 |
INTRODUCTION |
Free polymannose-type oligosaccharides (free
OS)1 are a by-product of the
biosynthesis of dolichol oligosaccharides (1-3) and the glycosylation
of glycoproteins (3, 4). Whereas phosphorylated free OS
(Man5GlcNAc2-P) are released from dolichol
oligosaccharides on the cytoplasmic face of the ER (5), neutral free OS
(Glc3-1Man9-8GlcNAc2) are
liberated from oligosaccharide-lipid in the lumen of the ER (1, 6).
After deglucosylation, these neutral free OS do not follow the
secretory pathway but are routed to the cytosol (7) by an ATP- and
calcium-requiring translocation process that can be inhibited by
mannose but not by N-acetylglucosamine (8). Glycopepetides
(9, 10) and misfolded glycoproteins (11, 12) are also translocated out
of the ER into the cytosol, where they can be deglycosylated by an
N-glycanase (13, 14), giving rise to further cytosolic free OS.
The rapid appearance of substantial quantities of free OS in the
cytosol of cells during the biosynthesis and quality control of
glycoproteins suggested that the cytosol must possess elements capable
of the disposal of these osmotically active components. In fact,
neutral free OS are subject to sequential processing (15-17) by a
previously characterized cytosolic endo-H-like activity (18) or
chitobiase (15) and cytosolic mannosidase (19-21) to yield a free OS
containing five residues of mannose and a single reducing
N-acetylglucosamine residue. It has now been demonstrated that this free OS is transferred to lysosomes in order to be degraded (17). The fate of cytosolic phosphorylated free OS is less clear, but
since this oligosaccharide possesses an identical configuration of
mannose residues to that of the free OS that undergoes
cytosol-to-lysosome translocation, it may be transferred directly into
the lysosome. The physiological importance of the capture of cytosolic
free OS by the lysosome is indicated by the fact that up to 40% of the
free OS recovered from the urine of people with the lysosomal storage
disease,
-mannosidosis, have oligosaccharide structures compatible
with their having been generated in the cytosol (22, 23). However,
despite the fact that the cytosol-to-lysosome translocation of free OS
must represent a vital housekeeping activity in cells engaged in the
biosynthesis of glycoproteins, this process remains to be characterized.
Although most molecules destined for degradation in the lysosome arrive
by way of a series of vesicular fusion steps after their initial
capture, either from the extracellular environment into endocytic
vesicles (24) or from within the cell into autophagic vacuoles
(25), it has become apparent that the lysosomal membrane itself may be
involved in the capture of cytosolic molecules. For instance, cytosolic
proteins containing the KFERQ sequence can be translocated into
lysosomes by a lysosomally situated translocation machinery comprising
both HSC73 and LGP96 molecules (26). In addition, lysosomes may contain
specific carriers responsible for the import of cysteine (27) and
methotrexate glutamates (28) from the cytosol. In vivo, the
cytosol-to-lysosome transfer of free OS was inhibited by agents that
reduce cellular ATP levels and by high swainsonine concentrations,
which also inhibited the cytosolic demannosylation of free OS (17).
However, apart from being able to rule out macroautophagic
sequestration as a possible mechanism to account for this phenomenon,
little could be inferred about this process.
Here, we demonstrate an ATP-dependent,
N-ethylmaleimide-sensitive, saturable import of free OS into
isolated rat liver lysosomes. Results are reported to show that the
lysosome possesses a novel oligosaccharide importer capable of
capturing an array of small polymannose oligosaccharides that possess a
reducing GlcNAc residue.
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EXPERIMENTAL PROCEDURES |
Materials--
The following compounds were purchased from
Sigma: D-(+)-glucose, D-(+)-mannose,
N-acetyl-D-glucosamine,
D-(+)-galactose,
-L-(
)-fucose, sodium
orthovanadate, sodium azide, FITC-dextran (60-70 kDa), valinomycin,
phloretin, and phloridzin. Benzyl
-D-mannopyranoside, Man(
1-4)GlcNAc, Gal(
1-4)GlcNAc, and
Man(
1-6)(Man(
1-3))Man(
1-6)(Man(
1-3))Man were from
Dextra Laboratories Ltd. (Reading, United Kingdom). The
-1,2-mannosidase was purchased from Oxford Glycosystems (Abingdon, UK). All chitooligosaccharides were purchased from Seikagaku America, Inc. (Rockville, MD), whereas
-benzyl chitobioside and
-benzyl GlcNAc were from Toronto Research Chemicals (Toronto, Canada). Cytochalasins B and D were from LC Laboratories (Laufelfingen, Switzerland). Concanamycin A (CCM A) was supplied by Dr. J. R. Green (Ciba Geigy Ltd.). The two standard isomers of
Man5GlcNAc were supplied by Dr. J-C Michalski (CNRS UMR
111, Lille, France). ATP
S, AMP-PNP, and AMP-PCP were from France
Biochem, Meudon, France.
Culture and Radiolabeling of Cells--
HepG2 cells (ECACC,
Porton Down, UK) were grown as described previously (29). The mutant
mouse lymphoma cell line, Thy
1 (provided by Drs. A. Conzelmann and P. Romagnoli, Institut de Biochimie, Université de
Lausanne), was cultivated in Dulbecco's modified Eagle's medium (Life
Technologies, Inc., Paisley, UK) containing 10% fetal calf serum (Life
Technologies) and was metabolically radiolabeled by incubating 30 × 108 cells in 7.5 ml of glucose-free Dulbecco's modified
Eagle's medium (Life Technologies) supplemented with 5% dialyzed
fetal calf serum, 2 mM glutamine, 5 mM fucose,
and 1 mM sodium pyruvate with 1.3 mCi of
[2-3H]mannose (30 mCi/mmol) for 4 h in the presence
of 10 nM concanamycin A.
Preparation and Purification of Free Oligosaccharides from
Metabolically Radiolabeled Lymphoma Cells--
Metabolically
radiolabeled neutral free oligosaccharides were prepared from
radiolabeled cells as described previously (16), and the resulting
[3H]free OS was resolved on plastic thin layer
chromatography plates coated with cellulose (Merck), which were
developed in pyridine/ethyl acetate/water/acetic acid (5:3:3:1) for
either 3 days or for a single day if monosaccharides were to be
examined. Resolved components were visualized by autoradiography prior
to their elution from the chromatography plates with water and passed
over coupled columns of AG 50 (H+ form) and AG 1 (acetate
form). Nonretained neutral components were loaded onto charcoal columns
as described previously (16), and after washing, free oligosaccharides
were eluted from the columns with 30% ethanol. Free oligosaccharide
phosphates (2, 30) were isolated from the upper phase of
chloroform/methanol/100 mM Tris-HCl, pH 7.4, containing 4 mM MgCl2 (3:2:1) extracts of Thy
1
cells. Material was dried down and passed over coupled columns of AG 50 over AG 1, and, whereas neutral free oligosaccharides were recovered
from the run-through and water washes, oligosaccharide phosphates were
eluted from the AG 1 with 2 M NaCl. After desalting this
material by Biogel P2 chromatography, it was subjected to thin layer
chromatography on silica-coated plates that were developed overnight in
1-propanol/acetic acid/water (3:3:2). Four components were resolved,
and after digestion with alkaline phosphatase it was found that the
slowest migrating component comigrated with Man5GlcNAc2.
Structural Analysis of Free Oligosaccharides--
Free
oligosaccharides were treated with 1 unit of jack bean
-mannosidase
(Sigma) overnight at 37 °C in 40 mM sodium acetate, pH
4.5, or digested overnight at 37 °C with 5 microunits of
-1,2-mannosidase (Oxford Glycosystems) in 100 mM sodium
acetate buffer, pH 5.0. The digestion products were then desalted as
described above, concentrated, and resolved by thin layer chromatography.
Preparation of [3H]Man9-8GlcNAc and
the Branched Isomer of
[3H]Man5GlcNAc--
Large free
oligosaccharides
([3H]Man9-8GlcNAc2) that were
generated in permeabilized HepG2 cells and recovered from the membrane-bound compartments (7) were treated with endoglycosidase H to
yield [3H]Man9-8GlcNAc. These
oligosaccharides were then treated with the
-1,2-mannosidase (see
above) to yield the branched isomer of
[3H]Man5GlcNAc.
Incubation of [3H]Free OS with Permeabilized HepG2
Cells--
HepG2 cells were harvested from cell culture flasks by
trypsinization and permeabilized with streptolysin O as described
previously (7). Permeabilized cells were then incubated with
[3H]free OS and 20 µM swainsonine (SW) in
transport buffer (31); 130 mM K+/glutamate, 10 mM NaCl, 2 mM EGTA, 1 mM
CaCl2, 2 mM MgCl2, 5 mM HEPES/KOH, (pH 7.3). Incubations were conducted in the presence of ATP
and an ATP-regenerating system (5 mM ATP, 3.5 mM creatine phosphate, 25 mg/ml creatine phosphokinase) and
6 mg/ml dialyzed rat liver cytosol.
Preparation of Rat Liver Lysosomes--
Male Wistar rats were
sacrificed, and livers were cut into small pieces before being
homogenized in 5 volumes of homogenization buffer (250 mM
sucrose, 1 mM EDTA, 20 mM HEPES/KOH, pH 7.3)
using a tight fitting Dounce apparatus. After centrifuging the
homogenate at 600 × g for 10 min, the supernatant was
removed and recentrifuged at 18,000 × gav.
The resulting mitochondrial/lysosomal pellet was resuspended in
homogenization buffer, 5 ml of this suspension was mixed with 3 ml of
an 80% Percoll solution (32), and the gradient was subsequently formed
by centrifugation for 35 min at 92,570 × gav (32). Material recovered from the bottom
third to quarter of the gradient was washed twice with HEPES-buffered sucrose (250 mM sucrose, 20 mM HEPES/KOH, pH
7.3). The final lysosomal pellet was resuspended in 1-1.5 ml of
HEPES-buffered sucrose.
The Transport Assay--
Radioactive substrates were dried down
into Eppendorf tubes, and the following additions were made, on ice, in
the indicated order: 5 µl of water (or, where indicated, test
substances); 5 µl of ATP and an ATP-regenerating system (50 mM ATP, 0.5 mg/ml creatine kinase, 70 mM
creatine phospate); 20 µl of incubation buffer (350 mM
sucrose, 125 mM KCl, 25 mM MgCl2,
20 mM HEPES/KOH, pH 7.3). After mixing, 20 µl of
lysosomes were added to make a final volume of 50 µl. Subsequent to
further mixing, the tubes were incubated on ice for 10 min prior to
being transferred to a water bath at 25 °C. Incubations were
terminated by the addition of 1 ml of ice-cold SH, and lysosomes were
recovered by centrifugation at 11,600 × gav for 15 min at 4 °C. The resulting
supernatant was removed, made 1% with respect to Triton X-100, and
kept for
-hexosaminidase determinations, and the lysosomal pellet
was washed with a further 1 ml of SH prior to being solubilized in 1 ml
of SH containing 1% Triton X-100. Radioactivity associated with the
lysosomal lysate was determined by scintillation counting. In
experiments where [3H]free OS transported into lysosomes
was to be analyzed, the transport assays were terminated by the
addition of 1 ml of ice-cold MH (250 mM mannitol, 20 mM HEPES/KOH, pH 7.3). Lysosomes were also washed, as
described above, with MH.
Measurement of Lysosomal pH--
Lysosomal pH was measured as
described previously (33). Briefly, rat liver lysosomes were loaded
with FITC-dextran in vivo prior to purification as described
above. Fluorescence measurements were made by incubating approximately
1 mg of lysosomes under conditions identical to those used for the
transport assay (see above).
Protein, Sugar, and Enzymatic Assays--
Lysosomal
-D-hexosaminidase activity was measured using
p-nitrophenyl N-acetylglucosamine as described
(34). Protein was measured by the bicinchoninic acid method using a kit
purchased from Sigma.
 |
RESULTS |
Preparation of Radiolabeled Substrates Suitable for Examining the
Cytosol-to-lysosome Transport of Free Oligosaccharides--
In order
to develop an in vitro assay for the lysosomal transport of
free oligosaccharides, we first sought an abundant source of
radiolabeled linear Man5GlcNAc, the component that has been demonstrated to be the major free OS that accumulates in the lysosomes of HepG2 cells treated with the vacuolar ATPase inhibitor CCM A (17).
Since the isomeric configuration of the mannose residues in this free
OS (17) is known to be the same as that occurring in the
Man5GlcNAc2 intermediate that appears during
the biosynthesis of dolichol oligosaccharides, we chose to harvest free
OS from the mutant mouse lymphoma cell line Thy
1 (35),
which, lacking functional dolichol-P-Man synthase, is unable to further
mannosylate this oligosaccharide-lipid intermediate (36, 37). Thin
layer chromatographic analysis of free OS isolated from
Thy
1 cells after metabolic radiolabeling with
[2-3H]mannose for 4 h, shown in Fig.
1A, reveals four
oligosaccharides (free OSa-d), the largest of which comigrates with
authentic Man5GlcNAc. Since it is known that free OS
isolated from mammalian cells possess either a
di-N-acetylchitobiose moiety or a single residue of
N-acetylglucosamine at their reducing termini (1, 4), we
wanted to verify that the free OS comigrating with
Man5GlcNAc was not Man4GlcNAc2 or a
mixture of these two components. This issue was addressed by digesting
free OSa with jack bean
-mannosidase, and Fig. 1A
(lane 6) shows that this treatment yielded
mannose and the disaccharide Man
1-4GlcNAc, confirming the parent
structure's identity as Man5GlcNAc and not
Man4GlcNAc2. Furthermore Fig. 1A
(lane 7) reveals that free OSa was sensitive to
an
-1,2-mannosidase, yielding mannose and a structure that
comigrated Man3GlcNAc, confirming that free OSa contains
two
-1,2-linked mannose residues. Taking into account these results
and what is known of the dolichol oligosaccharide biosynthetic pathway
in Thy
1 cells (36, 37), free OSa possesses the structure
shown in Fig. 1B. Similar structural analyses (results not
shown) of free OSb-d indicated that these three oligosaccharides are
likely to have the structures indicated in Fig. 1B. Because
the mixture of [3H]free OS comprised about 60%
Man5GlcNAc, initial transport assays were carried out using
the unfractionated oligosaccharide mixture, hereafter referred to as
[3H]free OS. In some instances, purified radiolabeled
[3H]free OSa was used as substrate and is referred to as
[3H]Man5GlcNAc.

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Fig. 1.
Structural analysis of [3H]free
OS isolated from mutant lymphoma
Thy 1 cells. A,
mutant lymphoma Thy 1 cells were metabolically
radiolabeled with [2-3H]mannose as described under
"Experimental Procedures." Total [3H]free OS were
recovered and analyzed by thin layer chromatography (lane
1), and the four species, labeled a,
b, c, and d, were eluted from the
plate and rechromatographed to assess their purity (lanes
2, 3, 4, and 5,
respectively). The chromatograph was developed for 3 days, and the
resolved components were detected by fluorography. The
[3H]free OS labeled a was subjected to jack
bean -mannosidase (lane 6) or
-1,2-mannosidase (lane 7) treatment, and the
resulting digestion products were resolved by thin layer
chromatography. The chromatograph was developed for 1 day.
M, mannose; MGN, Man 1-4GlcNAc;
MGN2, Man 1-4GlcNAc 1-4GlcNAc;
M3GN, Man3GlcNAc.
B, [3H]free OSa-d (A),
characterized as shown above, have been assigned the indicated
structures, and the amount of each component recovered from a typical
radiolabeling experiment is shown. The linkage position of the terminal
nonreducing mannose residue of [3H]free OSd was not
determined.
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|
Exogenously Applied [3H]Free OS Specifically
Associates with the Heavy Membranes of Permeabilized
Cells--
Initial experiments were carried out by incubating
permeabilized cells with [3H]free OS in the presence of a
preparation of dialyzed rat liver cytosol and ATP. SW, a potent
inhibitor of lysosomal mannosidases, was also included in assay
incubations in order to protect any [3H]free OS that
became internalized in lysosomes. Results demonstrated a
time-dependent association of [3H]free OS
with permeabilized cells that was abolished at 4 °C, stimulated
2-fold by ATP, and stimulated 1.25-fold by the inclusion of 6 mg/ml
dialyzed cytosol (results not shown). However, we only observed, while
trying to quench the reactions with 200 µM cold Man5GlcNAc, a modest inhibition of the association of
[3H]free OS with permeabilized cells, indicating a high
nonspecific component to this process. In order to examine this problem
in more detail, permeabilized cells were incubated with either
[3H]free OS or [3H]inulin in the presence
of ATP, SW, and cytosol; washed; and then, after homogenization,
fractionated on Percoll density gradients. Fig.
2 demonstrates that whereas both
[3H]free OS and [3H]inulin are recovered
from regions of the Percoll gradient (fractions 5-11) known to contain
light membranes such as those derived from the Golgi apparatus, ER, and
endosomal system (17, 32), only [3H]free OS associated
with more dense regions of the Percoll gradient containing the
lysosomal marker enzyme
-hexosaminidase. This association was
abolished when incubations were quenched with 200 µM
unlabeled Man5GlcNAc. These results, although suggesting a
specific association of [3H]free OS with lysosomal
membranes, indicated that because of the high nonspecific association
of radioactive components with permeabilized cells another assay system
would be required.

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Fig. 2.
Percoll density gradient fractionation of
permeabilized HepG2 cell homogenates. Permeabilized HepG2 cells
were incubated with either [3H]free OS (lower
panel) or [3H]inulin (upper
panel) as described under "Experimental Procedures."
Incubations with [3H]free OS were carried out in either
the absence (solid circles) or presence
(open circles) of 200 µM unlabeled
Man5GlcNAc. After terminating the incubations, the
permeabilized cells were washed and homogenized. Postnuclear
supernatants were mixed with Percoll and centrifuged as described under
"Experimental Procedures." The gradients were then separated into
20 fractions, and aliquots were taken for scintillation counting and
-hexosaminidase ( -HEX) assay.
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[3H]Free OS, but Not [3H]Inulin,
Associates with the Membranes of a Lysosome-enriched Particle Fraction
in an ATP-dependent Manner--
Percoll-purified rat liver
lysosomes were incubated with [3H]free OS or
[3H]inulin in the presence or absence of ATP at 37, 30, or 25 °C for the times indicated in Fig.
3. At each time point, the stability of
lysosomes was assessed by measuring the
-hexosaminidase associated with both the pelleted membranes and the supernatant. Results show that
at 37 °C there is, in contrast to the situation observed with
[3H]inulin, a rapid ATP-dependent association
of [3H]free OS with sedimentable membranes. Since there
was an approximately 50% loss of
-hexosaminidase from lysosomes
incubated at 37 °C for 80 min, further experiments were carried out
at 30 and 25 °C, and as can be seen, incubations performed at the
lower temperatures greatly increased the stability of lysosomes over
the longer incubation periods. Although these reduced incubation
temperatures lead to slower initial rates of association of
[3H]free OS with membranes, this association is more
sustained and reaches higher maximal levels at the two reduced
temperatures. These results demonstrate a time-, temperature-, and
ATP-dependent association of [3H]free OS, but
not [3H]inulin, with the membranes of a lysosome-enriched
particulate fraction. Because this association was ATP- and
temperature-dependent and appeared to correlate with the
intactness of lysosomes in the lysosomal preparation, it seemed
possible that [3H]free OS were being transported into
lysosomes.

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Fig. 3.
Association of [3H]free OS with
the particulate fraction of a partially pure preparation of lysosomes
is time-, temperature-, and ATP-dependent. The
Percoll-purified lysosomal fraction was incubated with either
[3H]free OS or [3H]inulin at 37, 30, or
25 °C for the indicated times in either the absence (open
circle) or presence (closed circle) of ATP. Incubations
were terminated by the addition of ice-cold SH, and the particulate
fraction was washed twice with this buffer prior to being suspended in
SH containing 1% TX-100. Aliquots of this material were taken for
scintillation counting (SEDIMENTABLE RADIOACTIVITY).
Aliquots of both the particulate fraction and the first wash
supernatant were assayed for -hexosaminidase activity in order to
compute the amount of this enzyme remaining associated with the
particulate fraction as a percentage of the total enzyme recovered from
both the particulate and supernatant fractions (% SEDIMENTABLE -HEX).
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[3H]Free OS Transported into Lysosomes--
If free
OS are being transported into functional lysosomes, they should be
subject to complete catabolism by lysosomal
- and
-mannosidases,
yielding mannose and N-acetylglucosamine. Since both of
these monosaccharides can be transported out of lysosomes (38, 39), the
observed transport of free OS into lysosomes should be greatly
increased in the presence of SW, a potent inhibitor of lysosomal
mannosidases. If, on the other hand, free OS are simply adsorbed onto
the outer surface of membrane-bound organelles, then this process would
not be expected to be affected by either the absence or presence of SW.
In order to distinguish between these two possibilities, we examined
the importance of SW in the assay mixtures, and as shown in Fig.
4A the association of
[3H]free OS with lysosomal membranes is greatly enhanced
when SW is included in the incubation medium. But does SW increase the association of [3H]free OS with membranes because it
protects input [3H]free OS from extralysosomal
mannosidases during the transport assay, or because it protects
transported substrate from latent (intralysosomal) mannosidases? This
question was addressed by looking at the stability of
[3H]Man5GlcNAc in both the supernatant and
particulate fractions obtained from transport assays that had been
conducted in either the absence or presence of SW. Fig. 4B
(upper part) shows that [3H]Man5GlcNAc, recovered from the
supernatant of transport assays conducted in the absence of SW, is
stable, suggesting that SW protects sedimentable
[3H]Man5GlcNAc from a latent mannosidase
activity. Taken together, these results show that
[3H]Man5GlcNAc is transported into functional
lysosomes.

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Fig. 4.
[3H]free OS is transported into
lysosomes. A, lysosomes were incubated with
[3H]free OS at 25 °C for 1 h in the presence of
ATP and increasing concentrations of swainsonine. Radioactivity
associated with the particulate fraction (solid
circles) was assayed as described for Fig. 3
(SEDIMENTABLE RADIOACTIVITY). The amount of
sedimentable -hexosaminidase has been expressed as a percentage of
that occurring in the absence of swainsonine. B, after
incubation of lysosomes with [3H]Man5GlcNAc,
radioactive components were recovered from the supernatant
(top panel), and particulate (bottom
panel) fractions derived from incubations conducted in
either the absence or presence of 100 µM swainsonine and
analyzed by thin layer chromatography.
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Lysosomal Import of [3H]Man5GlcNAc
Displays Michaelis-Menten-type Kinetics--
Since lysosomes are not
permeable to the disaccharide sucrose (40), this organelle would not be
expected to be permeable to larger free OS. Accordingly, the lysosomal
membrane should possess free OS transport machinery showing both
specificity and saturability. The saturability of the transport process
was examined by incubating lysosomes with increasing concentrations of
[3H]Man5GlcNAc (1 × 105
cpm/nmol). These incubations were performed at 30 °C for 10 min, conditions where transport was observed to be linear (see Fig. 3).
Results demonstrate (Fig. 5) that the
ATP-dependent transport of
[3H]Man5GlcNAc into lysosomes shows
saturability, and typical Michaelis-Menten saturation kinetics
(Km, 22.3 µM;
Vmax, 7.1 fmol/min/unit of
-hexosaminidase)
were observed.

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Fig. 5.
Kinetics of ATP-dependent
[3H]free OS transport into lysosomes.
Upper panel, lysosomes were incubated either in
the absence or presence of ATP at 30 °C for 10 min with increasing
concentrations of [3H]Man5GlcNAc (specific
activity 1 × 105 cpm/nmol). After terminating the
incubations and washing the particulate fraction twice, aliquots of the
lysed lysosomal pellet were taken for scintillation counting and
-hexosaminidase assay as described under "Experimental
Procedures." The saturable transport component (curve without
experimental points) was computed by subtracting the ATP-independent
sedimentable radioactivity from the ATP-dependent
sedimentable radioactivity. Lower panel, double
reciprocal analysis of the saturable, ATP-dependent,
transport component.
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Lysosomal Free OS Transport Shows Selectivity toward Trimmed,
Nonphosphorylated Free OS--
In order to examine the selectivity of
free OS transport into isolated rat liver lysosomes, we conducted
transport assays using the free OS that are generated physiologically
by the ER during glycoprotein biosynthesis. First, phosphorylated
Man5GlcNAc2 (Man5GlcNAc2-P; see Fig.
6A) released from
dolichol-PP-Man5GlcNAc2 on the cytosolic face
of the ER (5), was tested, and as can be seen in Fig. 6A we
were unable to detect lysosomal sequestration of this compound unless
it had been previously treated with alkaline phosphatase, indicating
that the specific activity of our preparation of phosphorylated
[3H]Man5 GlcNAc2 was
sufficiently high to observe transport. In addition, these results show
that free OS terminating with a reducing di-N-acetylchitobiose moiety can also be transported into
rat liver lysosomes. Second, we were not able to detect lysosomal import of the large free polymannose oligosaccharides
([3H]Man9-8GlcNAc2; see Fig.
6A) that are transported out of the ER during glycoprotein
biosynthesis (7, 8). Furthermore, treatment of these components with
endoglycosidase H, generating [3H]Man9-8GlcNAc, did not affect their
ability to be sequestered by lysosomes. However, treatment of this free
OS preparation with endoglycosidase H and an
-1,2-mannosidase
generated a branched isomer of
[3H]Man5 GlcNAc, which was found to be
transport-competent, indicating that the specific activity of the
parent [3H]Man9-8GlcNAc2 was
sufficient in order to observe lysosomal import. The above result
indicated that free OS uptake is not dependent upon the
-1,2-linked
mannoses of the free OS, suggesting that the Man4-3GlcNAc
structures shown in Fig. 1 may also be transport substrates. This is
confirmed in Fig. 6B, where it is shown that these
oligosaccharides are good transport substrates. In summary, these
results demonstrate that the free OS and phosphorylated free OS that
are generated by the ER during glycoprotein biosynthesis are not
efficiently imported into lysosomes unless they are processed by
cytosolic glycosidases and/or phosphatases.

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Fig. 6.
Phosphorylated and fully mannosylated free
oligosaccharides are poor substrates for lysosomal import.
A, standard transport assays were conducted using either an
aliquot of [3H]Man5GlcNAc2-P
(structure on upper left) or an aliquot of the
same preparation treated with alkaline phosphatase (Alk.
phosp'ase) as substrates. In a separate experiment, transport
assays were conducted on aliquots of a preparation of large free
polymannose-type oligosaccharides
([3H]Man9-8GlcNAc2; structure on
upper right), which were either untreated or
digested with endoglycosidase H (Endo H) alone to yield
[3H]Man9-8GlcNAc or endoglycosidase H and an
-1,2-mannosidase (the dotted lines in the
structure on upper right indicate the -1,2
linkages) to yield the branched isomer of
[3H]Man5GlcNAc. B, 50,000 cpm of
the indicated oligosaccharides were incubated with lysosomes under the
standard assay conditions for 0, 5, and 10 min. The oligosaccharide
structures and their abbreviations are defined in the legend to Fig.
1.
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The Cytosol-to-lysosome Free OS Transporter Recognizes a Reducing
N-Acetylglucosamine-containing Sugar Motif--
In order to compare
lysosomal free OS transport with other sugar transport processes, we
wanted to characterize the sugar motif that is recognized by the
lysosomal transporter. Knowing that the ER-to-cytosol transport of free
OS is inhibited by mannose and various mannosides (8), we conducted
lysosomal transport assays in the presence of several commonly
occurring monosaccharides. Fig.
7A shows that when lysosomes
are incubated with [3H]Man5GlcNAc in the
presence 50 mM glucose, galactose, mannose, fucose, or
N-acetylglucosamine (GlcNAc), each provokes a 15% loss of
-hexosaminidase from lysosomes; in contrast, whereas glucose, galactose, mannose, and fucose cause about 20% transport inhibition, GlcNAc completely abolishes [3H]Man5GlcNAc
transport. Furthermore, dose-response curves to GlcNAc, N-acetylgalactosamine, and N-acetylmannosamine
(see Table I), show that GlcNAc
(IC50, 1.2 mM) is 20 times more potent than
GalNAc (IC50, 26 mM) at inhibiting
[3H]Man5GlcNAc transport, whereas ManNAc has
little inhibitory activity. Since streptozotocin and glucosamine have
reduced [3H]Man5GlcNAc transport inhibitory
potencies when compared with that of GlcNAc, it is clear that the
N-acetyl group of the GlcNAc residue plays an important role
in oligosaccharide recognition by the transport machinery (Table I).
Fig. 7B and Table I demonstrate two important structural
features required for increased inhibitory potency of sugars. First,
the compound must possess an intact or free reducing GlcNAc residue;
accordingly, N-acetylglucosaminitol and
-benzyl GlcNAc
have reduced inhibitory capacities when compared with that of GlcNAc,
and di-N-acetylchitobiitol and
-benzyl
di-N-acetylchitobioside are much less potent than
di-N-acetylchitobiose at inhibiting [3H]Man5GlcNAc transport into lysosomes.
Second, substituting the reducing GlcNAc residue with Gal, Man, or
GlcNAc at position 4 results in 7-, 19-, and 240-fold increases,
respectively, in the inhibitory potency of the GlcNAc, indicating that
although a reducing GlcNAc moitey is the most important determinant of
the structural motif recognized by the transport machinery, a second
sugar linked
1-4 to the GlcNAc greatly enhances the interaction of
the substrate with the transporter. In addition, Table I shows that the
linear isomer of Man5GlcNAc is about 2-fold more efficient
at inhibiting [3H]Man5GlcNAc transport than
is Man
1-4GlcNAc, indicating that the transporter recognizes not
only this disaccharide unit but also other more distal mannose
residues. Finally, Table I shows that whereas the branched isomer of
Man5GlcNAc (see Fig. 6) interacts with the transporter less
efficiently than its linear counterpart, the same oligosaccharide
devoid of its reducing GlcNAc residue had no effect on
[3H]Man5GlcNAc transport over the
concentration range tested. In another type of experiment we treated
[3H]Man5GlcNAc with sodium borohydride and
found that the reduced oligosaccharide was no longer a substrate for
lysosomal import (results not shown). Taken together with the transport
specificity studies shown in Fig. 6 and the transport inhibition
experiments summarized in Table I, these results demonstrate that the
lysosomal transporter recognizes predominantly the reducing
Man
1-4GlcNAc disaccharide moiety of free OS.

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Fig. 7.
Lysosomal
[3H]Man5GlcNAc transport is inhibited by
N-acetylglucosamine and various
N-acetylglucosamine-containing disaccharides.
A, standard transport incubations were conducted in the
presence of the indicated monosaccharides (50 mM), and the
percentage of inhibition of [3H]Man5GlcNAc
transport was evaluated as described under "Experimental
Procedures." The percentage of sedimentable -hexosaminidase
present at the end of each incubation was evaluated by performing
-hexosaminidase assays on both the pellet and supernatant fractions
and comparing this value to that obtained in incubations performed in
the presence of ATP alone (100% sedimentable -hexosaminidase).
B, standard transport assays were performed in the presence
of increasing amounts of GlcNAc (GN), Man 1-4GlcNAc
(Man-GN), Gal 1-4GlcNAc (Gal-GN), and
GlcNAc 1-4GlcNAc (GN-GN).
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Lysosomal Free Oligosaccharide Transport Is Not Blocked by Various
Commonly Used Sugar Transport Inhibitors--
The selectivity of the
lysosomal free OS transport process suggested that it is carried out by
a novel transporter distinct from the hexose and GlcNAc/GalNAc
transporters already described in mammalian cells. In order to gain
more information on the characteristics of this transporter, we
conducted transport assays in the presence of various classical sugar
transport inhibitors. Cytochalasin B, unlike its close relative
cytochalasin D, inhibits the plasma membrane glucose carrier (41), the
lysosomal hexose carrier (42), and also the lysosomal
N-acetylhexosamine transporter (38). Phloretin and
phloridzin inhibit the plasma membrane glucose carrier and the
Na+-dependent active hexose transporter,
respectively (41, 43), whereas verapamil is an excellent substrate for
the multidrug resistance protein (44). However, as can be seen from the
upper portion of Table II, none of the
above compounds possess significant inhibitory action toward
[3H]free OS transport into lysosomes.
[3H]Free OS Transport into Isolated Rat Lysosomes
Requires the Hydrolysis of ATP and Is NEM-sensitive but Independent of
the Ability of the Vacuolar ATPase to Acidify Lysosomes--
In order
to further characterize the import of free OS into isolated rat liver
lysosomes, we have investigated the role of ATP in this process using
the in vitro transport assay. First, we tested whether free
OS transport into isolated rat liver lysosomes required the hydrolysis
of ATP, and as shown in Fig.
8A the nonhydrolyzable ATP
analogs ATP
S, AMP-PNP, and AMP-PCP were not efficient promoters of
free OS transport. We went on to verify that, under our transport assay
conditions, ATP hydrolysis leads to acidification of lysosomes, and, as
shown in Fig. 8B, the addition of ATP to FITC-dextran-loaded lysosomes led to a carbonyl cyanide
m-chlorophenylhydrazone-reversible quenching of
fluorescence, indicating lysosomal acidification (33); furthermore,
this acidification was inhibited by CCM A and NEM, two inhibitors of
the vacuolar ATPase (45, 46). Next, we examined the nature of the
ATPase activity responsible for the coupling of ATP hydrolysis to
lysosomal free OS import by testing a panel of well known ATPase
inhibitors in the transport assay. Table II shows that whereas
[3H]free OS transport is sensitive NEM, a nonspecific
thiol-reactive ATPase inhibitor, the specific vacuolar ATPase
inhibitor, CCM A, is without effect. Furthermore, Table II demonstrates
that whereas vanadate, a known inhibitor of P-type ATPases (47) and the
multidrug-resistant glycoprotein (44), weakly inhibits free OS
transport into lysosomes, sodium azide, a potent inhibitor of the
mitochondrial F0-F1-type ATPase (45), was able
to significantly potentiate [3H]free OS transport into
lysosomes. This effect may be due to the latter compound's ability to
inhibit phosphatases in the incubation mixtures, thereby protecting the
ATP and ADP during assays.

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Fig. 8.
ATP hydrolysis leads to both free OS
transport and activation of the proton pumping ATPase.
A, transport assays were carried out in the presence of 5 mM ATP or the nonhydrolyzable ATP analogues ATP S,
AMP-PNP, and AMP-PCP (all 5 mM). The transport of
[3H]free OS observed in the presence of 5 mM
ATP has been set at 100%. Lysosome disruption was monitored as
described in the legend to Fig. 6, and the percentage of sedimentable
-hexosaminidase observed in the presence of 5 mM ATP has
been set at 100%. B, FITC-dextran-loaded lysosomes were
prepared as described under "Experimental Procedures" and incubated
under standard transport assay conditions at room temperature
(22 °C) in a stirred cuvette. Fluorescence recordings were
initiated, and, after stabilization of the base line, water
(upper trace), NEM (1 mM final
concentration, middle trace), or CCM A (100 nM final concentration, lower trace)
was added prior to the addition of ATP (6.25 mM final
concentration). After observing the effect of ATP, the proton ionophore
carbonyl cyanide m-chlorophenylhydrazone was added (25 µM final). A downward deflection of the trace is a
measure of the fluorescence quenching caused by lysosomal
acidification, whereas an upward deflection represents an
alkalinization of the lysosomal interior.
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DISCUSSION |
Initial transport experiments, carried out using permeabilized
HepG2 cells (Fig. 2), suggested that lysosomal membranes have the
ability to interact with [3H]free OS and led us to test
the ability of partially purified rat liver lysosomes to sequester
these components. Our lysosome purification procedure led to a
30-40-fold enrichment of
-hexosaminidase activity over that
observed in the initial homogenate (results not shown), and in general,
greater than 90% of the
-hexosaminidase was found to be
sedimentable (see percentage of sedimentable
-hexosaminidase at
t = 0 in Fig. 3). In addition, we were able to show, as
have others (33), that lysosomes remain acidic during their
purification. Thus, the addition of either the proton ionophore,
carbonyl cyanide m-chlorophenylhydrazone, or valinomycin, a
potassium ionophore, individually to FITC-dextran-loaded lysosomes
resulted in modest alkalinizations, whereas the addition of both
reagents together induced a rapid and substantial alkalinization of
lysosomes (results not shown), indicating that the lysosomal membranes
in our lysosome preparation had low permeabilities to both hydrogen and
potassium ions and were more acid than the surrounding incubation
medium (33).
Much evidence indicates that not only do free OS associate with the
lysosomes in the lysosome-enriched preparation, but they are also
transported into functional lysosomes. First, there appeared to be a
correlation between association of free OS with the membranes of the
lysosomal preparation and the stability of the lysosomes in this
preparation (see Fig. 3). Second, we found that association of free OS
with the particulate fraction of the lysosomal preparation was greatly
enhanced by the addition of low concentrations of SW to the incubation
mixtures. As SW had no effect on the stability of free OS not
associated with membranes, we were able to conclude that SW is
protecting sedimentable free OS from latent mannosidase activities.
Since the lysosome is the only known organelle possessing enzymes
capable of completely hydrolyzing free OS into their constituent monosaccharides, we conclude that free OS are being transported into
the lysosomes of our lysosome-enriched fraction and not into other
contaminating organelles.
The Lysosomal Free OS Transporter Displays a Unique Specificity
That Distinguishes It from the ER-to-cytosol Free OS
Transporter--
Our results show that large polymannose structures
(Man9-8GlcNAc2), irrespective of whether they
are free or linked to peptide, protein, or lipid, interact poorly with
the transporter. On the other hand, less mannosylated free OS
(Man5-3GlcNAc) were found to be transport substrates,
indicating that the peripheral mannose residues of the larger
structures are inhibitory to transport. Inhibition studies with mono-
and disaccharides (Fig. 7 and Table I) revealed that the transporter
binds predominantly to the GlcNAc-containing reducing terminus of the
free OS and that a second sugar linked
-1,4 to the reducing GlcNAc
enhances affinity. Furthermore, since linear Man5GlcNAc was
better at quenching transport than Man
1-4GlcNAc and
[3H]Man3GlcNAc appeared to be a better
transport substrate than [3H]Man5GlcNAc,
either or both of the mannosyl residues linked
-1,3 and
-1,6 to
the Man
1-4-GlcNAc core must also add to the binding affinity. To
summarize, a possible hypothesis for the interaction of free OS with
the lysosomal transporter is that small free oligosaccharides are able
to enter a pocket in the transport molecule and, once inside, bind
tightly to the transporter via their reducing termini. More fully
mannosylated or branched free oligosaccharides, although possessing the
same high affinity binding motif, are sterically impeded from entering
the pocket and are therefore neither good substrates for transport nor
good transport inhibitors. Surprisingly, we noted that the most potent
inhibitors of [3H]free OS import into lysosomes were
small chitooligosaccharides containing 2-4 residues of GlcNAc, and the
parallelism of the dose-response curves shown in Fig. 7B
suggests that GlcNAc, chitobiose, N-acetyllactosamine, and
Man
1-4-GlcNAc inhibit free OS transport by a common mechanism.
These results suggest that the high affinity binding pocket of the
lysosomal free OS transporter may be similar to those of many
chitin-binding proteins and chitinases that possess a cysteine and
glycine-rich structural motif known as the hevein domain (48). Lectins
possessing this structural motif such as wheat germ agglutinin (49) and
Urtica dioca agglutinin (50) bind GlcNAc and small
chitooligosaccharides in the following order: chitotetraose
chitotriose > chitobiose
GlcNAc. These observations led
investigators to propose that the sugar binding sites of such lectins
comprise three closely situated subsites (49, 50). However, our
transport inhibition studies (Table I) demonstrate that
chitooligosaccharides inhibit [3H]free OS transport with
the following order of potency: chitotetraose
chitotriose
chitobiose
GlcNAc, suggesting that the free OS binding pocket
of the lysosomal transporter may possess only two high affinity sugar
binding sites. It is also noteworthy that both
- and
-N-acetylglucosaminisides bind more efficiently to wheat
germ agglutinin than does GlcNAc, whereas our transport inhibition
studies show that blocking of the reducing terminus of GlcNAc and
di-N-acetylchitobiose with
- and
-benzyl moieties, respectively, greatly reduced their capacities to quench free OS
transport (Table I), indicating further differences between the
oligosaccharide binding domain of the lysosomal free OS transporter and
that of hevein-type lectins. At present, it is not known whether or not
the lysosomal free OS transporter can import chitooligosaccharides into
lysosomes, but it is noteworthy that similar structures not only exist
in invertebrates and fungi but may exist in vertebrates and play a role
in their development (51, 52).
The ER-to-cytosol transport of large free polymannose-type OS can be
inhibited by mannose (IC50, 4.9 mM) and
mannosides (benzyl mannoside, IC50, 0.8 mM) but
neither by N-acetylglucosamine (50 mM) nor
di-N-acetylchitobiose (10 mM) (8), indicating
that the ER and lysosomal transporters recognize different structural motifs of free OS. Whereas the interaction of large free
polymannose-type oligosaccharides with the ER-to-cytosol transport
apparatus has similarities to ER-situated
N-glycan-lectin/enzyme interactions (calcium
dependence/inhibition by mannosides; see Ref. 8), the interaction of
free OS with the lysosomal transporter bears similarities to the
interactions between free OS and cytosolic glycosidases. Accordingly,
the cytosolic chitobiase (15) by definition interacts with the reducing
di-N-acetylchitobiose moiety of free OS, but more
surprisingly, the cytosolic mannosidase has been found to act only on
free OS bearing a single GlcNAc residue at their reducing termini (20).
Therefore, these two enzymes and the cytosol-to-lysosome free OS
transporter interact with free OS in a manner that involves
surveillance of the reducing terminus of the oligosaccharide. Perhaps
this system has developed to promote an ordered processing of free OS
in the cytosol and to ensure that glycoproteins, glycopeptides, and
dolichol oligosaccharides do not interfere with the sequestration of
free OS by lysosomes.
A Novel Transporter Is Responsible for the Import of Free
Oligosaccharides into Lysosomes--
How do the kinetic
characteristics of lysosomal free OS transport compare with those of
other lysosomal and plasma membrane sugar transporters? Here we
observed that lysosomal free OS sequestration was
temperature-dependent and displayed a
Q10 of about 2.5 (calculated from the
ATP-dependent transport observed after 5-min incubations at
37, 30, and 25 °C; see Fig. 3), which is similar to other lysosomal sugar transport systems (N-acetylhexosamines (38),
Q10 = 2.3; sialic acid (53),
Q10 = 2.4). We were unable to detect saturable free OS transport into lysosomes unless ATP was included in the incubation mixtures; furthermore, we show that free OS transport into
lysosomes is critically dependent upon ATP hydrolysis, yet independent
of a functional lysosomal proton pump. In contrast, other mammalian
lysosomal transporters influenced by ATP such as those described for
sialic acid and sulfate show saturability in the absence of ATP and
have been shown to be stimulated only about 2-fold by the addition of
ATP (39, 54). The ATP-dependent transport of free OS into
lysosomes showed Michaelis-Menten-type saturation kinetics displaying a
Kuptake of 22.3 µM. This value is
significantly lower than the Kuptake values
obtained for lysosomal hexose (Glc, 75-90 mM; Man, 50-75
mM (42)), N-acetylhexosamine (GlcNAc, 4.4 mM, GalNAc, 4.4 mM (38)), and sialic acid (0.24 mM (39)) carriers. Our Kuptake value
must be considered as an approximation, since 10-min incubations at
30 °C represent the limit of the linearity of the initial transport
rate; however, the time chosen was a compromise, since shorter
incubation times would have led to very low levels of radioactivity
becoming associated with lysosomes. Nevertheless, another cytosolic
activity implicated in the clearance of ER-generated free OS from the
cytosol, the neutral cytosolic endoglycosidase H-like activity, was
recently found to have a Km value of 25 µM for pyridylaminated Man6GlcNAc2 (55). Here, we demonstrate that
free OS import into lysosomes was sensitive to NEM, a thiol-reactive
drug that inhibits many ATPases (33, 56). In contrast, free OS import
into lysosomes was only weakly inhibited by vanadate, an inhibitor of
P-type ATPases (47) and not at all by the F0-F1
type ATPase inhibitor sodium azide (45). Furthermore, we were unable to
demonstrate inhibition of lysosomal free OS import by other well known
sugar transporters such as cytochalasin B, phloretin, and phloridzin. Taken together with the unique selectivity of the transport process, these results demonstrate that the sequestration of free OS into lysosomes is accomplished by a novel transporter. The ability of
lysosomes to continually clear ER-generated free polymannose-type oligosaccharides from the cytosol indicates that this free OS transporter must be considered an important housekeeping activity of
cells engaged in the biosynthesis of glycoproteins.