 |
INTRODUCTION |
B cells process and present antigens to T cells in order to
initiate T cell-dependent antibody responses. The antigen
processing involves the internalization of antigens, the transport of
antigens to the processing compartments, the proteolytic degradation of protein antigens, and the loading of the peptides onto major
histocompatibility complex
(MHC)1 class II molecules (1,
2). Recent studies have shown that antigen degradation and peptide
loading mainly occur in the endocytic system (3). The loading of
peptide onto MHC class II molecules has been reported to occur in both
the early (4-6) and late (7-13) parts of the endocytic pathway. The
location of peptide loading seems to vary with antigens, B cell lines
and MHC class II alleles. The MHC class II peptide-loading compartment
in the late part of the endocytic pathway (MIIC) has been better
characterized comparing to the others. This compartment is relatively
acidic (14), and contains class II molecules, DM (15), a class II-like molecule that catalyzes the peptide-exchange of class II molecules (16-19), and some of the late endosomal and lysosomal markers, such as
rab 7,
-hexosaminidase and LAMP-1 (9), but not the transferrin
receptor (10, 11). The peptide-class II complexes formed in this
compartment are capable of activating specific T cells in
vitro (9).
B cells, which are unique antigen-presenting cells, express clonally
specific antigen receptors on the cell surface. Membrane Ig (mIg) is
the antigen-binding domain of the B cell antigen receptor (BCR).
Disulfide-linked Ig
/Ig
heterodimer (Ig
/Ig
) noncovalently associates with the mIg (20). The cytoplasmic tails of the Ig
/Ig
contain conserved motifs (the immunoreceptor tyrosine-based activation motifs), which provide the BCR with the signal transducing ability (21,
22). B cells are very efficient antigen-presenting cells. The high
efficiency of B cells in antigen presentation relies on the BCR.
Whereas antigens can also enter the cells by fluid phase pinocytosis,
BCR-mediated antigen processing requires
th to
th the antigen as compared with antigen taken up by
fluid phase pinocytosis (23-25). Therefore, B cells are able to
efficiently present an antigen to T helper cells when the concentration
of the antigen is low. This is particularly critical for the initiation
of the secondary antibody response. Signals transduced through the BCR
also influence antigen processing. Earlier reports (26) showed that
both monovalent and divalent antigens are efficiently presented by
antigen-specific B cells, however, 10-fold more monovalent antigen is
required as compared with bivalent antigen. This observation suggests
that divalent antigens, which initiate the signal transduction cascade,
up-regulate the antigen presentation function of B cells. Indeed,
subsequent studies demonstrated that signaling through the BCR results
in biochemical changes in the MIIC (27) and the aggregation and fusion
of the late endosome and lysosome (28), which correlate with heightened
antigen processing. A recent report (29) showed that overexpression of
the dominant negative mutant of Syk, a key tyrosine kinase
in the BCR signal transduction pathway, affects antigen presentation
mediated by an Ig
chimera.
The BCR contributes to efficient antigen processing by its ability to
internalize and to deliver antigens to the processing compartment. Our
previous studies (30) demonstrated that in B cells mIgM and bound
antigens are internalized from the plasma membrane (PM) into the early
endosomes and are subsequently delivered to the MIIC, where they meet
newly synthesized class II molecules transported from the
trans-Golgi network. This result indicates that mIg is the
carrier that transports antigen to the MIIC. BCR signaling also
directly affects trafficking of the mIg and bound antigen to the MIIC.
Cross-linking mIgM by an antigen, which initiates a signal transduction
cascade, increases the internalization rate of the antigen and
accelerates the transport of mIg-antigen complexes to the MIIC. The
structural basis of such regulated trafficking is not known. Because
the cytoplasmic tails of mIgM and mIgD are only three amino acids long
and lack any identifiable trafficking motif, it is possible that the
Ig
/Ig
, a mIg-associated protein, controls the intracellular
trafficking of the BCR and bound antigens. Indeed, mIgMs, containing
mutations that disturb their interaction with the Ig
/Ig
, are not
able to facilitate antigen processing (31-33). The cytoplasmic tail of
either Ig
or Ig
can drive antigen processing mediated by chimeric
receptors, even though the cytoplasmic tail of Ig
targets the
chimeras to early endosomes (34). The deletion of the cytoplasmic tail
of Ig
blocks the constitutive internalization of the BCR (35). A
chimeric receptor of Ig
, with a point mutation of a tyrosine residue
in the immunoreceptor tyrosine-based activation motif and a 17-amino
acid deletion in its cytoplasmic tail, cannot facilitate the processing
and presentation of cryptic epitopes of antigens (29). All of these
studies indicate that Ig
/Ig
plays an important role in
BCR-mediated antigen transport. However, it is not known how the
Ig
/Ig
carries out this function. The intracellular trafficking
pathway of the Ig
/Ig
still remains to be determined. Thus, it is
unclear whether the single chain chimeras widely used in these studies
traffic through the same pathway as the native Ig
/Ig
and deliver
antigens into the same processing compartment as the BCR.
Herein, we describe the intracellular transport pathway of the
Ig
/Ig
heterodimer in B cells and show that the Ig
/Ig
constitutively traffics with mIg and bound antigen from the PM through
the endocytic pathway to the MIIC, where functional peptide-class II
complexes are formed.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Antibodies--
The B cell lymphoma CH27 was
generated and characterized by Haughton et al. (36) and is
an H-2k, IgM+, Fc
RIIB1
cell
line. The CH27 cells were cultured in Dulbecco's modified Eagle's
medium supplemented as described (37), containing 15% fetal calf serum
(15% complete medium). The mouse hybridoma 17.3.3s producing an
I-Ek-specific mAb (38) was obtained from the American Type
Culture Collection (Manassas, VA). The rat hybridoma ID4B producing a LAMP-1-specific mAb was obtained from the Development Studies Hybridoma
Bank (Iowa City, IA). Goat antibodies specific for mouse IgG (H+L)
(anti-Ig), goat antibodies specific for mouse µ chain (anti-µ), Fab
fragment of goat anti-µ antibody (Fab-anti-µ), horseradish peroxidase-conjugated goat anti-µ antibody (HRP-anti-µ), and
gold-labeled goat anti-rat antibody were purchased from Jackson
ImmunoReseach (West Grove, PA). Gold-labeled Fab-anti-µ was generated
as described previously (39). Ig
-specific polyclonal antibodies
(anti-Ig
) were generated in rabbits immunized with a synthetic
peptide construct, which consists of a 20-residue peptide of the Ig
protein C-terminal cytosolic tail connected by a
-turn to a potent T
cell epitope derived from tetanus toxoid. An Ig
-specific mAb
(anti-Ig
mAb) was generated in rats against the entire cytosolic
tail of the Ig
protein using a glutathione S-transferase
fusion protein. Ig
-specific polyclonal antibodies and the
glutathione S-transferase fusion protein of Ig
were gifts
from Dr. Marcus Clark (University of Chicago, IL). Gold-labeled protein
A and protein G were purchased from Sigma.
Surface Biotinylation--
CH27 cells were washed at 4 °C
with Hanks' balanced saline solution lacking phosphate and containing
20 mM sodium HEPES, pH 7.4, and incubated in the same
buffer containing 0.2 mg/ml sulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce) for 15 min at 4 °C. After 15 min of incubation, a freshly made biotin solution was added, and the incubation was extended for
another 15 min at 4 °C. The cells then were washed with Dulbecco's modified Eagle's medium containing 6 mg/ml bovine serum albumin and 20 mM MOPS, pH 7.4.
Subcellular Fractionation--
Subcellular fractionation was
conducted as detailed elsewhere (9). Briefly, cells (2 × 108) were washed and homogenized using a Dounce Tissue
Grinder (Wheaton, Millville, NJ). The postnuclear supernatant was
prepared and layered onto a Percoll gradient (1.05 g/ml). After
centrifugation, fractions (0.5 ml) were collected and pooled as
described (14): pool 1 (fractions 2-4), early endosomes and Golgi;
pool 2 (fractions 6-8), PM and endoplasmic reticulum; pool 3 (fractions 13-15), transport vesicles; pool 4 (fractions 20-22),
MIIC, dense late endosomes and lysosomes.
Immunoprecipitation--
Cells or fractions were lysed in 1%
Nonidet P-40 lysis buffer (1% Nonidet P-40, 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and
protease inhibitors). Ig
was immunoprecipitated from the lysate
using anti-Ig
antibody and protein A-Sepharose beads (Amersham
Pharmacia Biotech). Immunoprecipitates were analyzed by SDS-PAGE and
Western blotting. Biotinylated proteins were visualized with
streptavidin-HRP and ECL (NEN Life Science Products). For co-immunoprecipitation, cells or fractions were lysed in 1% digitonin (Calbiochem, San Diego, CA) lysis buffer (1% digitonin, 10 mM triethanolamine, pH 7.5, 150 mM NaCl, 1 mM EDTA) (40). Membrane IgM was immunoprecipitated from the
lysate using anti-µ antibody. The immunoprecipitates were analyzed by
SDS-PAGE and Western blotting. The Ig
in the anti-µ
immunoprecipitates was detected by anti-Ig
antibody.
HRP-mediated Cross-linking Assay--
Surface biotinylated cells
were pulsed with HRP-anti-µ for 15 min at 37 °C. After extensive
washing, the cells were incubated in 10% complete medium at 37 °C
for varying lengths of time. The HRP-mediated cross-linking reaction
was then conducted as previously detailed (41). Briefly, the cells were
washed and incubated in 3',3-diaminobenzidine (DAB) reaction buffer
(0.5 mg/ml DAB and 0.03% H2O2 in Hanks'
balanced saline solution lacking phosphate and containing 20 mM sodium HEPES, pH 7.4) at 4 °C for 45 min in the dark.
In a parallel experiment, H2O2 was omitted from
the DAB reaction buffer. Then the cells were washed and lysed with the
1% Nonidet P-40 lysis buffer. The cross-linked protein aggregates were
removed from the lysate by centrifugation at 16,000 × g for 30 min at 4 °C. The biotinylated Ig
was
immunoprecipitated from the lysates and analyzed as described above. In
the control experiment, cells were pulsed with HRP-anti-µ first and
incubated in 10% complete medium at 37 °C for 2 h to chase the
HRP-anti-µ into the dense compartments. Then, the surface of the
cells was biotinylated. After quenching, the cells were chased in 10%
complete medium for the second time at 37 °C. At the end of the
second chase, the cells were subjected to HRP-mediated cross-linking
reaction and processed as detailed above.
Immunoelectron Microscopy--
Cells were pulsed with
gold-labeled Fab-anti-µ for 10 min and then chased for 1 h at
37 °C. The cells were then washed with 0.1 M sodium
phosphate, pH 7.4 and fixed by 2% paraformaldehyde, 1% acrolein in
0.1 M sodium phosphate, pH 7.4 (freshly made) for 2 h
at room temperature. After being washed with 0.1 M sodium phosphate, the cells were embedded in 10% gelatin, immersed in 2.3 M sucrose in phosphate buffer for 2 h at 4 °C, and
snap frozen in liquid nitrogen. Ultrathin cryosections were collected
on a mixture of sucrose and methylcellulose (42). Ultrathin
cryosections were labeled with 17.3.3s, ID4B, anti-Ig
mAb, and
protein G- and protein A-conjugated colloidal gold (43) and examined in a Zeiss EM10CA electron microscope.
 |
RESULTS |
The Intracellular Degradation of the Ig
/Ig
Heterodimer--
To analyze the intracellular degradation of
Ig
/Ig
, the surface of CH27 cells was biotinylated. The
biotinylated cells were either treated with medium alone or treated
with anti-µ antibody for 30 min at 4 °C to cross-link the BCR and
then chased at 37 °C for up to 4 h. An equal number of cells
from each chase time point were lysed, and Ig
/Ig
s were purified
by immunoprecipitation and analyzed by SDS-PAGE and Western blotting.
The biotinylated proteins were detected by streptavidin-HRP. After
warming to 37 °C, the biotinylated Ig
gradually disappeared,
indicating the intracellular degradation of the Ig
(Fig.
1A). In the untreated cells,
the degradation of the Ig
did not start until the cells were
incubated at 37 °C for 1 h. By 4 h, 55% biotinylated
Ig
remained in the cells (Fig. 1B). In the cells treated
with anti-µ antibody, the degradation of the Ig
was significantly
rapid during the first hour, and after the first hour, the degradation
was reduced to a rate similar to the rate in the untreated cells. Only
25% of the biotinylated Ig
was left in the cells after 4 h
(Fig. 1B). The degradation rate of Ig
in the cells
treated with anti-µ antibody appears to have two phases, a rapid
degradation phase at the initial hour and a slower degradation phase at
the later time. The Ig
showed a very weak biotin signal that was only visible after long exposure. The presence of Ig
in the
anti-Ig
immunoprecipitates was determined by stripping the biotin
blots and reblotting the same Western blots with rabbit anti-mouse
Ig
antibody (data not shown). Because Ig
and Ig
exist as a
disulfide-linked heterodimer, the intracellular degradation and
movement of Ig
should reasonably reflect the behavior of the
Ig
/Ig
heterodimer. Compared with the turnover rate of mIgM
published previously (30), the Ig
/Ig
is degraded in the cells at
a rate similar to that of mIgM. Cross-linking BCR increases the
turnover rate of the Ig
/Ig
.

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Fig. 1.
Cross-linking the BCR increases the
intracellular degradation rate of the
Ig /Ig
heterodimer. The surface of CH27 cells was biotinylated at
4 °C. The cells were incubated with anti-µ antibody or medium
alone at 4 °C and chased at 37 °C for the times indicated. The
cells were then lysed and immunoprecipitated with anti-Ig antibody.
The immunoprecipitates were subjected to reducing SDS-PAGE and Western
blotting. Biotinylated Ig was detected by ECL, using
streptavidin-HRP. A, a representative blot is shown.
B, data from densitometry analysis are plotted as a
percentage of the biotinylated Ig at time 0. An average (± S.E.) of
the results of three independent experiments is shown.
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Intracellular Trafficking of the Ig
/Ig
Heterodimer--
Previously, using subcellular fractionation, we
isolated and characterized the MIIC (9) and described the intracellular transport pathway of mIg and bound antigen in B cells (30). Here, we
use this method to follow the trafficking of Ig
/Ig
through the
endocytic pathway. The surfaces of CH27 cells were biotinylated and the
cells were treated with medium alone or medium containing anti-µ
antibody at 4 °C for 30 min. The cells were incubated at 37 °C
for varying lengths of time to let biotinylated molecules move into the
cells. At the end of each time period, the cells were washed at 4 °C
and subjected to subcellular fractionation (9). The resulting fractions
were pooled into four membrane fractions (early endosomes/Golgi,
PM/endoplasmic reticulum, transport vesicles, and dense late
endosomes/lysosomes/MIIC), as previously characterized (14). The
biotinylated Ig
/Ig
s in the fractions were purified by
immunoprecipitation, analyzed by SDS-PAGE and Western blotting, and
detected using streptavidin-HRP. The Western blots (Fig.
2A) were analyzed by
densitometry (Fig. 2B). In the absence of chase, the
majority of the biotinylated Ig
was present in the fractions that
contain the PM, indicating that the biotinylation only occurred on the
cell surface at 4 °C, and the biotin reagent had been efficiently
removed and quenched before the cells were homogenized. Upon warming to
37 °C for 30 min, a portion of the biotinylated Ig
in both
anti-µ-treated and untreated cells was recovered from the fractions
containing the early endosomes. In the anti-µ-treated cells, a small
portion of the biotinylated Ig
was detected in the dense fractions
containing the MIIC. After 1 h at 37 °C, there was a
significant amount of the biotinylated Ig
in the dense fractions in
the anti-µ-treated cells. In contrast, in the untreated cells, the
biotinylated Ig
did not enter the dense fractions until 2 h
after warming to 37 °C. By 2 h, the relative amount of the
biotinylated Ig
in the dense fractions of the anti-µ-treated cell
slightly decreased, suggesting that the biotinylated Ig
was
degraded. However, we were unable to detect any biotinylated
degradation products in the dense compartments. This may be due to a
low biotin signal and a poor immunoprecipitation of degraded
products.

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Fig. 2.
Subcellular distribution of
Ig in anti-µ treated
and untreated cells. CH27 cells were biotinylated and treated with
anti-µ antibody and medium alone at 4 °C and chased at 37 °C
for the times indicated. The cells were washed, homogenized, and
applied to a Percoll density gradient (9). Fractions (0.5 ml) were
collected and combined as follows: lane 1, fractions 2-4
contain early endosomes and Golgi; lane 2, fractions 6-8
contain the PM and endoplasmic reticulum; lane 3, fractions
13-15 contain transport vesicles; lane 4, fractions 20-22
contain dense late endosomes, lysosomes, and the MIIC. Ig /Ig was
immunoprecipitated from the fractions and subjected to reducing
SDS-PAGE and Western blotting. Biotinylated Ig was detected by ECL,
using streptavidin-HRP. The blots were analyzed by densitometry.
A, representative blots of one of three individual
experiments are shown. B, data from the densitometry
analysis are plotted as a percentage of the total biotinylated Ig in
the cells. An average of the results of three independent experiments
is shown.
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|
These results show that the Ig
/Ig
is constitutively internalized
from the PM into the early endosomes en route to the dense endocytic
vesicles that include the MIIC. Cross-linking the BCR accelerates the
transport of the Ig
/Ig
to the dense vesicles.
Co-localization of the Ig
/Ig
Heterodimer and mIg-antigen
Complexes in the MIIC--
Using subcellular fractionation, we showed
that the Ig
/Ig
is internalized from the PM into the early
endosomes and subsequently moves to the dense vesicles that include the
MIIC. However, whether the Ig
/Ig
enters the MIIC was unclear. To
address this question, we next examined whether Ig
/Ig
co-localizes with mIg when mIg moves from the PM to the MIIC.
Previously, we developed an assay for delivery of mIg-antigen complexes
to the MIIC (30). Here, we used this assay to determine the transport
of Ig
/Ig
to the MIIC. We used HRP-anti-µ as an antigen and the
mIg and the Ig
/Ig
on the cell surface were labeled with biotin.
In the presence of DAB and H2O2, HRP catalyzes
nonspecific cross-linking of proteins in the same compartment,
resulting in large, detergent-insoluble protein polymers (41). Proteins
in the cross-linked polymers cannot be isolated by immunoprecipitation
and fail to enter SDS-polyacrylamide gels (11). Because HRP-anti-µ is
not membrane permeable, HRP-mediated cross-linking reactions only occur
in vesicles where HRP-anti-µ is present. A reduction in the amount of
the immunoprecipitated proteins indicates co-localization of these
proteins with HRP-anti-µ.
Surface biotinylated CH27 cells were pulsed with HRP-anti-µ for 15 min at 37 °C, washed, and chased at 37 °C for 0, 60, 120, and 180 min in the absence of HRP-anti-µ. The cells were washed and incubated
in a DAB reaction buffer at 4 °C to allow the HRP-mediated cross-linking reaction to proceed in HRP containing compartments. In a
parallel experiment, H2O2 was omitted from the
DAB reaction buffer as a control. The cells were lysed, and mIg and
Ig
/Ig
were immunoprecipitated with anti-Ig and anti-Ig
antibodies, respectively. The immunoprecipitates were analyzed by
SDS-PAGE and Western blotting. Biotinylated molecules were detected
with streptavidin-HRP. Previously we showed (30) that after the cells are pulsed with HRP-anti-Ig for 15 min at 37 °C, the majority of
HRP-anti-Ig accumulates in the early endosomes and on the PM. After
1-2 h of chase, most of HRP-anti-Ig reaches the MIIC, where it meets
with newly synthesized MHC class II molecules. If the Ig
/Ig
traffics with mIg-antigen complex all the way to the MIIC, the two
protein complexes should co-localize at all chase times. However, if
the Ig
/Ig
dissociates from the mIg-antigen complex after
internalization, the Ig
/Ig
and HRP-anti-µ complexes should only
co-localize at early chase times, but not at later chase times, when
the mIg bound HRP-anti-Ig reaches the MIIC. In the experiments in which
H2O2 was omitted from the DAB reaction buffer, both biotinylated Ig
and mIgM decreased with time indicating a
synchronized intracellular degradation (Fig.
3A). In the presence of
H2O2, there was a significant reduction in the
amount of biotinylated mIgM at all chase times up to 180 min (Fig. 3,
A and B), indicating that HRP-anti-µ associated
with mIgM throughout the transport pathway from the PM to the MIIC.
Moreover, there was a significant reduction of the biotinylated Ig
not only at the early chase times when HRP-anti-µ was still in the
early endosomes but also at the later chase times when HRP-anti-µ had
reached the MIIC (Fig. 3, A and B). These results
indicate that the Ig
/Ig
indeed enters the MIIC when the
mIg-antigen reaches there.

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Fig. 3.
Co-localization of the
Ig /Ig heterodimer
with mIgM. A, surface biotinylated CH27 cells were
pulsed with HRP-anti-µ at 37 °C for 15 min and chased for various
times as indicated. After the chase, the cells were incubated with DAB
reaction buffer with or without H2O2. The cells
were then lysed and centrifuged to remove cross-linked polymers. The
soluble Ig /Ig and mIgM molecules were immunoprecipitated with
anti-Ig antibody and anti-µ antibody, respectively. The
immunoprecipitates were subjected to SDS-PAGE and Western blotting.
Biotinylated Ig and mIgM were detected by ECL, using
streptavidin-HRP. The blots were analyzed by densitometry.
Representative blots are shown. B, data from densitometry
analysis are plotted as a percentage of the biotinylated Ig in the
control cells at time 0. An average (± S.E.) of the results of three
independent experiments is shown. C, cells were pulsed with
HRP-anti-µ first and chased at 37 °C for 2 h. Then, the
surface of the cells was biotinylated and chased at 37 °C for 10 or
60 min. After the chase, cells were incubated with the DAB reaction
buffer with or without H2O2. The cells were
treated as described in A. Representative blots of one of
four individual experiments are shown. D, densitometry
analysis of the blots from C.
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In the control experiment, CH27 cells pulsed with HRP-anti-µ for 15 min at 37 °C was chased at 37 °C for 2 h. By this time, HRP-anti-µ had reached the MIIC (30). The surface of the cells was
then biotinylated at 4 °C and the cells were chased again at
37 °C for 0, 10, and 60 min. The mIgM and the Ig
/Ig
were immunoprecipitated from cells. As shown in Fig. 3, C and
D, in the presence of H2O2, there
was no detectable reduction in the amount of the biotinylated mIgM and
Ig
until after 1 h of chase time. This shows that HRP-anti-µ
in the MIIC and the dense vesicles was unable to cross-link the
biotinylated mIgM and Ig
/Ig
on the PM and in the early endosomes.
Therefore, the results shown in Fig. 3, A and B,
were not caused by the leakage of the HRP from vesicles, and they
indicate that the mIg-antigen complexes co-localize with the
Ig
/Ig
heterodimer throughout the entire antigen transport pathway.
Our biochemical data provided evidence that the Ig
/Ig
co-localizes with mIg and bound antigen from the PM, through the early endosomes to the MIIC. The MIIC has been characterized as
multivesicular bodies and multilamellar vesicles containing class II
molecules and later endosomal markers, such as LAMP-1. To directly
examine the distribution of mIgM and Ig
/Ig
in the MIIC using
electron microcopy, ultrathin cryosections were prepared from CH27
cells that were pulsed with gold-labeled Fab-anti-µ (8 nm) and chased at 37 °C for 1 h to label the MIIC. The ultrathin cryosections were labeled with the class II I-Ek-specific mAb (17.3.3s),
the LAMP-1-specific mAb (ID4B), or anti-Ig
mAb and detected using
gold-labeled protein A (5 nm) or protein G (4 nm). Most of the
Fab-anti-µ accumulated in the multivesicular structures located in
the perinuclear area of the cells (Fig. 4). Both class II-specific (Fig.
4B) and LAMP-1-specific (Fig. 4C) antibodies
labeled multivesicular structures containing the Fab-anti-µ,
indicating that after 1 h of chase, the Fab-anti-µ, the BCR
internalized antigen, accumulated in the MIIC. As shown in Fig.
4A, the multivesicular structures in which the Fab-anti-µ accumulated were also labeled by anti-Ig
mAb. Thus, at the steady state, the Ig
/Ig
s are present in the MIIC.

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Fig. 4.
Co-localization of the
Ig /Ig heterodimer
with class II molecules, LAMP-1, and BCR internalized antigen in the
MIIC. CH27 cells were pulsed with 8 nm gold-labeled Fab-anti-µ
antibody for 10 min and chased for 1 h at 37 °C. Then the cells
were fixed, perfused with sucrose, and snap frozen in liquid nitrogen
for thin section. ~100-nm sections were collected. The sections were
stained with anti-Ig mAbs (A), 17.3.3s (class II)
(B), and ID4B (LAMP-1) (C) and gold-labeled
protein A (5 nm) and protein G (4 nm). N, nuclei;
M, mitochondria. The bar represents 0.1 µm.
Fab-anti-µ (8 nm); arrowheads, Ig (4 nm);
straight arrows, class II (5 nm); curved arrows,
LAMP-1 (4 nm).
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Association of the Ig
/Ig
Heterodimer with mIg--
The
Ig
/Ig
noncovalently associates with mIg to form the BCR
complexes. This association can be maintained in a mild detergent, digitonin (40). In order to determine whether the Ig
/Ig
remains associated with mIg during trafficking, we carried out
co-immunoprecipitations of mIg and Ig
using a digitonin lysis buffer
(40). Surface biotinylated CH27 cells were chased at 37 °C for up to
4 h. The cells were then lysed in 1% digitonin lysis buffer. The
lysates were subjected to immunoprecipitation using anti-µ antibody.
The immunoprecipitates were analyzed by SDS-PAGE and Western blotting. The Ig
in the immunoprecipitates was identified with anti-Ig
antibody (Fig. 5B). The
biotinylated Ig
and mIgM was visualized with streptavidin-HRP (Fig.
5A). As shown in Fig. 5A, biotinylated Ig
/Ig
was recovered in the anti-µ immunoprecipitates throughout the entire 4 h chase period. By 4 h, the amount of both mIgM
and the co-immunoprecipitated Ig
/Ig
started to decrease
indicating their degradation.

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Fig. 5.
Co-immunoprecipitation of
Ig with mIgM. Surface biotinylated CH27
cells were washed and chased at 37 °C for the times indicated. The
cells were lysed in 1% digitonin lysis buffer. Membrane Ig was
immunoprecipitated from the cell lysates with anti-µ antibody. The
immunoprecipitates were analyzed by SDS-PAGE and Western blotting. The
Ig in the anti-µ immunoprecipitates was detected with anti-Ig
antibody (B). The biotinylated mIgM and Ig were detected
using streptavidin-HRP (A).
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|
To confirm that Ig
/Ig
still associates with mIg when it reaches
the MIIC, CH27 cells were washed, homogenized, and applied to a Percoll
density gradient. The fractions from the Percoll gradient were
subjected to immunoprecipitation with anti-Ig
antibody in the
Nonidet P-40 lysis buffer, which disrupts the association between mIg
and the Ig
/Ig
(Fig. 6, top
panel), and with anti-µ antibody in the digitonin lysis buffer,
which reserves the association (Fig. 6, bottom panel). The
anti-Ig
immunoprecipitation of the fractions lysed by Nonidet P-40
reflected the steady-state distribution of the Ig
in the cells. The
result in the top panel of Fig. 6 showed that at steady
state, there is a significant amount of the Ig
/Ig
s in the dense
vesicles, which is consistent with the results we showed early in this
report. The Ig
in the anti-µ immunoprecipitation was detected with
anti-Ig
antibody. As shown in the bottom panel of Fig. 6,
Ig
/Ig
in the dense vesicle was co-immunoprecipitated by
mIg-specific antibody.

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Fig. 6.
Subcellular distribution of mIg-associated
Ig /Ig . CH27
cells were washed, homogenized, and applied to a Percoll density
gradient (9). The fractions (1 ml) from the Percoll gradient were
collected and subjected to immunoprecipitation with anti-Ig antibody
in the 1% Nonidet P-40 lysis buffer (A) and with anti-µ
antibody in 1% digitonin lysis buffer (B). The
immunoprecipitates were analyzed by SDS-PAGE and Western blotting and
blotted with anti-Ig antibody.
|
|
Taken together, these results indicate that the Ig
/Ig
heterodimers remain in association with mIgM when they are on the cell
surface and until they reach the processing compartment to be degraded.
 |
DISCUSSION |
The BCR transports antigen to the processing compartments and
transduces signals for accelerated BCR movement, which enhance the
antigen processing ability of B cells. It is not clear how this
receptor directs and regulates the internalization and the intracellular transport of antigen. Membrane IgM and mIgD, on the
surface of resting B cells, have only three positively charged amino
acids in their cytoplasmic tails. This makes them unlikely candidates
as the only driver for the intracellular transport of antigen. Data
generated with chimeric proteins of Ig
or Ig
(29, 32-35) and the
mutated BCR lacking the associated Ig
/Ig
(31, 33) indicate that
BCR-mediated antigen transport depends on the Ig
/Ig
heterodimer.
However, it is not known how Ig
and Ig
as a heterodimer traffic
in the cell to carry out this function. Here, we studied the
intracellular trafficking of the endogenous Ig
/Ig
in B cells, and
we provide evidence that Ig
/Ig
constitutively traffics with mIg
and bound antigen from the PM through the early endosomes to the MIIC.
Our data show that Ig
/Ig
degrades at a similar rate and follows
the same intracellular trafficking pathway as mIgM. Using biochemical
co-localization and immunoelectron microscopy, we show that upon
entering cells, Ig
/Ig
moves to the MIIC containing class II
molecules, the late endosomal marker, LAMP-1, and the internalized
mIgM. The data from co-immunoprecipitation experiments demonstrate that
Ig
/Ig
remains associated with mIg on its way to the MIIC.
Although class II peptide-loading occurs in both the early (4-6) and
the late (7-13) endocytic system, the MIIC, located in the late part
of the endocytic system, offers a low pH environment that increases the
activities of protein hydrolases and releases DM from the negative
control of DO molecules (44, 45). Moreover, the invariant chain, which
targets class II molecules to the MIIC (46, 47), is proteolytically
degraded there (14). This allows peptides to be loaded onto class II
molecules. Therefore, the transport of antigens to the MIIC is directly
related to the antigen presenting efficiency of B cells and also
influences the generation of antigen epitopes presented by B cells. The
results from this and previous studies (11, 30) suggest that BCR
internalized antigens pass through the early endosomal compartment
transitively and are eventually accumulated in the MIIC. Mitchell
et al. (33) also showed that although the interaction
between mIg and Ig
/Ig
is disrupted by mutations, the mIg is still
able to internalize antigen, but the internalized antigen is not
efficiently presented. Using a group of chimeric proteins of mIg,
Aluvihare et al. (48) recently demonstrated that the
internalization of antigen is not sufficient for presentation. These
results suggest that BCR-facilitated antigen presentation requires
rapid movement of antigens through the early endosomes to the MIIC. The
early endosomal compartment is a major sorting location for
internalized proteins. After being internalized from the cell surface,
proteins either recycle back to the PM or move to the later endosomes.
Transporting receptors, such as the transferrin receptor (49) and the
low density lipoprotein receptor (50), constitutively internalize from
and recycle back to the PM. After internalization into endosomes the
bound ligands are released, iron from the transferrin receptor and low
density lipoprotein from the low density lipoprotein receptor, and the empty receptors then recycle back to the PM for another round of
transport. In contrast, mIg, as the antigen transporter of B cells,
does not dissociate from antigen after internalization (30). The
results from this study show that Ig
/Ig
heterodimers traffic with
mIg-antigen complexes from the PM all the way to the MIIC. This
suggests that the Ig
/Ig
heterodimer not only plays a role in the
internalization of antigen, but also in the targeting of the BCR and
bound antigen from the early endosomes to the MIIC. Therefore, the
association of the Ig
/Ig
heterodimer with mIg is important for
the entire antigen transport pathway of the BCR.
Significant observations made in this study are that in the absence of
BCR cross-linking, the Ig
/Ig
constitutively internalizes and
moves to the MIIC and that cross-linking BCR accelerates its transport.
These observations suggest that the antigen transport function of the
Ig
/Ig
heterodimer is partially independent of its signaling
function; BCR-mediated signaling regulates rather than initiates the
internalization and transport. Thus, in addition to the signaling
motifs, the cytoplasmic tails of Ig
/Ig
heterodimers also contain
information for protein targeting. Batista and Neuberger (51) recently
reported that antigen presentation efficiency of B cells depends on the
binding affinity of antigen to the BCR and the dissociation rate of
antigen from the BCR, which is consistent with our observations.
Because the BCR constitutively internalizes and moves to the MIIC,
antigens that bind to the BCR in a higher affinity and remain
associated with the BCR during the intracellular trafficking should be
processed and presented more efficiently. Recent studies on BCR and T
cell antigen receptor (52, 53) show that the fidelity of
receptor/antigen interaction can fine tune signals transduced by
receptors that subsequently regulate various cellular activities
including BCR-mediated antigen transport. The BCR constitutively moves
from the cell surface to the processing compartment, which ensures its
capability of facilitating the processing and presentation of
monovalent antigens. Cross-linking BCR by multivalent antigens
increases the binding avidity (51) of the antigens to the BCR and
initiates a signaling cascade that accelerates the BCR transport (30),
which subsequently will increase the presenting ability of B cells.
BCR-facilitated antigen processing might be necessary for selectively
amplifying those B cells expressing high affinity receptors during
affinity maturation in the germinal center.
We propose that the Ig
/Ig
heterodimer has dual functions in
antigen processing, targeting antigen to the MIIC, and transducing signals to accelerate antigen transport. Future studies on the interrelationship between the targeting and signaling functions of the
Ig
/Ig
heterodimer should provide new information on the molecular
mechanism of antigen processing.