The EMBO Journal vol.6 no.9 pp.2677-2681, 1987
Mr 46 000 mannose 6-phosphate specific receptor: its role in
targeting of lysosomal enzymes
Martin Stein, Jos E.Zijderhand-Bleekemolen',
Hans Geuze', Andrej Hasilik and Kurt von Figura
Physiologisch-Chemisches Institut, Universitiat Munster, Waldeyerstrasse 15,
D-4400 Munster, FRG, and 'Centre for Electron Microscopy, Medical
Faculty, University of Utrecht, Nic. Beetstraat 22, 3511 HG Utrecht, The
Netherlands
Communicated by B.Dobberstein
Antibodies that block the ligand binding site of the cationdependent mannose 6-phosphate specific receptor (Mr 46 000
MIPR) were used to probe the function of the receptor in
transport of lysosomal enzymes. Addition of the antibodies
to the medium of Morris hepatoma 7777 cells, which express
only the Mr 46 000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized
lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation-independent mannose 6-phosphate specific
receptor (Mr 215 000 MPR) in addition to the Mr 46 000
MPR, antibodies against the Mr 46 000 MPR inhibited the
intracellular retention of newly synthesized lysosomal enzymes
only when added to the medium together with antibodies
against the Mr 215 000 MPR. Morris hepatoma (M.H.) 7777
did not endocytose lysosomal enzymes, while U937 monocytes,
which express both types of MPR, internalized lysosomal
enzymes. The uptake was inhibited by antibodies against
the Mr 215 000 MPR, but not by antibodies against the Mr
46 000 MPR. These observations suggest that Mr 46 000
MPR mediates transport of endogenous but not endocytosis
of exogenous lysosomal enzymes. Internalization of receptor
antibodies indicated that the failure to mediate endocytosis
of lysosomal enzymes is due to an inability of surface Mr
46 000 MPR to bind ligands rather than its exclusion from
the plasma membrane or from internalization.
Key words: mannose 6-phosphate specific receptor/lysosomal
enzymes/targeting
Introduction
Targeting of newly synthesized lysosomal enzymes depends in
many cell types on mannose 6-phosphate residues in lysosomal
enzymes and their recognition by mannose 6-phosphate specific
receptors (MPR) (von Figura and Hasilik, 1986; Komfeld, 1986).
A mannose 6-phosphate binding protein with a subunit molecular
size of 215 000 has been isolated from bovine liver by Sahagian
et al. (1981) and demonstrated in many tissues (reviewed in von
Figura and Hasilik, 1986). This receptor is a transmembrane
glycoprotein (von Figura et al., 1984; Sahagian and Steer, 1985)
that binds its ligands in the absence of divalent cations. Recently,
Hoflack and Kornfeld (1985a,b) characterized a second mannose
6-phosphate binding protein in membranes from bovine liver and
murine macrophages, which requires divalent cations for ligand
binding. This second MPR is a glycoprotein with a subunit molecular size of 46 000 and is immunologically distinct from the
Mr 215 000 MPR. With the aid of antibodies blocking the ligand binding site of the Mr 215 000 MPR it has been shown
IRL Press Limited, Oxford, England
previously that this receptor functions in targeting of newly synthesized lysosomal enzymes as well as in endocytosis of exogenous lysosomal enzymes (von Figura et al., 1984; Gartung et al.,
1985). In the present study we have utilized antibodies blocking
the ligand binding site of the Mr 46 000 MPR to examine its
role in transport of lysosomal enzymes in cells expressing either
both types of MPR or only the Mr 46 000 MPR.
Results
Blocking antibodies against the Mr 46 000 MPR
The experimental approach for examining the role of Mr 46 000
MPR in the transport of lysosomal enzymes depended on the
availability of antibodies blocking the binding site of the receptor.
We examined the effect of receptor antibodies (Ig fraction of a
polyclonal antiserum) on the binding of [125I]pentamannosyl-6phosphate bovine serum albumin (PMP-BSA) to membranes of
M.H. 7777 cells and P388D1 cells. Both cells are known to
express the M, 46 000 MPR, but not the Mr 215 000 MPR
(Stein et al., 1987; Hoflack and Kornfeld, 1985a). PMP-BSA
is a neoglycoprotein with 35 pentamannose 6-phosphate
residues per polypeptide with high affinity to MPR (Braulke et
al., 1987). Membranes prepared from both cell types bound
PMP-BSA in a mannose 6-phosphate-dependent manner (Table
I). The binding was only partially sensitive to inhibition by
EGTA. We assume that the strict cation requirement of binding
of lysosomal enzymes to the Mr 46 000 MPR (Hoflack and
Kornfeld, 1985a, b) is compensated for by the high degree of
substitution of PMP-BSA with pentamannose 6-phosphate
residues. Treatment of membranes with anti-Mr 46 000 MPR
Ig inhibited the binding of PMP-BSA to a similar extent as mannose 6-phosphate, while treatment with anti-Mr 215 000 MPR
Ig did not inhibit the binding. The slight increase in ligand binding observed in the presence of anti-Mr 215 000 MPR Ig was
due to non-specific binding. This increase in binding was neither
sensitive to EGTA nor to mannose 6-phosphate (not shown). The
binding of PMP-BSA to membranes of U937 cells, which contain the Mr 46 000 MPR and the Mr 215 000 MPR (Stein et al.,
1987) was inhibited to 62% of control by anti-Mr 46 000 MPR
Ig (not shown).
Mr 46 000 MPR does not mediate endocytosis of lysosomal
enzymes
It was previously shown that M.H. 7777 cells do not internalize
lysosomal enzymes (Mainferme et al., 1985). In these earlier
experiments endocytosis may have been missed due to the rather
high concentrations of divalent cations required for binding of
ligands to the Mr 46 000 MPR. Therefore, uptake of lysosomal
enzymes by M.H. 7777 cells was followed in culture medium
supplemented with 5 mM MgCl2. Under these conditions, M.H.
7777 cells also failed to internalize lysosomal enzymes (not
shown). Furthermore, M.H. 7777 cells in the medium containing
5 mM MgCl2 neither bound nor internalized [1251]PMP-BSA.
Membranes from M.H. 7777 cells suspended in culture medium
containing 5 mM MgCl2 and 0.5% saponin bound [251I]PMP-
2677
M.Stein et al.
Table I. Effect of anti-Mr 46 000 MPR Ig on binding of [1251]PMP-BSA to
membranes of M.H. 7777 cells and P388D, macrophages
Additions
20 mM MgC12
20 mM MgCl2+10 mM
mannose 6-phosphate
10 mM EGTA
20 mM MgCl2
20 mM MgCl2
Table Im. Uptake of
Cell-associated [1251]anti-Mr
46 000 MPR Ig (ng/mg cell
['251]PMP-BSA bound
Ig
(3 mg/ml)
(ng/mg protein)
M.H. 7777 P388D1
Control
Control
26.8
4.2
23.6
3.6
Control
19.6
2.1
31.0
15.0
3.4
26.6
protein)
0°C
A
B
A-B
A
B
A-B
370C
Anti-Mr 46 000 MPR
Anti-Mr 215 000 MPR
All values are the means of duplicates (maximal deviation of the mean was
9%). For experimental details see Materials and methods.
Table H. Binding of ['25I]PMP-BSA by U937 monocytes
Addition
['251]PMP-BSA (ng/mg cell
protein)
Cell surface
-
5 mM Man6P
Anti-Mr 215 000 MPR Ig, 10 Ag
Anti-Mr 46 000 MPR Ig, 10 jg
Anti-Mr 215 000 MPR +
anti-Mr 46 000 MPR Ig, 10 ltg each
82.3
16.6
41.8
89.6
48.1
Internalizeda
Cctitlpsin
K-- - - -
BSA in amounts comparable with membranes suspended in Hepes
buffer supplemented with 20 mM MgCl2 (Table 1). This finding
excluded the possibility that the failure of M.H. 7777 cells to
bind (and to internalize) PMP-BSA was due to conditions unsuitable for ligand binding. Furthermore, incubation of the M.H.
7777 cells for 15 min at 0°C with 5 mM mannose 6-phosphate
prior to the binding assay did not result in subsequent binding
of PMP-BSA. This indicated that the lack of the binding of PMPBSA was not due to an interference of endogenous ligands. U937
monocytes, which contain both types of MPR, bound and internalized [1251]PMP-BSA (Table H). In the presence of 5 mM
mannose 6-phosphate, binding was reduced to 20% and uptake
to 3% of control. Antibodies against the Mr 215 000 MPR
decreased the binding to 51 % and the uptake to 26% of control,
while antibodies against the Mr 46 000 MPR had no effect on
binding and uptake.
From the inability of M.H. 7777 cells to bind and internalize
mannose 6-phosphate-containing ligands and from the failure of
blocking antibodies against the Mr 46 000 MPR to inhibit binding and uptake of the ligands by U937 monocytes we concluded
that the Mr 46 000 MPR is not involved in endocytosis of
ligands.
Receptor-mediated uptake of anti-Mr 46 000 MPR Ig
A small fraction of Mr 46 000 MPR is located at the cell surface. In U937 monocytes the Mr 46 000 MPR at the cell surface
accounts for 3% of the total receptor (Stein et al., 1987). Preliminary immunocytochemical data suggest that in M.H. 7777
cells 10% of the Mr 46 000 MPR is located at the cell surface
(H.Geuze, unpublished). We examined the ability of fibroblasts
to internalize antibodies against the Mr 46 000 MPR. Fibroblasts were incubated for 4 h at 0 and 370C with '25I-labelled
anti-Mr 46 000 MPR Ig. The experiments were performed in
2678
i 'v1
*CIet-
27.6
0.7
7.1
28.1
10.3
aThe values are the means of duplicates. For experimental details see
Materials and methods.
5.4
3.8
1.6
14.9
4.4
10.5
(3.4)
(2.7)
(4.1)
(2.6)
Cells were incubated for 4 h at 0 or 37°C in medium containing 250 ng/ml
iodinated antibody and 20% pre-immune serum (A) or 20% anti-Mr 46 000
MPR serum (B). The amount of cell-associated radioactivity was
determined. The numbers in parentheses refer to the radioactivity that could
be released from the cells by incubation with pronase for 1 h at 0°C.
Specific association is defined as the difference between A and B. All
values are the means of duplicates.
bounda
-
[1251]anti-Mr 46 000 MPR Ig by fibroblasts
4
D
:
I'
PS1'ii
__----_--_
'
- - j ;Cl!i -I')-
,*
S
9;;.4
*1 *~;-
I
s~~~~~~~~~~~~" 9
_
-e
-
4
.4
.4
Ar"
;,
-4-301
;vI.-O a&
,
9.
-
9 78
86 73
I4 f5
5)C
Fig. 1. Effect of anti-Mr 46 000 MPR serum on sorting of cathepsin C and
cathepsin D in M.H. 7777 cells. M.H. cells were labelled with
[35S]methionine in the presence of up to 10% anti-Mr 46 000 MPR serum
(a-46). The final concentration of serum was adjusted to 15% with control
rabbit serum. Cathepsins D and C were immunoprecipitated from extracts of
cells and medium. Precursor (P) and mature (M) forms of cathepsin D are
indicated at the left margin, that of cathepsin C (4) at the right margin.
Mature cathepsin C is represented by at least 14 polypeptides with Mr
values ranging from 73 000 to 12 000 (Mainferme et ai., 1985). The
numbers below the lanes give the percentage of intracellular retained 35Slabelled cathepsins D and C.
the presence of an excess of unlabelled pre-immune serum or
anti-Mr 46 000 MPR serum. Specific association of the 125ilabelled antibodies to the cells was defined as that inhibitable by
the anti-Mr 46 000 MPR serum. About 6 times more radioactivity associated with the cells at 37°C than at 0°C (Table III).
A similar amount of radioactivity was released from the cells
that had been incubated at 0 and 370C with the '251-labelled antibodies during a 1-h incubation at 0°C with pronase. The pronase-
Role of Mr 46 000 MPR in targeting of lysosomal enzymes
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Fig. 2. Effect of antisera against the Mr 46 000 MPR and the Mr 215 000
MPR on targeting of cathepsin D, (3-hexosaminidase and arylsulfatase A in
fibroblast and HepG2 cells. Fibroblasts and HepG2 cells were labelled with
[35S]methionine in the presence of 10% anti-Mr 46 000 MPR serum (a-46)
and/or 10% anti-Mr 215 000 MPR serum (a-215). The final concentration
of the serum was adjusted to 20% with control rabbit serum. The lysosomal
enzymes were sequentially immunoprecipitated from extracts of cells and
medium. The precursor (P), intermediate (I) and mature (M) forms of
cathepsin D, the precursor (pa, p,B) and mature forms (ma, m13) of the aand 13-chain of hexosaminidase and arylsulfatase A (arrow) are indicated.
The secreted lysosomal enzyme precursors are shown only for cathepsin D.
The numbers below the lanes give the percentage of intracellularly retained
35S-labelled lysosomal enzyme.
releasable fraction is assumed to derive from the cell surface.
In cells incubated at 0°C with '25I-labelled antibodies it accounted for about two-thirds of cell-associated radioactivity. We conclude from these results that the higher association of radioactivity
at 370C results from uptake of the antibodies mediated by the
cell surface Mr 46 000 MPR.
Role of Mr 46 000 MPR in targeting of endogenous lysosomal
enzymes
M.H. 7777 cells were metabolically labelled in the presence of
pre-immune serum or antiserum against the Mr 46 000 MPR.
Extracts of cells and media were analysed for labelled cathepsin
C and cathepsin D (Figure 1). In cells that had been incubated
in the presence of receptor antiserum, the fraction of intracellular
retained cathepsin C and cathepsin D was lower (50 and 78%)
than in the control cells (86 and 93%). The secreted enzymes
were represented exclusively by the precursor forms, while the
cell-associated enzymes were represented largely by the mature
(lysosomal) forms of cathepsin C and cathepsin D. The inhibitory
effect of the receptor antiserum on intracellular retention of newly
synthesized lysosomal enzyme precursors was reproducible, although subject to variation. Occasionally only 30% or less of
the newly synthesized cathepsin C and cathepsin D was retained
intracellularly. The inhibitory effect of the receptor antiserum
on the targeting of newly synthesized cathepsin C and cathepsin
D indicates that the Mr 46 000 MPR mediates transport of
newly synthesized lysosomal enzymes to lysosomes. The effect
of the receptor antiserum is tentatively explained by the assumptions that (i) binding of the antibodies to receptors at the cell
surface functionally inactivates the receptors and (ii) internal
receptors are subject to the inactivation due to an equilibrium
of internal and cell surface receptors. The functional inactivation
of the receptors could be due to the blocking of the ligand binding site (see above) and/or the sequestration of antibody-tagged
receptors into a pool excluded from targeting of newly synthesized lysosomal enzymes.
The effect of the anti-receptor serum on intracellular retention
of newly synthesized lysosomal enzymes was also examined in
human skin fibroblasts and HepG2 cells. These cells express both
the Mr 46 000 MPR and the Mr 215 000 MPR (Stein et al.,
1987). When fibroblasts and HepG2 cells were metabolically
labelled in the presence of 10% of anti-Mr 46 000 MPR serum,
the retention of newly synthesized fl-hexosaminidase and arylsulfatase A was unaffected (Figure 2, lanes 1 and 2). Only in
fibroblasts a small, but reproducible increase in secretion was
noted for cathepsin D (Figure 2, lanes 1 and 2). Incubation of
fibroblasts and HepG2 cells in the presence of 10% of anti-Mr
215 000 MPR serum significantly decreased the intracellular
retention of cathepsin D, 3-hexosaminidase and arylsulfatase A
(Figure 2, lane 3) as has been reported earlier for fibroblasts (von
Figura et al., 1984; Gartung et al., 1985). Increasing the concentration of the anti-receptor sera to 20% did not further decrease
the retention of the newly synthesized lysosomal enzymes. When
fibroblasts and HepG2 cells were incubated in the presence of
a combination of the antisera against the Mr 215 000 MPR and
Mr 46 000 MPR, the retention of the three lysosomal enzymes
was significantly lower than in the presence of anti-Mr 215 000
MPR antiserum alone (Figure 2 lane 4).
Discussion
The targeting of newly synthesized lysosomal enzymes to lysosomes was inhibited when M.H. 7777 cells were exposed to
antiserum against the Mr 46 000 MPR. This indicated that the
Mr 46 000 MPR participates in transport of endogenous lysosomal enzymes. We assume that blocking antibodies bind to
receptors at the cell surface and that a deficiency of functionally
active receptors at the sorting site results from equilibrium of
antibody-tagged cell surface receptors with internal receptors.
In contrast to the anti-Mr 215 000 MPR serum the anti-Mr
46 000 MPR serum was ineffective in inhibiting the targeting
of lysosomal enzymes in cells that express both the Mr 46 000
MPR and Mr 215 000 MPR. In these cells the anti-Mr 46 000
MPR serum exerted an inhibitory effect on targeting of newly
synthesized lysosomal enzymes only in combination with anti2679
M.Stein et al.
L
L
L
L-R1
Rl
Rl
R1-L
R2
Rl
L
R2
-L
L-R
R2
R2-L
R2
L
R2-L
L
A
B
C
Fig. 3. Models for targeting of lysosomal enzymes (L) by two receptors
(RI, R2). Binding of the lysosomal enzyme to RI and R2 (models A and
B) as well as binding to RI, dissociation from RI and binding to R2 (model
C) is assumed to occur in the secretory pathway. Dissociation from the RI
and R2 (models A and B) or R2 (model C) is assumed to occur after
segregation from the secretory route.
Mr 215 000 MPR serum. This suggests that the Mr 46 000
MPR contributes to targeting only when the Mr 215 000 MPR
is blocked or that antibody-mediated inactivation of Mr 46 000
MPR can be compensated for by the Mr 215 000 MPR.
Receptor-dependent targeting is assumed to depend on the
binding of the newly synthesized lysosomal enzymes to the receptors within the secretory route, segregation of the MPR-ligand
complexes into specific vesicles, delivery of the ligands to elements of the endocytic route and recycling of the receptors to
the binding site (von Figura and Hasilik, 1986). Several models
are conceivable to explain the function of the two types of MPR
within the sorting compartment of a cell. Three models representing extreme views are schematically depicted in Figure 3 (the
receptors are designated RI and R2, the lysosomal enzyme precursors L). In model A the ligand binds either to Rl or R2. If
ligands bind at the same intracellular site to either receptor, compensation of the inactivation of one receptor depends on the
transport capacity of the other receptor. If binding to RI and R2
occurs at different sites, only the receptor located more distal
along the secretory route could compensate for a functional loss
of the other receptor. In the latter case compensation of the inactivation of the Mr 46 000 MPR in fibroblasts and HepG2 cells
by the Mr 215 000 MPR would therefore imply that the Mr
46 000 MPR is located proximal to the Mr 215 000 MPR. According to model B the two receptors are located at different sites
and RI, the receptor located more proximally along the secretory
route, is responsible for the transport of the bulk of ligands. This
model predicts that functional inactivation of R2 has only a
marginal effect on targeting of ligands and that functional inactivation of RI may be compensated for by R2. If this model holds
true, the Mr 215 000 MPR would correspond to proximal receptor RI and the Mr 46 000 MPR to the distal receptor R2,
since inactivation of the Mr 46 000 MPR had only a minor
effect on targeting. In model C the two receptors operate in sequence in transporting the same ligands. Inactivation of either
of the two receptors should produce the same effect. Model C
is not compatible with the observation that inactivation of the
Mr 46 000 MPR inhibited targeting only when the Mr 215 000
MPR was inactivated simultaneously. The available data are compatible with models A and B. Knowledge of the subcellular distribution of the Mr 46 000 MPR and the Mr 215 000 MPR
2680
among the internal membranes of one cell type may help in
understanding how the two types of MPR function in the targeting
of newly synthesized lysosomal enzymes.
Although present at the cell surface, the Mr 46 000 MPR did
not mediate endocytosis of mannose 6-phosphate-containing ligands such as lysosomal enzymes or the neoglycoprotein PMPBSA. The inability to mediate endocytosis distinguishes the M,
46 000 MPR from the Mr 215 000 MPR, which mediates endocytosis of exogenous lysosomal enzymes (Gartung et al., 1985).
The failure of the cells to internalize ligands via the M, 46 000
MPR correlated with the inability of the cell surface Mr 46 000
MPR to bind ligands. Our results indicate that the failure to bind
ligands to the cell surface receptors is not due to the composition
of the incubation medium or to interference of the endogenous
ligands. Several observations suggested that the receptors at the
surface are in equilibrium with internal receptors, e.g. the inhibitory effect of receptor antibodies on the targeting of endogenous lysosomal enzymes is supposed to depend on the exchange
of cell surface receptors with internal receptors. Furthermore,
antibodies bound to cell surface Mr 46 000 MPR are subject to
internalization.
It is unclear why Mr 46 000 MPR at the cell surface do not
bind ligands. It is conceivable that exposure of the receptors at
the cell surface alters the conformation or subunit arrangement
[the Mr 46 000 MPR occurs in membranes mostly as a dimer,
Stein et al. (1987)], in a manner that is not compatible with ligand
binding. In summary, the results presented in this study provide
evidence for a function of the Mr 46 000 MPR in targeting of
endogenous lysosomal enzymes. In cells that simultaneously express the Mr 46 000 MPR and Mr 215 000 MPR, both receptors
are involved in targeting of endogenous lysosomal enzymes.
Materials and methods
Materials
1251-labelled PMP-BSA sp. act. 2100-3000 c.p.m./mg protein was kindly provided by Dr T.Braulke of this institute. The ['2I]PMP-BSA has a Kd of 0.53 x 10-9 M for the Mr 215 000 MPR (Braulke et al., 1987).
Antibodies
The antibodies against rat cathepsin C were kindly provided by Dr F.Mainferme,
University of Namur (Mainferme et al., 1985) and the antibodies against rat
cathepsin D by Dr Baccino, University of Torino. The antisera and affinity-purified
antibodies, human Mr 215 000 MPR (von Figura et al., 1984), human (-hexosaminidase (Hasilik and Neufeld, 1980), human cathepsin D (Gieselmann et al.,
1983) and human arylsulfatase A (Waheed et al., 1982) were those described.
The antiserum against the Mr 46 000 MPR purified from human liver according
to Hoflack and Komfeld (1985a) was raised in rabbits. The antiserum and affinitypurified immunoglobulins were monospecific for the Mr 46 000 MPR as shown
in immunoblots and by immunoprecipitation of the receptor from extracts of
metabolically labelled cells (Stein et al., 1987).
Cell culture and metabolic labelling
Fibroblasts, U937 monocytes, HepG2 cells (all of human origin), rat M.H. 7777
cells were grown and metabolically labelled as described (Stein et al., 1987),
murine P388D, macrophages according to Hoflack and Kornfeld (1985a). The
labelling period with [35S]methionine (sp. act. 29.7 TBcq/mmol) was 12 h.
Where indicated the fetal calf serum in the labelling medium was replaced by
rabbit serum (control serum or antiserum against the Mr 46 000 MPR and Mr
215 000 MPR). The rabbit sera were heat inactivated (56°C for 30 min) and
dialysed overnight against serum-free medium (Gorham and Waymouth, 1965,
modified, as formulated in the catalogue of GIBCO) or against RPMI 1640 if
used for labelling of U937 monocytes.
Endocytosis of labelled lysosomal enzymes
Radioactive secretions were prepared from human skin fibroblasts incubated in
the presence of [35S]methionine and 10 mM NH4Cl as described (von Figura
et al., 1983). Recipient M.H. 7777 cells were incubated in medium supplemented
with the radioactive secretions and 5 mM MgCl2 for 24 h and analysed for
internalized fl-hexosaminidase and arylsulfatase A (von Figura et al., 1983).
Role of Mr 46 000 MPR in targeting of lysosomal enzymes
Immunoprecipitation
Extracts of cells and medium of fibroblasts, HepG2 cells, U937 monocytes
(Gieselmann et al., 1983) and M.H. 7777 cells (Mainferme et al., 1985) were
prepared and subjected to sequential immunoprecipitation of the lysosomal enzymes
as described. The immunoprecipitates were solubilized in the presence of SDS
and dithiothreitol (except for rat cathepsin C, which was solubilized with SDS
only), separated by electrophoresis in 12.5-15% polyacrylamide gels (Laemmli,
1970) and visualized by fluorography (Laskey and Mills, 1975). Bands visible
in fluorograms were quantified by densitometry.
Binding and uptake of iodinated anti-Mr 46 000 MPR Ig
The affinity-purified receptor antibodies were iodinated using Na125I (17 Ci/mg
I, Amersham) and lodogen (Pierce Chemical Co., Rockford), accroding to Parker
and Strominger (1983) to a specific activity of 4000 c.p.m./ng protein. Fibroblasts
were incubated for 4 h at 0°C (placed on ice water) or at 37°C in the respective
culture medium containing 250 ng/ml of the iodinated antibody. The fetal calf
serum of the culture medium was replaced by 20% heat-inactivated (56°C for
30 min) rabbit serum (pre-immune or anti-Mr 46 000 MPR serum). After incubation the cells were washed five times with ice-cold Hank's balanced salt solution and incubated for 1 h at 0°C with 0.1% pronase (Calbiochem). The
radioactivity solubilized with pronase from cells incubated with the ligand at 37°C
represents receptor antibodies associated with the cell surface, while the radioactivity resistant to pronase represents by and large the internalized receptor antibodies.
Binding and uptake of [1251]PMP-BSA by cells
Cells were incubated for 2 h at 37°C in medium supplemented with 10% heatinactivated fetal calf serum, 5 mM MgCl2 and 10-8 M [1251]PMP-BSA (425 000
c.p.m./ml). As indicated the medium was supplemented with 5 mM mannose
6-phosphate, 5 mM glucose 6-phosphate or affinity-purified antibodies against
the Mr 46 000 MPR or Mr 215 000 MPR. Cell surface-associated [125I]PMPBSA was released by incubation with Hank's balanced salt solution adjusted to
pH 3.0 for 15 min. This incubation was repeated once. The radioactivity remaining
with the cells was resistant to solubilization with trypsin and represented the faction
of internalized [1251]PMP-BSA.
Binding of [1251]PMP-BSA by membranes
A mixture of cell surface and internal membranes from M.H. 7777 cells and
P388D, macrophages was prepared and incubated with ligand following the procedure of Hoflack and Kornfeld (1985b). The membranes, 300 ug protein, were
incubated for 90 min on ice in 0.15 mi of 50 mM Hepes pH 7.8, containing
0.15 M NaCl, 0.5% saponin, 0.17 tiypsin inhibitor units aprotinin, 5 mM sodium
,B-glycerophosphate, the additions (MgCl2, mannose 6-phosphate, EGTA) and
3 mg/ml Ig as indicated. The Ig were purified with the aid of protein A-Sepharose
4B (Pharmacia). Then [125I]PMP-BSA (150 ng) was added. After incubation for
90 min on ice, the membranes were collected by centrifugation for 20 min at
50 000 g and washed twice with Hepes buffer prior to determination of radio-
Sahagian,G.G., Distler,J. and Jourdian,G.W. (1981) Proc. Natl. Acad. Sci. USA,
78, 4289-4293.
Stein,M., Braulke,T., Krentler,C., Hasilik,A. and von Figura,K. (1987) Biol.
Chem. Hoppe-Seyler, 36, 937-947.
von Figura,K. and Hasilik,A. (1986) Annu. Rev. Biochem., 55, 167-193.
von Figura,K., Steckel,F. and Hasilik,A. (1983) Proc. Natl. Acad. Sci. USA,
80, 6066-6070.
von Figura,K., Gieselmann,V. and Hasilik,A. (1984) EMBO J., 3, 1281-1286.
von Figura,K., Gieselmann,V. and Hasilik,A. (1986) Biochem. J., 225, 543-547.
Waheed,A., Hasilik,A. and von Figura,K. (1982) Hoppe Seyler's Z. Physiol.
Chem., 363, 425-430.
Received on March 11, 1987; revised on May 15, 1987
activity.
Other methods
Protein was detenmined according to Peterson (1977) using bovine serum albumin
as standard.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds
der Chemischen Industrie.
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1735-1742.
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