER. Class I
-mannosidases cleave the four
1,2-linked mannose
residues, converting Man9GlcNAc2 to
Man5GlcNAc2 in the ER and Golgi complex.
Historically, the only class II
-mannosidase thought to be involved
in N-glycan processing was Golgi
-mannosidase II. This
enzyme requires the prior action of
N-acetylglucosaminyltransferase I and cleaves the terminal
1,3- and
1,6-linked mannose residues from
GlcNAcMan5GlcNAc2 producing
GlcNAcMan3GlcNAc2. Golgi
-mannosidase II was
considered to be essential for the conversion of N-glycan precursors to complex structures. However, when the mouse Golgi
-mannosidase II gene was inactivated, most cell types still produced complex N-glycans (6). This observation revealed that mice have at least one additional processing class II
-mannosidase, which
compensated for the absence of Golgi
-mannosidase II. Furthermore, although null mouse cell extracts could not hydrolyze
GlcNAcMan5GlcNAc, they could convert Man5GlcNAc
to Man3GlcNAc. This suggested that N-glycan
processing was mediated by an
-mannosidase activity, termed
-mannosidase III, which produced a novel
Man3GlcNAc2 intermediate in these cells.
However, the enzyme(s) responsible for this activity were not purified,
nor was the gene encoding this enzyme identified.
In a previous study, we cloned and characterized a cDNA encoding a
class II
-mannosidase from the lepidopteran insect cell line,
Sf9 (7). The deduced amino acid sequence of this enzyme is
similar to those of various mammalian Golgi
-mannosidases II. Like
the latter enzymes, the Sf9
-mannosidase is an integral membrane glycoprotein with type II topology. It can hydrolyze p-nitrophenyl
-D-mannopyranoside
(pNP-
-Man), and it is inhibited by swainsonine. In the present
study, we further examined the catalytic properties of this Sf9
class II
-mannosidase to more clearly determine its relationship to
mammalian Golgi
-mannosidase II. The results showed that this enzyme
is actually distinct from Golgi
-mannosidase II as its activity is
stimulated by cobalt and it can hydrolyze various substrates containing
terminal mannose residues but not
GlcNAcMan5GlcNAc2. In view of these properties, we have designated this enzyme Sf9
-mannosidase III
(SfManIII), and we propose that it functions in an alternate
N-glycan processing pathway in Sf9 cells.
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EXPERIMENTAL PROCEDURES |
Cells--
Sf9 cells are derived from
IPLB-Sf21-AE, a cell line originally isolated from
Spodoptera frugiperda (fall armyworm) ovaries (8).
Sf9 cells were maintained in suspension between 0.3 and 3.0 × 106 cells/ml in TNM-FH medium (9) supplemented with 10%
(v/v) heat-inactivated fetal bovine serum (HyClone, Logan, UT) and
0.1% (w/v) pluronic F68 (BASF Wynandotte Corp., Parsippany, NJ; Ref. 10).
Isolation of a Recombinant Baculovirus Expressing a Glutathione
S-transferase (GST)-tagged, Secreted Form of SfManIII--
A cDNA
fragment encoding the SfManIII ectodomain (amino acids 35-1130) was
subcloned into pAcSecG2T (PharMingen, San Diego, CA). The resulting
plasmid, pAcGST-SfManIII, encoded a hybrid protein consisting of a
cleavable signal peptide, the GST protein, and the SfManIII ectodomain
under the transcriptional control of a baculovirus polyhedrin promoter.
This plasmid was used to produce a recombinant baculovirus,
AcGST-SfManIII, which expresses the GST-tagged, secreted form of
SfManIII during the very late phase of infection. AcGST-SfManIII was
produced by homologous recombination (9, 11) with
Bsu36I-digested BacPAK6 viral DNA (12), plaque-purified
twice, amplified in Sf9 cells, and titered by plaque assay
(9).
Expression and Purification of GST-SfManIII--
Sf9
cells were seeded at a density of 12 million cells/75-cm2
flask and infected at a multiplicity of infection of 5 plaque-forming units/cell with either AcGST-SfManIII or wild type baculovirus. The
cells were cultured for 18 h in TNM-FH medium containing 10% (v/v) heat-inactivated fetal bovine serum, then the medium was removed,
and the cells were washed and fed with Grace's medium (13) containing
0.5% (v/v) heat-inactivated fetal bovine serum. At 36 h post
infection, the medium was harvested and clarified by centrifugation for
5 min at 1,000 × g. The supernatant was harvested, and
virus particles were removed by centrifugation for 30 min at
20,000 × g. This second supernatant was adjusted to
0.5% (v/v) Triton X-100 and applied at room temperature to a column
containing 400 µl of immobilized glutathione-agarose (Pierce)
equilibrated with buffer A (100 mM NaMES, pH 6.3, 0.5% Triton X-100). The flow-through was passed back over the column, then
the column was washed with 10 ml of buffer A, and the packing was
removed, resuspended in a final volume of 1 ml of buffer A, and used
for enzyme assays.
For SDS-polyacrylamide gel electrophoresis (14) analyses, samples of
the starting material, second flow-through, and wash were mixed with an
equal volume of 2× protein sample buffer. A sample of the bound
material was obtained by treating glutathione-agarose beads with an
equal volume of 2× protein sample buffer. All samples were heated at
70 °C for 15 min and clarified by centrifugation at 13,000 × g for 5 min prior to the analysis. Proteins were visualized by either Coomassie Blue staining or immunoblotting. For immunoblots, proteins were electrophoretically transferred from the polyacrylamide gel to an ImmobilonTM-P membrane (Millipore Corp., Bedford, MA; Ref.
15). The membrane was then probed with rabbit anti-GST (Sigma) and
alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega, Inc.,
Madison, WI), and antibody binding was detected by a color reaction
(16).
Mannosidase Activity Assays with pNP-
-Man--
Hydrolysis of
pNP-
-Man was assayed in a 37 °C incubator-shaker using 5 µl of
GST-SfManIII in a final volume of 200 µl of buffer containing 50 mM NaMES, 0.25% (v/v) Triton X-100, and 5 mM
pNP-
-Man. The enzyme was assayed in triplicate in each experiment and the results were presented as averages with standard deviations. To
examine the effects of metal ions, SfManIII activity assays were done
at pH 6.3 in the presence of 1 mM EDTA or various
concentrations of CoCl2, MnCl2,
ZnCl2, or NiCl2. For these assays, the enzyme was preincubated with EDTA or the metal ions for 2 h at 37 °C prior to the addition of pNP-
-Man. To determine the influence of pH,
assays were done at various pH values in the presence of 1 mM CoCl2. To measure the effect of swainsonine,
the assays were done at pH 6.3 in the presence of 1 mM
CoCl2 and various concentrations of swainsonine (Genzyme
Corp., Cambridge, MA). All assays were terminated by adding 800 µl of
a buffer containing 133 mM glycine, 67 mM NaCl,
and 83 mM Na2CO3 (pH 10.4). After
removing the beads by centrifugation for 3 min at 13,000 × g, the absorbance of the supernatant was measured at 405 nm
using a Beckman model DU® 7400 spectrophotometer (Beckman Instruments).
Mannosidase Activity Assays with Glycan
Substrates--
Hydrolysis of various pyridylamine (PA)-tagged glycans
was assayed in a 37 °C incubator-shaker using 200 µl of
GST-SfManIII in a final volume of 207 µl of buffer A supplemented
with 1 mM CoCl2 and 100 pmol of substrate.
After various incubation times, the beads were removed by
centrifugation for 3 min at 13,000 × g, and the
supernatants were boiled for 3 min. The reaction products were then
resolved on a Hypersil APS-2 NH2 HPLC column as previously described (17).
Isolation of a Recombinant Baculovirus Expressing a GFP-tagged
Form of SfManIII--
A cDNA fragment encoding full-length
SfManIII was subcloned into the immediate early baculovirus transfer
plasmid, pAcP+E1TV3 (18). The sequence encoding enhanced green
fluorescent protein, a mutant form of GFP with enhanced fluorescence at
507 nm (CLONTECH, Palo Alto, CA), was then inserted
immediately downstream, in-frame with the SfManIII coding sequence. The
resulting plasmid, pAcP+IE1-SfManIII-GFP, encoded a hybrid protein
consisting of the full-length SfManIII protein with GFP fused to its C
terminus under the transcriptional control of a baculovirus immediate
early promoter. This plasmid was used to produce a recombinant
baculovirus, AcP+IE1-SfManIII-GFP, which expresses the enhanced green
fluorescent protein-tagged SfManIII protein during the immediate early
phase of infection. AcP+IE1-SfManIII-GFP was produced by homologous
recombination (9, 11) with Bsu36I-digested BacPAK6 viral DNA
(12), plaque-purified twice, amplified in Sf9 cells, and titered
by plaque assay (9).
Confocal Microscopy--
Sf9 cells were seeded at a
density of 300,000 cells/chamber into LAB-TEK® two-chamber
slides (Nalge Nunc International Corp., Napeville, IL), infected with 3 plaque-forming units/cell of AcP+IE1-SfManIII-GFP, and cultured for
18 h in TNM-FH medium containing 10% (v/v) heat-inactivated fetal
bovine serum. The cells were then incubated with either Golgi- or
lysosome-specific dyes, rinsed with phosphate-buffered saline, and
examined with a Leica TSD-4D CSLM confocal microscope (Leica,
Heidelberg, Germany). To stain the Golgi, the cells were treated for
40-120 min at 27 °C with Grace's medium containing 0.5% (v/v)
heat-inactivated fetal bovine serum and 250 nM
BODIPY®TR ceramide (Molecular Probes Inc., Eugene, OR). To
stain the lysosomes, the cells were treated for 4 min at room
temperature with Grace's medium containing 0.5% (v/v)
heat-inactivated fetal bovine serum and 25 nM LysoTrackerTM
Red DND-99 (Molecular Probes).
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RESULTS |
Expression and Purification of GST-SfManIII--
AcGST- SfManIII
is a recombinant baculovirus that encodes a GST-tagged, secreted form
of SfManIII. Sf9 cells were infected with either this
recombinant virus or a wild type control, and the extracellular media
were harvested and analyzed by immunoblotting with rabbit anti-GST as
described under "Experimental Procedures." The results revealed
that there were two specifically immunoreactive proteins in the
extracellular growth medium from the AcGST-SfManIII-infected cells
(Fig. 1A, lane 1).
Neither was detected in the medium from wild type virus-infected cells
(data not shown). The major protein had an apparent molecular
mass of ~150 kDa suggesting that it is intact GST-SfManIII,
which has a calculated molecular mass of 157.5 kDa. The other,
relatively minor protein had an apparent molecular mass of
~130 kDa and is probably a degradation product. Another, weakly
immunoreactive protein of ~68 kDa was also detected, but its presence
in the negative controls (data not shown) indicated that it was not
specific. Coomassie Blue staining revealed that the growth medium
contained at least three proteins, including the 68-kDa protein, which
were present in vast excess relative to the 150- and 130-kDa
immunoreactive proteins (Fig. 1B, lane 1).

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Fig. 1.
Affinity purification of GST-SfManIII.
Extracellular medium from Sf9 cells infected for 36 h with
AcGST-SfManIII was used for affinity purification as described under
"Experimental Procedures." Equivalent samples of medium
(lanes 1), flow-through (lanes 2), wash
(lanes 3), and bound material (lanes 4) were
analyzed by SDS-polyacrylamide gel electrophoresis followed by
immunoblotting with polyclonal anti-GST (panel A) or
Coomassie Blue staining (panel B). A 50-fold excess of the
Coomassie Blue-stained GST-SfManIII preparation is shown in panel
B, lane 5. The positions of protein markers are
indicated by their sizes (kDa) on the left.
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Affinity chromatography on an immobilized glutathione column was used
to isolate the extracellular immunoreactive proteins as described under
"Experimental Procedures." The affinity column retained the
specifically immunoreactive proteins detected in the growth medium
(compare Fig. 1A, lanes 1 and 4), but
not the nonspecifically immunoreactive and non-immunoreactive proteins detected by Coomassie Blue staining (compare Fig. 1B,
lanes 1 and 4). About half of the
-mannosidase
activity in the medium was retained by the column as determined by
pNP-
-Man assays (data not shown). Multiple attempts to elute active
GST-SfManIII from the column with reduced glutathione were
unsuccessful, but the column-bound enzyme had high levels of
pNP-
-Man activity (data not shown). Therefore, the immobilized
glutathione-agarose beads were removed from the column, resuspended in
buffer A, and used as the source of GST-SfManIII for this study. The
bound GST-SfManIII retained its activity for at least 1 week at 4 °C
but was inactivated by freezing.
The Coomassie Blue-stained gel profile of an excess amount of the
material extracted from the immobilized glutathione-agarose beads is
shown in lane 5 of Fig. 1B. This analysis
revealed that the SfManIII preparations consisted of a major protein
with an apparent molecular mass of ~150 kDa, a minor protein
with an apparent molecular mass of ~130 kDa, and no other
detectable proteins. Presumably, the two proteins stained by Coomassie
Blue corresponded to those detected by immunoblotting.
Biochemical Characterization of GST-SfManIII--
A series of
enzyme assays was performed to characterize the biochemical properties
of GST-SfManIII. One set of assays was designed to evaluate the
influence of various metal ions on GST-SfManIII activity (Table
I). The addition of 1 mM EDTA
reduced hydrolysis of pNP-
-Man to less than 10% of control levels
indicating that GST-SfManIII has a divalent cation requirement. Indeed,
GST-SfManIII activity was greatly enhanced by the addition of
CoCl2 and, to a lesser extent, MnCl2.
NiCl2 had little effect, and GST-SfManIII activity was
inhibited by ZnCl2. The effect of calcium was not examined
because TNM-FH and Grace's media contain high concentrations of
CaCl2.
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Table I
Influence of metal ions on GST-SFMANIII activity
pNP- -Man assays were performed in the presence of the indicated
supplements, as described under "Experimental Procedures." Activity
levels are expressed as percentages of the level measured in control
assays containing no supplements.
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Another set of assays was performed to examine the influence of pH on
GST-SfManIII activity against pNP-
-Man in the presence of 1 mM CoCl2 (Fig.
2). GST-SfManIII activity was negligible
at pH 5.5, increased at pH 5.7 and 5.9, decreased at pH 6.1, and finally rose to a plateau at pH 6.5-6.7. These data showed that GST-SfManIII had significantly more activity around neutral pH than at
acidic pH, which is consistent with the idea that it is a processing,
not a lysosomal, enzyme. The reason for the reproducible decline in
activity observed at pH 6.1 is unknown.

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Fig. 2.
Influence of pH on GST-SfManIII
activity. GST-SfManIII activity against pNP- -Man was measured
at various pH values as described under "Experimental Procedures."
The error bars in this and subsequent figures indicate the
standard deviations obtained with triplicate samples.
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Finally, a set of pNP-
-Man assays was performed to
determine the effect of swainsonine on GST-SfManIII activity
(Fig. 3). GST-SfManIII was clearly
sensitive to swainsonine in assays performed at pH 6.3 in the
presence of 1 mM CoCl2. The IC50
was ~10 nM, and 1.5 µM swainsonine
reduced GST-SfManIII activity to less than 0.5% of control
values.

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Fig. 3.
Influence of swainsonine on GST-SfManIII
activity. GST-SfManIII activity against pNP- -Man was measured
in the presence of various concentrations of swainsonine
(SW) as described under "Experimental Procedures."
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Thus, GST-SfManIII shares some biochemical properties with Golgi
-mannosidase II including its neutral pH optimum and swainsonine sensitivity, but its dependence on cobalt clearly distinguishes this
enzyme from Golgi
-mannosidase II.
Substrate Specificity of GST-SfManIII--
Whereas it was well
established that Golgi
-mannosidase II hydrolyzes
GlcNAcMan5GlcNAc2 to
GlcNAcMan3GlcNAc2 (2), the natural substrate
specificity of SfManIII had not been determined. Therefore, experiments
were undertaken to examine the action of GST-SfManIII on various glycan
substrates. GST-SfManIII had little effect on
GlcNAcMan5GlcNAc2-PA even after extended
incubation times (Fig. 4). Thus,
GST-SfManIII failed to hydrolyze the natural substrate of Golgi
-mannosidase II. On the other hand, GST-SfManIII effectively
hydrolyzed Man5GlcNAc2-PA through a
Man4GlcNAc2-PA intermediate to
Man3GlcNAc2-PA (Fig.
5). This activity was not due to
contamination with endogenous
-mannosidases as there was no
detectable hydrolysis when Man5GlcNAc2-PA was
incubated for 72 h with a mock affinity-purified enzyme
preparation from wild type baculovirus-infected Sf9 cells (Fig.
5, Mock). Thus, GST-SfManIII can hydrolyze the proposed
substrate for
-mannosidase III, the enzyme thought to mediate the
alternate N-glycan processing pathway in mice (6).

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Fig. 4.
Action of GST-SfManIII on
GlcNAcMan5GlcNAc2.
GlcNAcMan5GlcNAc2-PA (top right) was
incubated for the indicated times with GST-SfManIII, and the
oligosaccharide products were analyzed by HPLC (left-hand
panels) as described under "Experimental Procedures." The
panel on the bottom right shows the results of a control
reaction in which GlcNAcMan5GlcNAc2-PA was
incubated for 72 h with a mock-purified enzyme preparation made
from cells infected with wild type baculovirus. The arrows
mark the positions of glycan standards. GnM5 and
GnM4 refer to GlcNAcMan5GlcNAc2-PA
and GlcNAcMan4GlcNAc2-PA, respectively.
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Fig. 5.
Action of GST-SfManIII on
Man5GlcNAc2.
Man5GlcNAc2-PA (top right) was
incubated for the indicated times with GST-SfManIII, and the
oligosaccharide products were analyzed by HPLC (left-hand
panels) as described under "Experimental Procedures." The
panel on the bottom right shows the results of a control
reaction in which Man5GlcNAc2-PA was incubated
for 72 h with a mock-purified enzyme preparation made from cells
infected with wild type baculovirus. The arrows mark the
positions of glycan standards. M5 to M2 refer to
Man(5-2)GlcNAc2-PA.
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Considering its unusual substrate specificity, we also evaluated the
ability of GST-SfManIII to hydrolyze other glycans, which had terminal
1,2-linked mannose residues (Fig. 6).
GST-SfManIII was incubated for 72 h with
Man9GlcNAc2-PA,
Man8GlcNAc2-PA (mixed isomers) or
Man6GlcNAc2-PA (isomer C with one
1,2-linked
mannose residue on the middle arm). Over half of the
Man9GlcNAc2-PA was consumed producing
Man8GlcNAc2-PA and a small amount of
Man7GlcNAc2-PA. Similarly, most of the
Man8GlcNAc2-PA was consumed producing
Man7GlcNAc2-PA and small amounts of
Man6GlcNAc2-PA and
Man5GlcNAc2-PA. Finally, Man6GlcNAc2-PA was completely consumed
producing mainly Man5GlcNAc2-PA and small
amounts of Man4GlcNAc2-PA and
Man3GlcNAc2-PA. None of these activities were
due to contaminating endogenous
-mannosidases as there was no
detectable hydrolysis of any of these substrates when they were
incubated for 72 h with a mock affinity-purified enzyme
preparation from wild type baculovirus-infected Sf9 cells (Fig.
6, Mock column). These results demonstrated that
GST-SfManIII can hydrolyze a variety of glycan substrates containing
terminal
1,2-linked mannose residues in addition to
Man5GlcNAc2-PA. Interestingly, SfManIII
produced much less Man7GlcNAc2-PA from the
Man9GlcNAc2-PA substrate than from the
Man8GlcNAc2-PA (mixed isomer) substrate. The
reason why Man9GlcNAc2-PA was not completely
converted to Man7GlcNAc2-PA is unclear. It is
possible that this simply reflects the relatively slow conversion of
Man9GlcNAc2-PA to
Man8GlcNAc2-PA (Fig. 6, top left
panel). Alternatively, it is possible that this reflects the
composition of the Man8GlcNAc2-PA (mixed
isomer) substrate, which consisted of ~75% isomer A, 20% isomer B,
and 5% isomer C (data not shown). This mixture might be a better
substrate for SfManIII because isomers A and B are better substrates
for SfManIII. In contrast, SfManIII digestion of
Man9GlcNAc2-PA could yield a totally different mixture of
Man8GlcNAc2-PA isomers, which is relatively
resistant to further digestion by SfManIII. These possibilities were
not examined in this study.

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Fig. 6.
Action of GST-SfManIII on
Man(9-6)GlcNAc2.
Man9GlcNAc2-PA,
Man8GlcNAc2-PA (mixed isomers), or
Man6GlcNAc2-PA (isomer C) were incubated for
72 h with GST-SfManIII, and the oligosaccharide products were
analyzed by HPLC (left-hand panels) as described under
"Experimental Procedures." The right-hand panels show
the results of control reactions in which each glycan substrate was
incubated for 72 h with a mock-purified enzyme preparation made
from cells infected with wild type baculovirus. The arrows
mark the positions of glycan standards. M9-M3 refer to
Man(9-3)GlcNAc2-PA.
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All of the assays of GST-SfManIII activity against glycan
substrates were performed in the presence of 1 mM
CoCl2. Therefore, additional assays were performed to
specifically evaluate the cobalt requirement for
Man5GlcNAc2-PA hydrolysis by
GST-SfManIII. Hydrolysis was greatly reduced when CoCl2 was
not added to the assays and was completely abolished in the presence of
5 mM EDTA (data not shown) indicating that cobalt is
required for the hydrolysis of Man5GlcNAc2-PA
by GST-SfManIII. The effect of swainsonine on Man5GlcNAc2-PA hydrolysis by GST-SfManIII was
also evaluated, and the results were similar to those obtained in the
pNP-
-Man assays (data not shown) indicating that hydrolysis of
Man5GlcNAc2-PA by GST-SfManIII is sensitive to
swainsonine, as well.
Intracellular Distribution of SfManIII-GFP--
All of the
properties of SfManIII were consistent with the interpretation that
this enzyme plays a role in N-glycan processing in
Sf9 cells. If SfManIII has this role in vivo, it
should reside in the Golgi compartment. To examine its intracellular
distribution, confocal microscopy was used to examine live Sf9
cells infected with a baculovirus vector encoding a full-length,
GFP-tagged version of SfManIII. This baculovirus expression vector
expresses SfManIII-GFP under the transcriptional control of a
baculovirus promoter that is active immediately after infection and
provides relatively low expression levels (18). This promoter was used
to circumvent the potential problem of aberrant localization of
SfManIII-GFP, which might happen if it had been overexpressed.
Sf9 cells were infected with this baculovirus for 18 h, and
then labeled with the red fluorescent dyes BODIPY®TR
ceramide to stain the Golgi (Fig. 7,
top panels) or LysoTrackerTM Red DND-99 to stain the
lysosomes (Fig. 7, bottom panels). Confocal microscopy revealed that the SfManIII-GFP was localized in punctate fluorescent structures dispersed throughout the cytoplasm but excluded
from the nuclei (Fig. 7, left panels). This staining pattern
overlapped significantly with the pattern obtained with the Golgi dye
(Fig. 7, top middle panel) but not with the pattern obtained
with the lysosomal dye (Fig. 7, bottom middle panel). It is
noteworthy that a previous study established the dispersed nature of
the Golgi elements in Sf9 cells (19). Thus, the confocal microscopy results described above indicate that SfManIII is localized in the Golgi compartment of Sf9 cells.

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Fig. 7.
Intracellular distribution of
GST-SfManIII. Sf9 cells were infected for 18 h with
AcP(+)IE1-SfManIII-GFP, then labeled with red-fluorescent dyes specific
for the Golgi (top panels) or lysosomes (bottom
panels), and unfixed cells were examined by confocal microscopy.
The panels on the left show the pattern of fluorescence
obtained with SfManIII-GFP, whereas those on the right show
the pattern obtained with the dyes. The panels in the middle
show overlays of the panels on either side. Bar, 10 µ m.
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DISCUSSION |
This study demonstrated that Sf9 cells encode a
class II
-mannosidase that is distinct from Golgi
-mannosidase II
and has a unique set of properties. Based on these properties, and in accordance with the nomenclature used by Chui et al.(6), we named this enzyme SfManIII. Unlike Golgi
-mannosidase II, SfManIII is activated by cobalt, it can hydrolyze
Man(9-5)GlcNAc2, and it can convert
Man5GlcNAc2 to
Man3GlcNAc2 but cannot hydrolyze GlcNAcMan5GlcNAc2. On the other hand,
SfManIII has the same neutral pH optimum and intracellular distribution
as Golgi
-mannosidase II. These properties, together with the fact
that Man3GlcNAc2 can be elongated by
N-acetylglucosaminyltransferase I (20, 21) and further
processed to complex structures, suggest that SfManIII functions in
N-glycan processing in Sf9 cells. However, the
current study did not establish whether or not SfManIII actually plays this role in vivo.
The early steps of N-glycan processing, including mannose
trimming, appear to be similar or identical in Sf9 and mammalian cells (22, 23). A cDNA encoding a class I
-mannosidase has been
cloned from Sf9 cells, and its product has been extensively characterized (19, 24, 25). The results of these studies have shown
that SfManI is virtually identical in structure, function, and
intracellular distribution to the mammalian
-mannosidases IA and IB.
In addition, Sf9 cells have a class II
-mannosidase that acts
on GlcNAcMan5GlcNAc2 without a metal ion
requirement and appears to be functionally identical to mammalian Golgi
-mannosidase II (26). In contrast, whereas SfManIII has the
biochemical properties of Class II mannosidases, a mammalian ortholog
with similar catalytic characteristics has not yet been purified or
cloned (5).
Considering the similarities between the other processing
-mannosidases of Sf9 and mammalian cells, there is probably a
mammalian ortholog of SfManIII as well. One likely candidate is the
cobalt-activated
-mannosidase activity(ies) that convert
Man5GlcNAc to Man3GlcNAc in null mice lacking
Golgi
-mannosidase II (6). This activity, termed
-mannosidase
III, compensates for the absence of Golgi
-mannosidase II in these
mice by providing an alternate pathway for N-glycan
processing. However, it is currently impossible to unequivocally
decipher the relationship between this activity and SfManIII because
the mouse enzyme(s) that provide this activity have not yet been
identified. In fact, several enzymes probably contribute to the
-mannosidase III activity detected in mouse cell lysates including
both swainsonine-resistant enzymes that are not involved in
N-glycan processing, as well as one or more swainsonine-sensitive, processing enzymes.
One mammalian enzyme that could account for the
Man5GlcNAc-hydrolyzing activity in the null mouse cell
lysates is an
-mannosidase first characterized in BHK cells
(27) and later purified from rat liver (28, 29). This BHK/rat liver
enzyme is cobalt-activated, relatively resistant to swainsonine, and
can convert Man5GlcNAc2 to
Man3GlcNAc2 but cannot hydrolyze pNP-
-Man.
This enzyme does not appear to be a processing enzyme because there is
no complex N-glycan biosynthesis in BHK cells treated with
swainsonine (30) indicating that all of the processing class II
-mannosidases in these cells are swainsonine sensitive. But, like
the null mice discussed above, mutant BHK cells lacking detectable
Golgi
-mannosidase II activity can still synthesize some complex
N-glycans (31). Thus, these cells must have another,
swainsonine sensitive
-mannosidase III activity that provides the
alternate N-glycan processing pathway.
A second mammalian enzyme that could account for the
-mannosidase
III activity is an isozyme of Golgi
-mannosidase II that has been
termed
-mannosidase IIx (32). This enzyme shares
sequence similarity with both the human and insect class II
-mannosidases, but its N-glycan substrate specificity has
not yet been characterized.
There also is another group of mammalian enzymes, generally classified
as ER/cytosolic
-mannosidases, which could account for the
Man5GlcNAc-hydrolyzing activity detected in the null mouse cell lysates. These enzymes are activated by cobalt, are relatively resistant to swainsonine, and can convert Man5GlcNAc to
Man3GlcNAc. However, they act efficiently only on glycans
with a single GlcNAc residue on their reducing end and appear to
function in N-glycan catabolism rather than processing
(33-36).
SfManIII is clearly distinct from both the swainsonine resistant
BHK/rat liver enzyme and the ER/cytosolic
-mannosidases. SfManIII is
sensitive to swainsonine, can hydrolyze pNP-
-Man, and can
efficiently hydrolyze glycans with chitobiose cores. Considering these
properties and the fact that Sf9 cells have another
-mannosidase, which is functionally equivalent to mammalian Golgi
-mannosidase II (26), SfManIII is probably equivalent to the enzyme
providing the alternate pathway in the null mice (6) and mutant BHK
cells (31). However, an unequivocal test of this hypothesis awaits purification of the mammalian enzyme(s) responsible for the
-mannosidase III activity.