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Biochemistry and Biophysics |
Department of Biochemistry, University of Western Australia, Nedlands WA 6907, Australia [K. L., P. T., J. L. C., L. M.], and Zentrum für Angewandte Genetik, Universität für Bodenkultur Wien, A-1190 Vienna, Austria [S. C., L. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Intracellular trafficking of cathepsins to lysosomes depends on the presence of M6P5 in the carbohydrate moieties of the proteinases. These residues mediate binding to specific M6P receptors (7) . Mutant cells with either impaired synthesis of the M6P recognition marker or a M6P receptor deficiency fail to retain their newly synthesized lysosomal hydrolases (8) . Two distinct M6P-binding proteins occur in mammalian cells, the 300 kDa M6P/IGF2R and the 46 kDa cation-dependent MPR46. Besides representing the main targeting receptor for lysosomal enzymes, M6P/IGF2R binds IGF-II and TGF-ß precursor (9) . M6P/IGF2R participates in the degradation of IGF-II, a potent growth stimulant and mitogen that is often overproduced in tumors (9 , 10) . Furthermore, the receptor facilitates activation of latent TGF-ß, a growth-suppressing cytokine (11) . Because of this dual role in the control of cellular growth, M6P/IGF2R is considered a tumor suppressor (10) . Indeed, the M6P/IGF2R gene is frequently mutated in liver, breast, and gastrointestinal cancers (12, 13, 14) . Recently, direct evidence for a growth-suppressive role of M6P/IGF2R in tumors has been provided (15) . However, the impact of M6P/IGF2R on tumor invasion and metastasis has not yet been investigated.
We have shown previously that the invasive properties of murine SCC-VII squamous carcinoma cells are in part attributable to the extracellular action of the major lysosomal cysteine proteinase cathepsin B (16) . In this report, we demonstrate that SCC-VII cells are deficient in M6P/IGF2R, causing hypersecretion of cathepsin B and other lysosomal proteinases. Our results indicate that loss of M6P/IGF2R may promote the invasiveness of malignant tumor cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies.
The production and characterization of a rabbit antiserum against
purified human liver cathepsin B has been reported previously
(17)
. The antiserum has been shown to cross-react with all
forms of the murine enzyme (16)
. Rabbit antiserum against
rat M6P/IGF2R was generously supplied by Dr. Thomas Braulke
(Georg-August University Göttingen, Germany). Dr. Braulke also
provided an anti-peptide antiserum against the COOH terminus of murine
MPR46, with the kind permission of Dr. Annette Hille-Rehfeld from the
same department. Antisera against mouse cathepsins D and L were donated
by Dr. John S. Mort (Shriner’s Hospital for Crippled Children,
Montreal, Quebec, Canada) and Dr. Ann H. Erickson (University of North
Carolina, Durham, NC), respectively.
Cell Culture.
Murine SCC-VII squamous carcinoma cells were propagated in Minimal
Essential Medium supplemented with 10% fetal bovine serum, 2
mM glutamine, 100 µg/ml penicillin, and 100 µg/ml
streptomycin at 37°C as described (16)
. Human HepG2
hepatoma cells and normal human GM5522 skin fibroblasts were obtained
from the American Type Culture Collection (Rockville, MD) and the Human
Genetic Mutant Cell Repository (Camden, NJ), respectively. Both cell
lines were cultured as outlined above. Murine 3T3-L1 fibroblasts,
supplied by Dr. David James (University of Queensland, Brisbane,
Queensland, Australia), were maintained in DMEM supplemented with 10%
bovine calf serum, 2 mM glutamine, 100 µg/ml penicillin,
and 100 µg/ml streptomycin at 37°C. Murine NIH 3T3 fibroblasts were
obtained from the American Type Culture Collection and propagated in
DMEM supplemented with 10% fetal bovine serum, 2 mM
glutamine, 100 µg/ml penicillin, and 100 µg/ml streptomycin at
37°C. All tissue culture reagents were purchased from Life
Technologies, Inc. (Gaithersburg, MD).
Metabolic Labeling and Immunoprecipitation.
Confluent cell monolayers were metabolically labeled for 1 h with
[35S]methionine (100 µCi/ml) or
[33P]Pi (250 µCi/ml)
and were subsequently chased for 4 h as described
(17)
. When added, NH4Cl (10
mM) and chloroquine (30 µM) were present
throughout the entire experimental procedure. Immunoprecipitation of
antigens from labeled cell and medium extracts, followed by SDS-PAGE
and fluorography, was performed as reported (17
, 18)
. To
determine the amount of label incorporated into individual
polypeptides, the corresponding gel areas were excised and solubilized
in 30% H2O2/0.25%
NH3 prior to quantification by liquid
scintillation counting (17)
. The raw data were corrected
for the number of methionine residues present in each polypeptide. The
complete sequences of mouse procathepsins B, D, and L are available
from the National Center of Biotechnology
Information.6
Endocytosis Experiments.
Confluent layers of 3T3-L1 and SCC-VII cells were incubated with
NH4Cl-induced secretions of
[35S]methionine-labeled HepG2 cells
(3 x 106
cpm/ml) for 20 h at
37°C. Internalization of HepG2 procathepsins B and D by the
respective recipient cells was determined by immunoprecipitation,
followed by SDS-PAGE and fluorography as reported (18)
.
Subcellular Fractionation.
HepG2- and SCC-VII cells were incubated in complete culture medium
containing 2 mg/ml HRP for 5 min at 37°C. The cells were then washed
prior to a 10-min chase in HRP-free medium to label endosomes.
Postnuclear supernatants were obtained and fractionated by Percoll
density gradient centrifugation as described (19)
. The
activity of lysosomal ß-N-acetylhexosaminidase was
determined spectrophotometrically with
4-nitrophenyl-ß-N-acetylglucosaminide (17)
.
Cathepsin B was assayed spectrofluorometrically with
benzyloxycarbonyl-arginyl-arginyl-7-amido-4-methylcoumarin (Bachem,
Bubendorf, Switzerland) as substrate (6)
. The activity of
galactosyltransferase, a Golgi enzyme, was measured with
UDP-[3H]galactose and chicken ovalbumin as
reported by Rome et al. (20)
with minor
modifications. Lysosomal acid phosphatase was assayed with
4-nitrophenyl phosphate according to Saftig et al.
(21)
. HRP activity was determined at ambient temperature
in 0.1 M sodium acetate buffer (pH 5.0),
containing 50 µg/ml 3,3',5,5'-tetramethylbenzidine and 0.01%
H2O2. The reactions were
stopped with 2 M sulfuric acid and analyzed by
spectrophotometry at 450 nm. The buoyant density of each gradient
fraction was determined gravimetrically, using calibrated glass
constriction pipettes (19)
.
Western Blotting Analysis.
Confluent cell monolayers were harvested and disrupted essentially as
described (17)
, and the homogenates were then centrifuged
for 10 min at 10,000 x g. The soluble
fractions were recovered, and the membrane pellets were extracted with
20 mM sodium phosphate buffer (pH 7.4), 150
mM NaCl, 0.5% (w/v) Triton X-100, 1 µg/ml
leupeptin, and 1 µg/ml E-64 for 30 min at 4°C prior to
recentrifuging. Soluble fractions and membrane extracts (100 µg of
protein each) were separated by SDS-PAGE and electrophoretically
transferred onto a nitrocellulose membrane (Hybond-C; Amersham
Pharmacia Biotech) as reported (18)
. The membrane was
probed with rabbit antibodies to the cytoplasmic domain of murine
MPR46. Bound immunoglobulins were detected with HRP-conjugated goat
anti-rabbit IgG immunoglobulins (Accurate, Westbury, NY) and Enhanced
Chemiluminescence Western blotting reagents (16)
.
Analysis of Phosphorylated Oligosaccharides.
SCC-VII cells (8 x 106
) were
metabolically labeled for 8 h with
[3H]mannose (1 mCi/ml) in 5 ml of glucose-poor
culture medium (1 mM glucose) containing 10 mM
NH4Cl. Medium proteins were collected by
precipitation with 0.5 g/ml ammonium sulfate, redissolved in 2 ml of 20
mM sodium acetate buffer (pH 5.5), 5 mM sodium
ß-glycerophosphate, and dialyzed against the same buffer. The
retentate was concentrated by ultrafiltration (10 kDa cutoff) and
incubated with 5 milliunits of endoglucosaminidase H as described
(5)
. 3H-labeled oligosaccharides
thus released were isolated by ultrafiltration, desalted on a
1.5 x 7-cm column of Sephadex G-10 eluted with water,
and finally fractionated on diethyl(2-hydroxypropyl)aminoethyl-Sephadex
according to (5)
. To remove phosphomonoester groups,
3H-labeled oligosaccharides were treated with 1
unit of alkaline phosphatase in 0.1 M Tris/HCl buffer (pH
8.0) for 1 h at 37°C. Phosphodiester linkages were cleaved by
incubation in 2 M acetic acid for 2 h at 80°C.
Immunofluorescence Staining.
SCC-VII cells and NIH 3T3 fibroblasts grown on glass coverslips were
fixed by incubation for 10 min in 4% paraformaldehyde in PBS. After
blocking with PBS containing 2 mg/ml BSA for 1 h, the cells were
incubated for 1 h with rabbit anti-mouse cathepsin D antiserum
(diluted 1:50 in PBS containing 0.1% saponin). Nonimmune rabbit serum
was used as a negative control. After a second blocking step in PBS
containing 0.1% saponin and 5% fetal bovine serum (1 h), bound
primary antibodies were detected by incubation for 1 h with
fluorescein-conjugated, affinity-purified goat anti-rabbit
immunoglobulin antibodies (Sigma) at a concentration of 30 µg/ml in
PBS containing 0.1% saponin. All steps were performed at room
temperature. The immunostained cells were examined using a Zeiss
Axiovert 35 microscope with the appropriate filter combination.
Other Methods.
Enzymatic deglycosylation of polypeptides was carried out with
endoglucosaminidase H and peptide N-glycosidase F as
described (6
, 18)
. Sensitivity of protein-bound phosphate
groups to alkaline phosphatase was tested according to
(22)
. Total protein was determined by the Lowry method
with the Bio-Rad DC Protein Assay kit (Bio-Rad, Richmond, CA) using BSA
as a standard.
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RESULTS |
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).
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). The heterogeneity of mature
SCC-VII cathepsin B (32–36 kDa) was found to be attributable to
differences in glycosylation, because removal of N-linked
oligosaccharides by treatment with peptide N-glycosidase F
yielded a discrete 31 kDa band (Fig. 1B
). Interestingly,
procathepsin B secretion by SCC-VII cells was not significantly
enhanced by NH4Cl (44%). Similar results were
obtained with chloroquine (not shown). However, procathepsin B
maturation was incomplete (86%) when SCC-VII cells were treated with
either base, indicating that endosomal/lysosomal alkalinization had
indeed occurred. Therefore, the residual retention of cathepsin B by
SCC-VII cells is apparently mediated by a pH-insensitive mechanism that
differs from the classic M6P receptor pathway.
Biosynthesis of Cathepsin L in SCC-VII Cells and 3T3-L1
Fibroblasts.
The most prominent mammalian lysosomal cysteine proteinase besides
cathepsin B is cathepsin L. In 3T3-L1 cells, only 16% of newly
synthesized procathepsin L (37 kDa) was secreted into the culture
medium, with the remainder being intracellularly retained. Unlike
cathepsin B, the retained procathepsin L was processed in these cells
via a single-chain intermediate (29 kDa) into the mature double-chain
enzyme, as indicated by the appearance of its heavy chain (20 kDa).
NH4Cl substantially stimulated procathepsin L
secretion (62%). As for cathepsin B, residual intracellular
procathepsin L did not undergo proteolytic maturation in the presence
of the base (Fig. 2A
).
|
). Our results demonstrate that SCC-VII cells and 3T3-L1 fibroblasts sort procathepsin L less efficiently than procathepsin B to lysosomes. This discrepancy may be attributable to the intrinsic low affinity of procathepsin L for M6P receptors (5) or indicate the involvement of distinct M6P-independent sorting receptors, with preference for cathepsin B over cathepsin L.
Biosynthesis of Cathepsin D in SCC-VII Cells and 3T3-L1
Fibroblasts.
Cathepsin D, the major aspartic proteinase in mammalian lysosomes, was
found to be initially synthesized by SCC-VII cells and 3T3-L1
fibroblasts as a latent 46 kDa precursor. 3T3-L1 cells released <1%
of newly made procathepsin D into the medium. All proenzymes retained
by the cells were converted to mature single-chain cathepsin D (44
kDa). Treatment with NH4Cl induced secretion of
the latent proenzyme to some extent (21%), but most cathepsin D
remained inside the cells even in the presence of the lysosomotropic
agent. In contrast to cathepsins B and L, proteolytic maturation of
intracellular procathepsin D in these cells was not significantly
affected by NH4Cl (Fig. 2B
).
SCC-VII cells secreted substantial amounts (24%) of their newly
synthesized procathepsin D. Any intracellularly retained proenzyme was
processed into the mature single-chain form of the proteinase.
Procathepsin D secretion was only slightly stimulated by
NH4Cl (33%). As in 3T3-L1 cells,
NH4Cl did not inhibit intracellular procathepsin
D processing (Fig. 2B
).
Our results indicate that SCC-VII cells and 3T3-L1 fibroblasts preferentially retain cathepsin D as compared with cathepsins B or L. This finding is consistent with an alternative, M6P-independent lysosomal targeting mechanism for cathepsin D, as it has been reported for human breast cancer cells (23) .
Phosphorylation of Lysosomal Enzymes in SCC-VII Cells.
Because SCC-VII cells hypersecrete, in addition to cathepsins B, D, and
L, also the lysosomal marker ß-N-acetylhexosaminidase (not
shown), we investigated whether the cells are capable of forming M6P
residues on lysosomal enzymes. Secreted procathepsin B (42 kDa) was
immunoprecipitated and then treated with endoglucosaminidase H to test
for the presence of high-mannose-type, phosphorylated
N-linked oligosaccharides (5)
. Treatment with
endoglucosaminidase H resulted in the appearance of two novel
polypeptides of 40 and 38 kDa reflecting the loss of one and two
N-glycans, respectively. Complete deglycosylation of
procathepsin B with peptide N-glycosidase F reduced the
apparent molecular mass of the protein to 36 kDa, consistent with the
removal of three N-linked oligosaccharide side chains.
Similar results were obtained for procathepsin B synthesized in the
presence of NH4Cl. These data indicate that one
or two of the three carbohydrate side chains of procathepsin B contain
M6P residues (Fig. 3A
).
|
). Similar
results were obtained for procathepsin B (not shown).
When 33P-labeled procathepsin L was treated with
peptide N-glycosidase F, all radioactivity associated with
the protein was lost, demonstrating that the radiolabel had been indeed
incorporated into the carbohydrate moiety of the proenzyme. Incubation
with peptide N-glycosidase F led to complete deglycosylation
of procathepsin L, because the treatment shifted the apparent molecular
mass of the 35S-labeled cathepsin L precursor to
35 kDa. The 33P-label of procathepsin L also
proved sensitive to alkaline phosphatase (Fig. 3C
). The same
results were obtained for procathepsin B (not shown).
To comprehensively assess the status of phosphorylated N-linked oligosaccharides, SCC-VII cells were metabolically labeled with [3H]mannose. 3H-Labeled glycoproteins secreted into the culture medium were isolated and treated with endoglucosaminidase H. By means of anion-exchange chromatography, the N-glycans thus released were found to consist of neutral sugars (51.9%) and oligosaccharides containing either one (9.8%) or two (4.2%) phosphodiesters, both one phosphomonoester and one phosphodiester (6.3%), or either one (5.4%) or two (20.1%) phosphomonoesters. The identity and composition of each fraction were verified by mild acid treatment (which converts phosphodiesters into phosphomonoesters) and alkaline phosphatase treatment (which cleaves exclusively linkages involving phosphomonoester groups). These results demonstrate that 69% of the phosphorylated N-linked oligosaccharides synthesized by SCC-VII cells contain phosphomonoester groups that serve as high-affinity ligands for M6P receptors (7) . This is in good agreement with data reported for human fibroblasts (22) . Thus, it appears that phosphorylation of lysosomal enzymes is fully functional in SCC-VII cells.
Expression of M6P Receptors in SCC-VII Cells.
Mammalian cells usually synthesize both known M6P receptors, M6P/IGF2R
and MPR46. We have investigated the expression of both receptors in
SCC-VII cells and 3T3-L1 fibroblasts. Upon immunoblotting with
antibodies against MPR46, a diffuse 45-kDa protein was detected in
membrane extracts of SCC-VII cells. A slightly smaller immunoreactive
polypeptide (43 kDa) was present in 3T3-L1 membranes. No reaction was
observed with the corresponding soluble protein fractions (Fig. 4A
). It has been established that differences in glycosylation
may account for cell type-specific molecular forms of MPR46
(24)
.
|
). Although both M6P receptors contribute to intracellular sorting of lysosomal enzymes, receptor-mediated uptake of exogenous M6P-containing ligands is exclusively mediated by M6P/IGF2R (7) . We have shown previously that procathepsin B synthesized by human hepatoma HepG2 cells is efficiently internalized by autologous recipient cells and human fibroblasts in a M6P/IGF2R-dependent manner (18) . As a functional test for the presence of M6P/IGF2R, NH4Cl-induced secretions of HepG2 cells labeled with [35S]methionine were offered to unlabeled SCC-VII cells and 3T3-L1 fibroblasts for receptor-mediated endocytosis. Although procathepsin B was internalized by 3T3-L1 cells in a M6P-inhibitable manner, no uptake was detectable in SCC-VII cells. Identical results were obtained for procathepsin D (data not shown). We conclude that SCC-VII cells lack any endogenous synthesis of M6P/IGF2R, which induces hypersecretion of newly synthesized lysosomal enzymes as well as impairs their recapture via receptor-mediated endocytosis.
The Formation of Mature Lysosomes Is Impaired in SCC-VII Cells.
Recent studies have shown that M6P/IGF2R plays a key role in the
biogenesis of lysosomes (8)
. To assess the status of
lysosomes in SCC-VII cells, postnuclear organelles were separated by
density-gradient centrifugation. When the distribution of cathepsin B
in the gradient fractions was determined, a marked peak of enzyme
activity was observed at a buoyant density of 1.06 g/ml (Fig. 5A
). The same distribution was found for the lysosomal marker
ß-N-acetylhexosaminidase and acid phosphatase, a lysosomal
enzyme transported to these compartments in a M6P-independent manner
(7
, 21)
. In contrast, the bulk of the cathepsin B (and
ß-N-acetylhexosaminidase) activity of control HepG2 cells
was located in compartments with a density of >1.08 g/ml, as typical
for mature lysosomes (Fig. 5B
). However, the subcellular
distribution of lysosomal enzymes in SCC-VII cells overlaps
significantly with the locations of the Golgi marker
galactosyltransferase and the endocytic tracer HRP (Fig. 5A
), a feature reminiscent of late endosomal/prelysosomal
compartments. In fact, mutant human fibroblasts with a defect in the
formation of dense lysosomes exhibited the same sedimentation pattern
as observed for SCC-VII cells (19)
.
|
). In contrast, immunocytochemical detection of cathepsin D
in SCC-VII cells revealed numerous vesicles distributed throughout the
cytoplasm (Fig. 6B
). A similar subcellular distribution of
lysosomal markers was observed in M6P/IGF2R-deficient murine
fibroblasts, where the labeled structures were found to resemble late
endocytic compartments (8)
. Thus, SCC-VII "lysosomes"
display at least some characteristics of late endosomes/prelysosomes,
indicating impaired formation of mature lysosomes in these cells as a
direct consequence of the absence of M6P/IGF2R.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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M6P/IGF2R plays a pivotal role in the formation of mature lysosomes (7 , 8) . SCC-VII cells fail to synthesize the receptor and thus lack a functional set of these organelles. Consequently, these cells store lysosomal enzymes in compartments with properties reminiscent of late endosomes/prelysosomes. Similar results were obtained for other M6P/IGF2R-negative tumor cell lines, such as murine J774 myeloid leukemia cells (28) and Morris 7777 hepatoma cells (29) . Interestingly, cathepsin B was relocated to endosomal vesicles in human MCF-10A breast epithelial cells upon transfection with an oncogenic version of the c-Ha-ras gene (30) . Furthermore, transformation of mouse NIH 3T3 fibroblasts with Kirsten murine sarcoma virus leads to redistribution of cathepsin L and other lysosomal enzymes to endosomes/prelysosomes (31) . Because both latter cell types are not deficient in M6P/IGF2R,7 impaired formation of lysosomes may also occur in M6P/IGF2R-positive tumor cells.
Despite the lack of M6P/IGF2R, SCC-VII cells retain a large proportion of their newly synthesized lysosomal proteinases. Although the other known mammalian M6P receptor, MPR46 (8) , is present in SCC-VII cells, the insensitivity of the intracellular transport of cathepsins B, D, and L to lysosomal alkalinization suggests that M6P-independent sorting of lysosomal enzymes may take place in these cells. Similarly, M6P-independent targeting mechanisms have been proposed for procathepsins D and L in normal and transformed mouse NIH 3T3 fibroblasts (32) and for procathepsin D in human breast cancer cells (23) . Interestingly, procathepsin D transiently associates during its biosynthesis with prosaposin (33) , a protein delivered to lysosomes via interaction with the low-density lipoprotein receptor-related protein (34) . Thus, interaction of procathepsin D with endogenous prosaposin may account for its preferential retention in SCC-VII cells as compared with cathepsins B and L.
It has been reported recently that M6P/IGF2R interacts with uPAR through a domain distinct from its M6P- and IGF-II binding sites (35) . In the absence of M6P/IGF2R, uPAR is not efficiently internalized, effectively increasing the number of surface binding sites for plasminogen activators, which may cause enhanced focal proteolysis. Because SCC-VII cells produce plasminogen activators (16) , deficient uPAR internalization could further add to the proteolytic load in the pericellular environment of the cells. Hence, M6P/IGF2R deficiency may enhance the invasiveness of tumor cells by at least two means, elevated secretion of matrix-degrading cathepsins and increased amounts of cell-surface plasminogen activators. Interestingly, uPAR may also promote tumor cell invasion in a protease-independent manner through stimulation of integrin-mediated cell migration (36) .
The M6P/IGF2R gene is often inactivated in human and animal tumors. Rodents appear particularly susceptible because M6P/IGF2R displays monoallelic expression in mice and rats, with the paternal allele being repressed. In contrast, this genomic imprinting of M6P/IGF2R is a polymorphic trait in humans, with both alleles being transcribed in most humans. Thus, inactivation of the M6P/IGF2R locus generally requires two genetic events in humans but only one in mice (10) . Interestingly, loss of one M6P/IGF2R allele is a common feature of human hepatocellular and breast cancers (12 , 13) . M6P/IGF2R contains several microsatellite sequences in its coding region, and deletions within the remaining M6P/IGF2R allele arise frequently as a consequence of microsatellite instability (14) . These mutations generally cause frameshifts that result in premature termination of translation, giving rise to the synthesis of truncated, soluble receptors (12) . However, M6P/IGF2R-related polypeptides were not detectable in the culture supernatants of SCC-VII cells, thus indicating that the molecular defect in the M6P/IGF2R gene of these cells is probably not directly related to microsatellite instability.
M6P/IGF2R exerts at least two growth-suppressive functions: (a) the receptor accounts for the internalization and degradation of IGF-II, a powerful growth-stimulating factor; and (b) M6P/IGF2R binds latent TGF-ß1 and stimulates activation of the growth-suppressive cytokine by recruitment of plasminogen and uPAR-bound plasminogen activators (37) . In M6P/IGF2R-mutant gastrointestinal tumors, IGF-II degradation and TGF-ß1 activation are significantly reduced (38) . Recently, transfection of wild-type M6P/IGF2R cDNA into M6P/IGF2R-mutant human SW48 colorectal carcinoma cells was shown to suppress growth and induce apoptosis (15) . Because our results indicate that M6P/IGF2R-negative tumor cells display an increased potential to degrade extracellular matrix components, inactivation of the M6P/IGF2R gene may support growth as well as metastasis of malignant cancers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by Australian
Research Council Grants 04/15/412/170, 04/15/412/284, and 04/15/412/343
(to L. M.). 
2 These authors contributed equally to this
study.
3 Present address: Department of Animal Housing,
Baxter AG, A-2304 Orth, Austria.
4 To whom requests for reprints should be
addressed, at Zentrum für Angewandte Genetik, Universität
für Bodenkultur Wien, Muthgasse 18, A-1190 Vienna, Austria.
Phone: 43-1-36006-6360; Fax: 43-1-36006-6392; E-mail: lmach{at}edv2.boku.ac.at
5 The abbreviations used are: M6P, mannose
6-phosphate; IGF-II, insulin-like growth factor II; IGF2R, IGF-II
receptor; HRP, horseradish peroxidase; MPR46, 46-kDa cation-dependent
M6P receptor; TGF, transforming growth factor; uPAR, urokinase-type
plasminogen activator receptor.
6 Internet address: http://www.ncbi.nlm.nih.gov/.
7 P. Ton and L. Mach, unpublished data.
Received 11/ 2/99. Accepted 5/30/00.
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), cathepsin B activity (
), and galactosyltransferase activity
(•). Enzyme activities are expressed as the percentage of the total
activity recovered in the fractions of each gradient. In
A, endocytosed HRP emerged as a broad peak in fractions
9–12. In B, HRP distribution was essentially the same
as for galactosyltransferase, with maximum activity in fraction 9.