From the
Glycoprotein 330 (gp330) is a member of a family of endocytic
receptors related to the low density lipoprotein receptor. gp330 has
previously been shown to bind a number of ligands in common with its
family member, the low density lipoprotein receptor-related protein
(LRP). To identify ligands specific for gp330 and relevant to its
localization on epithelia such as in the mammary gland, gp330-Sepharose
affinity chromatography was performed. As a result, a 70-kDa protein
was selected from human milk and identified by protein sequencing to be
apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed
that gp330 bound to apoJ with high affinity (K
The endocytic receptor gp330
Efforts to identify ligands for gp330 have
revealed that gp330, like LRP, is capable of binding in vitro to a number of different ligands including RAP, a 39-kDa protein
that copurifies with gp330 and LRP (Kounnas et al., 1992b;
Orlando et al., 1992; Christensen et al., 1992),
lipoprotein lipase (Willnow et al., 1992; Kounnas et
al., 1993), apolipoprotein E-enriched
While
the overlapping ligand binding specificity of LRP and gp330 might
suggest that gp330 is part of a redundant receptor system, the very
different pattern of expression of the two receptors suggests that
gp330 has a unique function with respect to LRP. Not only is gp330
expression much more restricted than is LRP (Zheng et al.,
1994; Kounnas et al., 1994), but in those cells where both
receptors are expressed the two proteins have distinct subcellular
distribution patterns (Kounnas et al., 1994). gp330 is
strictly confined to apical portions of cells within epithelial layers,
whereas LRP seems to be more basolaterally distributed within
gp330-expressing cells. In addition, gp330 can be found expressed
exclusive of LRP such as in the epithelial cells of epicardium and
renal proximal tubules.
In an effort to understand the function of
gp330 we have sought to identify gp330 ligands present in bodily fluids
that are normally in contact with gp330-expressing epithelial cells.
Given that gp330 has been shown to be expressed by mammary epithelial
cells (Zheng et al., 1994), we have examined milk as a source
for gp330 ligands. Herein we report on the identification of a milk
protein known as apolipoprotein J (apoJ)
While we have identified gp330 as a receptor capable of
mediating apoJ endocytosis and degradation, we do not yet know the
biological significance for such a process. ApoJ has been reported to
bind to several proteins including the
A
particularly important feature of apoJ is that its expression is
up-regulated at sites undergoing tissue remodeling occurring in
conjunction with apoptosis or following injury (Jenne and Tschopp,
1992). Increased levels of apoJ transcripts and/or protein have been
measured in rat hippocampus following experimental injury (May et
al., 1990), in prostate epithelial cells that atrophy following
castration (Buttyan et al., 1989), in myocardiocytes adjacent
to ischemic or inflammatory lesions of the heart (Vakeva et
al., 1993),
ApoJ has been found to be widely expressed
by cells of reproductive, endocrine, and nervous tissues (Sylvester
et al., 1991; Aronow et al., 1993; Sensibar et
al., 1993; French et al., 1993; Laslop et al.,
1993). For example, apoJ has been detected in ovary, prostate, seminal
vesicle, testis, epididymis, kidney, liver, eye, and brain. ApoJ has
also been detected in many body fluids including urine, saliva, seminal
plasma, cerebral spinal fluid, and plasma (Blaschuk et al.,
1983; Watts et al., 1990). There is a striking correlation
between sites of apoJ expression and known sites of gp330 expression.
Prominent expression of gp330 has been observed in the ependyma,
choroid plexus, ciliary, and pigmented retinal epithelium, lung,
kidney, yolk sac epithelium, and tissues of the male and female
reproductive systems (Zheng et al., 1994; Kounnas et
al., 1994). The colocalization of apoJ and gp330 strengthens the
possibility that the two proteins interact with one another in
vivo.
The average equilibrium dissociation
constants (K
= 14.2 nM). Similarly, gp330 bound to apoJ
transferred to nitrocellulose after SDS-polyacrylamide gel
electrophoresis. LRP, however, showed no binding to apoJ in either type
of assay. The binding of gp330 to apoJ could be competitively inhibited
with excess apoJ as well as with the gp330 ligands apolipoprotein E,
lipoprotein lipase, and the receptor-associated protein, a 39-kDa
protein that acts to antagonize binding of all known ligands for gp330
and LRP. Several cultured cell lines that express gp330 and ones that
do not express the receptor were examined for their ability to bind and
internalize
I-apoJ. Only cells that expressed gp330
endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells
treated with retinoic acid and dibutyryl cyclic AMP to increase
expression levels of gp330 displayed an increased capacity to
internalize and degrade apoJ. Cellular internalization and degradation
of radiolabeled apoJ could be inhibited with unlabeled apoJ,
receptor-associated protein, and gp330 antibodies. The results indicate
that gp330 but not LRP can bind to apoJ in vitro and that
gp330 expressed by cells can mediate apoJ endocytosis leading to
lysosomal degradation.
(
)
is
expressed by many specialized epithelial cells such as those of renal
proximal tubules, ependyma, lung alveoli, and mammary gland epithelium
(Kerjaschki and Farquhar, 1983; Zheng et. al.; 1994, Kounnas
et al., 1994). gp330 is typically found on the apical surfaces
of these epithelia, which are exposed to fluid-filled spaces. It is
believed that gp330 functions to mediate endocytosis of ligands based
on its cytoplasmic domain having consensus endocytosis signal motifs
(Raychowdhury et al., 1989; Saito et al., 1994), that
it has been localized within clathrin-coated pits and vesicles
(Kerjaschki and Farquhar, 1983; Kerjaschki et al., 1987), and
that it is structurally related to a family of receptors including the
low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP)
(Raychowdhury et al., 1989; Saito et al., 1994;
Korenberg et al., 1994) that mediate cellular internalization
of their ligands.
- very low density
lipoproteins (Willnow et al., 1992), complexes of tissue-type
plasminogen activator and plasminogen activator inhibitor-1 (tPA:PAI-1)
(Willnow et al., 1992), and complexes of urokinase plasminogen
activator and PAI-1 (uPA:PAI-1) (Moestrup et al., 1993b). Each
of the proteins that bind to gp330 have been shown to also bind LRP.
However, LRP binds several additional ligands, such as
-macroglobulin-protease complexes (Ashcom et
al., 1990) and Pseudomonas aeruginosa exotoxin A (Kounnas
et al., 1992a) that are not shared with gp330. To date no
ligand has been identified that can bind gp330 exclusively.
(
)
as a
novel gp330 ligand. Furthermore we have used gp330-expressing cell
lines to demonstrate that gp330 mediates internalization and
degradation of apoJ.
Proteins
gp330 from human urine or porcine
kidney brush border membrane extracts was isolated by RAP-Sepharose
affinity chromatography as described previously (Kounnas et
al., 1992b, 1993). LRP was isolated from human placenta by
-macroglobulin/methylamine-Sepharose affinity
chromatography as described previously (Ashcom et al., 1990).
Human RAP, expressed as a glutathione S-transferase fusion
protein in bacteria, was prepared (free of glutathione
S-transferase) as outlined by Williams et al. (1992).
ApoJ was isolated from human plasma by immunoaffinity chromatography
and reverse phase high performance liquid chromatography (de Silva
et al., 1990b). Apolipoprotein E3 (apoE3) was purified as
described by Kelly et al.(1994). Pro-urokinase (pro-uPA) and
uPA were provided by Dr. Jack Henkin (Abbott Laboratories, Abbott Park,
IL). PAI-1 was purchased from Molecular Innovations (Royal Oak, MI).
Human lactoferrin was provided by Dr. David Mann (American Red Cross,
J. H. Holland Laboratory, Rockville, MD). The 18-kDa carboxyl-terminal
fragment of human lipoprotein lipase (residues 313-448) was from
Dr. David Chappell (University of Iowa College of Medicine, Iowa City,
IA). uPA:PAI-1 complexes were generated by incubation of a 1:1 molar
ratio of active uPA with active PAI-1 for 30 min at room temperature.
Bovine serum albumin-fraction V, and ovalbumin were purchased from
Sigma.
Antibodies
The mouse monoclonal antibody to human
apoJ designated 1D11 has been described previously (de Silva et
al., 1990a). The mouse monoclonal antibody to rat gp330 designated
1H2 was provided by Dr. Robert McCluskey (Harvard/Massachusetts General
Hospital, Boston, MA). This antibody has previously been shown to cross
react to both porcine (data not shown) and human gp330
(Kounnas et al., 1993). The mouse monoclonal antibody to the
515-kDa heavy chain of human LRP designated 8G1 has been described
previously (Strickland et al., 1991). IgG was purified on
protein G-Sepharose from each of the mouse ascitic fluids. IgG was
isolated from rabbit polyclonal anti-gp330 serum (rb239) (Kounnas
et al., 1994) and anti-LRP serum (rb777 and rb810) (Strickland
et al., 1991) by affinity chromatography on either gp330- or
LRP-Sepharose (1-2 mg of protein/ml of resin) followed by
absorption on RAP-Sepharose.
gp330-Sepharose Affinity Chromatography of Human Breast
Milk
An affinity matrix of gp330-Sepharose was prepared by
coupling porcine gp330 to activated CNBr-activated Sepharose (Pharmacia
Biotech Inc.) at 1-2 mg of protein/ml of resin. Human breast milk
(12-20 weeks postdelivery) was centrifuged for 1 h at 100,000
g in the presence of the following proteinase
inhibitors: phenylmethylsulfonyl fluoride (1 mM), leupeptin (4
µg/ml), pepstatin (66 ng/ml), and
D-Phe-Pro-Arg-CH
Cl (4 µg/ml). After
centrifugation, the milk supernatant (200 ml) was preabsorbed on CL-4B
Sepharose (20 ml bed volume) and applied to the gp330-Sepharose
affinity matrix (5 ml bed volume). The column was then washed with 50
volumes of 50 mM Tris, 150 mM NaCl, pH 7.4 (TBS), and
bound protein was eluted with 2 column volumes of 8 M urea, 50
mM Tris, pH 7.4. Eluted fractions were electrophoresed in the
presence of SDS on 4-12% polyacrylamide gradient gels (Novex, San
Diego, CA). Protein bands were excised from SDS-PAGE gels, extracted in
0.1% SDS, 0.1 M Tris, pH 8.0, and sequenced on a Hewlett
Packard (model Gl000S) protein sequencing instrument.
Solid Phase Binding Assays
Enzyme-linked
immunosorbent assays (ELISA) were carried out as described previously
(Kounnas et al., 1993) except that 0.5% ovalbumin was used in
the blocking buffer instead of bovine serum albumin. ELISA data was
analyzed using a form of the binding isotherm as described by Ashcom
et al.(1990). Both homologous and heterologous ligand
displacement assays were conducted according to methods already
outlined (Williams et al., 1992). Briefly, microtiter wells
were coated with gp330, LRP, or ovalbumin (5 µg/ml) in TBS, 5
mM CaCl, pH 8.0, for 4 h at 37 °C, and
nonspecific sites were blocked with 0.5% ovalbumin, TBS.
Receptor-coated wells were then incubated with radiolabeled ligand in
0.5% ovalbumin, TBS, 5 mM CaCl
, 0.05% Tween-20 in
the presence of unlabeled competitor at varying concentrations for 18 h
at 4 °C. For binding assays, apoJ was labeled with
I
using IODO-GEN (Pierce Chemical Co., Rockford, IL) and used within 24 h
at a concentration of 0.5-2 nM (specific activity ranged
from 1 to 5 mCi/mg of protein). Binding data was analyzed, and
dissociation constants (K
) and inhibition
constants (K
) were determined using the
computer program LIGAND (Munson and Rodbard, 1980).
Gel Blot Overlay Assays
ApoJ and RAP (0.5 µg
each) were electrophoresed in the presence of SDS and under nonreducing
conditions on 4-12% polyacrylamide gradient gels (Novex, San
Diego, CA) and then transferred to nitrocellulose membranes and blocked
with 3% nonfat milk, which lacks detectable levels of apoJ (data
not shown), in TBS (blocking buffer). The membranes were then
incubated with unlabeled gp330 or LRP (25 nM) in blocking
buffer with 5 mM CaCl and 0.05% Tween-20. After a
1-h incubation, the membranes were washed with TBS, 0.05% Tween-20 and
then probed with
I-labeled monoclonal antibodies to gp330
(1H2) or LRP (8G1) (0.1 nM) for 1 h at 25 °C. Filters were
washed and exposed to Kodak X-Omat AR film (Eastman Kodak Co.) at
-70 °C.
Cell-mediated Ligand Internalization and Degradation
Assays
Cell assays were carried out according to procedures
described previously (Kounnas et al., 1993). Mouse embryonal
carcinoma F9 cells (ATCC CRL1720) were grown in Dulbecco's
modified Eagle's medium, supplemented with 10% fetal calf serum,
100 units/ml penicillin, 100 µg/ml streptomycin (Life Technologies,
Inc.) in tissue culture plates (Corning, Corning, NY) precoated with
0.1% gelatin. To augment expression of gp330, the F9 cells were induced
to differentiate by treatment with 0.1 µM retinoic acid
(Calbiochem, San Diego, CA) and 0.2 µM dibutyryl cAMP
(DBC, Sigma, St. Louis, MO) in complete Dulbecco's modified
Eagle's medium for 7 days. After 7 days of treatment, the cells
were transferred to gelatin-coated 6-well dishes at 3-5
10
cells/well (60-80% confluence) and allowed to grow
for 24 h. Prior to the addition of radioactive ligands, the cells were
washed and incubated in Dulbecco's modified Eagle's medium
containing 20 mM HEPES, Nutridoma serum substitute (Boehringer
Mannheim), penicillin/streptomycin, and 1.5% bovine serum albumin
(assay medium) for 1 h at 37 °C. Radioiodinated apoJ (5-10
nM) in assay medium was added to the cells in the presence or
absence of increasing concentrations of RAP or unlabeled apoJ
(1.8-450 nM) and incubated for 18 h at 37 °C.
Typically, 200 µg of apoJ was radioiodinated using IODO-GEN
(specific activity of 1-5 mCi/mg protein) and used within 24 h.
For assays evaluating the effect of gp330 antibodies on
I-apoJ internalization and degradation, the cells were
preincubated with antibodies to gp330 (rb239) or antibodies to LRP
(rb777) for 1 h at 37 °C prior to the addition of
I-apoJ. Antibodies were also included during the
subsequent incubation with labeled ligand. Radioactivity in the cell
medium that was soluble in 10% trichloroacetic acid was taken to
represent degraded ligand (Goldstein and Brown, 1974). Total ligand
degradation was corrected for any degradation that occurred in
radioligand-containing medium lacking cells, typically this amount was
5-10% of the total counts added. To inhibit lysosomal protease
activity, the cells were treated with 0.1 mM chloroquine
(Sigma) throughout the duration of the internalization and degradation
assays. To determine the amount of
I-apoJ that was
internalized, the cells were washed 3 times with isotonic
phosphate-buffered saline and then treated with serum-free medium
containing 0.5 mg/ml trypsin, 0.5 mg/ml proteinase K (Sigma), and 0.5
mM EDTA for 2-4 min at 4 °C. The washing and
trypsin/proteinase K/EDTA treatment were found to remove >80% of the
surface-associated
I-apoJ (data not shown). The
cells were then centrifuged at 6000
g for 2 min, and
the amount of radioactivity in the cell pellet was measured.
gp330-Sepharose Chromatography of Human Milk Selects
ApoJ
The fact that gp330 expression in vivo is
restricted to epithelial cells in contact with fluids prompted us to
examine biological fluids for the presence of gp330 ligands. Human
breast milk was available in larger quantities than other potential
gp330 ligand-containing fluids like cerebral spinal fluid and seminal
plasma. An affinity matrix of gp330 coupled to Sepharose was used to
select gp330-binding proteins from human skim milk. The skim milk was
preabsorbed on a column of Sepharose and then applied to a column of
gp330-Sepharose. Elution of the gp330-Sepharose column with a buffer
containing urea released a polypeptide that when analyzed by SDS-PAGE
had a molecular mass of 70 kDa (Fig. 1). The 70-kDa protein was
subjected to protein sequence analysis, which revealed two sequences
identical to the first 10 residues of the - and
-subunits of
human apolipoprotein J, a known 70-kDa milk protein (de Silva et
al., 1990a, 1990b). In immunoblotting analysis, monoclonal apoJ
antibodies were found to react with the 70-kDa protein eluted from the
gp330 affinity matrix (data not shown). The results show that
apoJ can be isolated from milk by gp330-Sepharose affinity
chromatography and suggest that gp330 interacts with apoJ.
Figure 1:
Selection of apoJ by gp330-Sepharose
affinity chromatography. Human skim milk was passed over
gp330-Sepharose, the column was washed with 50 mM Tris, 0.15
M NaCl, pH 8.0, and the bound fractions were eluted with 2
column volumes of 8 M urea, 50 mM Tris, pH 7.4.
Lanes 1-6 represent SDS-PAGE analysis of sequentially
eluted fractions from the affinity column stained with Coomassie Blue.
The migration positions of molecular mass standards are indicated on
the right in kDa.
ApoJ Binds with High Affinity to gp330 but Not to
LRP
We next investigated the ability of apoJ to bind to gp330 by
using several types of solid-phase binding assays. In ELISA, apoJ bound
to gp330 (urine or kidney derived) immobilized on microtiter well
plastic with half-saturating levels of binding achieved at 2.8
nM for porcine gp330 and 3.8 nM for human gp330
(Fig. 2). Homologous ligand competition assays were performed to
obtain quantitative information regarding the apoJ-gp330 interaction.
As shown in Fig. 3A, I-apoJ bound to
immobilized gp330, and the binding could be inhibited with increasing
concentrations of unlabeled apoJ. The binding data could be fit to a
model containing a single class of sites with a
K
of 10 nM. In these assays, we
also investigated the ability of apoJ to bind to LRP, a receptor
closely related to gp330 in structure and function. However, no binding
of
I-apoJ to immobilized LRP was observed
(Fig. 3A). In addition, no binding was observed between
apoJ and LRP in ELISA (data not shown). The functionality of
the LRP used in the assays was confirmed by its ability to promote
binding of
I-apoE, a known LRP ligand (Beisiegel et
al., 1989; Kowal et al., 1989) (Fig. 3B).
In these experiments
I-apoE also bound to gp330 with a
K
of 50.7 nM, which was similar
to that for LRP, K
= 53.6
nM. The apoJ-gp330 interaction was also evaluated by using a
gel blot-overlay assay in which apoJ, transferred onto nitrocellulose
filters after SDS-PAGE, was incubated with buffer containing either
gp330 or LRP. Bound receptor was then detected by using radiolabeled
monoclonal receptor antibodies. As shown in Fig. 4A,
lane 4, gp330 bound to immobilized apoJ, whereas LRP did not
(Fig. 4B, lane 4). A control for the
functionality of both receptors in this assay was their ability to bind
to the 39-kDa protein RAP (Fig. 4, lane 2 in panels
A and B). The results of both the solid phase microtiter
well assays and gel blot overlay assays show that apoJ binds to gp330
but not to LRP.
Figure 2:
Binding of apoJ to gp330 as measured by
ELISA. Increasing concentrations of apoJ were incubated with microtiter
wells coated with human gp330 (), porcine gp330 (
), or
ovalbumin (
) (5 µg/ml). Bound apoJ was detected with the
monoclonal anti-apoJ IgG 1D11. The half-saturation values determined
for porcine and human gp330 were 2.8 and 3.8 nM, respectively.
The data presented are representative of three experiments each
performed in duplicate.
Figure 3:
Binding of I-apoJ and
I-apoE3 to gp330. In panel A,
I-labeled apoJ (1 nM) was incubated for 18 h at
4 °C in wells coated with gp330 (
), LRP (
), or ovalbumin
(
) in the presence of increasing concentrations of unlabeled
apoJ (0.2-450 nM). After incubation, wells were washed
and counted. The data were analyzed by the program LIGAND, and the
curve represents the best fit of the data to a single class of sites
with a K of 10 nM. The data presented are
representative of six experiments having a mean K =
14.2 ± 8.6 nM). In panel B,
I-labeled apoE3 (2 nM) was incubated with gp330,
LRP, or ovalbumin-coated wells in the presence of increasing
concentrations of unlabeled apoE3 (0.2-450 nM) for 18 h
at 4 °C. The K values measured for the binding of apoE3 to
gp330 and LRP were 50.7 and 53.6 nM,
respectively.
Figure 4:
I-gp330 binding to apoJ
immobilized on nitrocellulose membranes after SDS-PAGE. ApoJ (0.5
µg, lane 2) or RAP (0.5 µg, lane 4) were
electrophoresed on 4-12% polyacrylamide gradient gels under
nonreducing conditions and then transferred to nitrocellulose
membranes. In panel A, the membrane was probed with unlabeled
gp330 (25 nM), and the bound receptor was detected with
I-labeled monoclonal anti-gp330 IgG 1H2. In panelB, the membrane was probed with unlabeled LRP (25
nM), and bound receptor was detected with
I-labeled monoclonal anti-LRP IgG 8G1. Both membranes
were washed and exposed to x-ray film. The migration positions of
molecular mass standards are indicated on the right in
kDa.
The gp330 Ligands RAP, ApoE3, and Lipoprotein Lipase
Carboxyl-terminal Receptor-binding Fragment Block Binding of ApoJ to
gp330
We next evaluated the ability of several previously
described gp330 ligands to inhibit the binding of apoJ to the receptor.
Heterologous ligand competition assays were performed using RAP, apoE3,
or lipoprotein lipase carboxyl-terminal receptor-binding fragment as
competitors for radiolabeled apoJ binding to gp330-coated microtiter
wells. As shown in Fig. 5, each of these ligands competed
effectively for the I-apoJ binding (Fig. 5). By
curve fitting the data, inhibition of binding constants
(K
) were determined for each ligand
(). Inhibition constants were also derived for several
other gp330 ligands including pro-uPA, uPA:PAI-1, and lactoferrin, and
the values are shown in (binding curves not
shown). While apoJ, RAP, apoE, and lipoprotein lipase carboxyl-terminal
receptor-binding fragment were able to compete for
I-apoJ
binding to gp330, the other gp330 ligands pro-uPA, uPA:PAI-1, and
lactoferrin were not able to inhibit the binding. The results indicate
that apoJ, apoE3, and lipoprotein lipase carboxyl-terminal
receptor-binding fragment may share a common binding site on gp330 or
have binding sites in close proximity to one another, whereas the other
ligands bind to separate sites on the receptor. Similar results and
interpretations have been reported for the binding of many of these
ligands to LRP (Moestrup et al., 1993a).
Figure 5:
The
binding of I-apoJ to gp330 can be inhibited by RAP,
lipoprotein lipase carboxyl-terminal receptor-binding fragment, and
apoE3.
I-labeled apoJ (1 nM) was incubated
overnight at 4 °C in wells coated with gp330 (5 µg/ml) in the
presence of increasing concentrations of unlabeled apoJ (
), RAP
(
), lipoprotein lipase carboxyl-terminal receptor-binding
fragment (
), or apoE3 (
). Following incubation, the wells
were washed, and bound radioactivity was measured. The binding data was
analyzed using the program LIGAND. The data presented are
representative of two experiments, each performed in duplicate. The
curves represent the best fit data for a single class of sites, and the
derived inhibition constants (K) are indicated in Table I. The
binding of
I-apoJ, in the presence of increasing
concentration of unlabeled apoJ, to wells coated with ovalbumin is
shown as a control (
).
gp330 Mediates the Internalization of ApoJ in F9
Cells
We studied the uptake of I-apoJ by
gp330-expressing embryonal carcinoma F9 cells in order to determine if
gp330 could mediate its cellular endocytosis and degradation. The F9
cells were grown in the presence of RA and DBC since this treatment can
increase gp330 expression levels 20-40-fold (Fig. 6,
inset) while having no effect on LRP, very low density
lipoprotein receptor, or LDLR levels.
(
)
As shown
in Fig. 6, RA/DBC-treated F9 cells internalized 17-fold greater
levels of
I-gp330 monoclonal antibody (1H2) than
untreated cells. Similar results were observed with
I-gp330 polyclonal antibodies (data not shown).
The increased ability of the RA/DBC-treated cells to endocytose gp330
antibody is comparable in magnitude to the increase in gp330 protein
levels in the treated cells as measured by immunoblotting analysis of
F9 cell extracts (Fig. 6, inset). Both the untreated and
RA/DBC-treated F9 cells were found to internalize and degrade
I-apoJ (Fig. 7). The levels of uptake and
degradation of
I-apoJ were respectively 5- and 10-fold
greater in the RA/DBC-treated F9 cells as compared with the untreated
cells. Several cells lines that do not express gp330 but are known to
express LRP such as HepG2 and Chinese hamster ovary cells were found to
be unable to mediate
I-apoJ internalization and
degradation (data not shown). RAP, a competitor of apoJ
binding to gp330 (Fig. 5), and unlabeled apoJ both acted to
inhibit the F9 cell-mediated uptake and degradation of radiolabeled
apoJ in a dose-dependent manner (Fig. 7). Lactoferrin, a gp330
ligand (Willnow et al., 1992) that was a poor competitor of
gp330 binding to apoJ (), did not inhibit the degradation
of apoJ (Fig. 7, C and D). The results show
that cellular catabolism of apoJ correlates with the expression of
gp330.
Figure 6:
Internalization of I-gp330
monoclonal antibody by F9 cells. F9 cells were cultured in the presence
or absence of RA and DBC for 7 days. Duplicate wells each containing 3
10
cells (RA- and DBC-treated or untreated) were
incubated with
I-gp330 monoclonal antibody 1H2 IgG (1.2
nM) for 4 h at 37 °C, 5% CO
in the presence or
absence of 200 molar excess unlabeled 1H2 IgG. The values indicated
represent the amount of antibody specifically internalized as
determined by subtracting the amount of
I-1H2 IgG
internalized in the presence of a 200 molar excess unlabeled 1H2 IgG
from the amount of
I-1H2 IgG internalized minus any
unlabeled 1H2 IgG. These data are representative of five experiments,
each performed in triplicate. Shown in the inset are
immunoblotting analyses of detergent extracts from F9 cells (lane
1) and F9 cells treated for 7 days with RA/DBC (lane 2),
which are stained with monoclonal anti-gp330
1H2.
Figure 7:
Internalization and degradation of
I-apoJ by F9 cells. Wells containing 3
10
F9 cells that had been either RA and DBC treated (panels B and D) or untreated (panels A and C) as
described under ``Materials and Methods'' were incubated with
I-apoJ (5 nM) for 18 h at 37 °C in the
presence of increasing concentrations (1.8-450 nM) of
unlabeled apoJ (
), RAP (
), or lactoferrin (
).
PanelsA and B show
I-apoJ
internalization data, and panels C and D show
I-apoJ degradation data.
To provide further evidence for the role of gp330 as a
mediator of apoJ endocytosis and degradation, we tested the effects of
gp330 polyclonal antibodies (r239) on F9-mediated catabolism of
I-apoJ. These antibodies have been shown to efficiently
block the uptake and degradation of other gp330 ligands (data
not shown). As shown in Fig. 8, both internalization (panel
A) and degradation (panel B) of
I-apoJ by
RA/DBC-treated F9 cells were inhibited by gp330 antibodies, whereas
antibodies to LRP had no effect. In addition, the degradation of
I-apoJ was inhibited by chloroquine (Fig. 8,
panel B), a drug that alters the pH of lysosomes leading to
inhibition of lysosomal proteinase activity. These results suggest that
gp330 functions to mediate endocytosis of apoJ, which leads to its
degradation by lysosomal proteases.
Figure 8:
Antibodies to gp330 inhibit
internalization and degradation of I-apoJ by F9 cells. F9
cells treated for 7 days with RA/DBC were incubated with
I-apoJ (5 nM) for 18 h at 37 °C in the
presence of RAP (450 nM), polyclonal gp330 antibodies (100
mg/ml), polyclonal LRP antibodies (100 mg/ml), or chloroquine (100
mM). PanelA shows
I-apoJ
internalization data, and panelB shows
I-apoJ degradation data. The observed effect of
chloroquine on the level of apoJ internalization (panel A) is
consistent with accumulation of the ligand within
lysosomes.
A4 peptide of the
Alzheimer's precursor protein (Ghiso et al., 1993), a
subclass of high density lipoprotein (de Silva et al., 1990a,
1990b; James et al., 1991; Stuart et al., 1992) and
the membrane-attack complex C5b-C9 (Murphy et al., 1989; Jenne
and Tschopp, 1989). It is conceivable that gp330 mediates clearance of
apoJ in complex with these or other molecules. In this regard, apoJ
would function like several other ligands for the LDLR family. For
example, apoJ may function to target lipoproteins for clearance as does
apoE or lipoprotein lipase (Kowal et al., 1989; Beisiegel
et al., 1991; Chappell et al., 1992). ApoJ has also
been shown to act as an inhibitor of the cytolytic activity of the
complement membrane-attack complex C5b-C9 (Jenne and Tschopp, 1989;
Kirszbaum et al., 1989). This interaction may facilitate
clearance of the inactive complexes via gp330 in a manner analogous to
the way in which proteases are inactivated by
-macroglobulin or plasminogen activator inhibitor-1
and then cleared by LRP (Strickland et al., 1994).
(
)
in the regressing interdigital
regions of the developing limb (Buttyan et al., 1989) and in
retinitis pigmentosa-affected retinas (Jones et al., 1992;
Jomary et al., 1993). The function that apoJ serves in these
situations is not yet known. French et al.(1992) have proposed
that apoJ may be involved in the removal of debris resulting from
apoptosis or that it may protect by-stander cells from cytolysis by
cell debris-activated complement. They also point out that in the
thymus, where extensive apoptotic cell death normally occurs,
inflammation is absent perhaps owing to the action of apoJ expressed by
nondying cells in the medulla of the tissue. The typical absence of
inflammation in areas that undergo apoptosis and the observed enhanced
expression of apoJ in such areas makes it tempting to speculate that a
primary function of apoJ is to restrict inflammation. Consistent with
any of the postulated roles of apoJ is a role for a receptor such as
gp330 to endocytose apoJ in complex with complement components or lipid
debris/apoptotic bodies.
Table:
Summary of binding constants for gp330-ligand
interactions derived from homologous and heterologous ligand
displacement experiments
) and average inhibition
constants (K
) were derived from the best
fit of the data to a single class of sites by using the computer
program LIGAND (Munson and Rodbard, 1980). The
K
shown for gp330 binding to uPA:PAI-1 is
in agreement with that reported by Moestrup et al. (1993b).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.