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Trafficking of the Igα/Igβ Heterodimer with Membrane Ig and Bound Antigen to the Major Histocompatibility Complex Class II Peptide-loading Compartment*

The binding of antigen to the B cell antigen receptor (BCR) initiates two major cellular events. First, upon cross-linking by antigen, the BCR induces signal transduction cascades leading to the transcription of a number of genes associated with B cell activation. Second, the BCR internalizes and delivers antigens to processing compartments, where processed antigenic peptides are loaded onto major histocompatibility complex (MHC) class II molecules for presentation to T helper cells. The BCR consists of membrane Ig (mIg) and Igα/Igβ heterodimer (Igα/Igβ). The Igα/Igβ, the signal transducing component of the BCR, has been indicated to play a role in antigen processing. In order to understand the function of the Igα/Igβ in antigen transport, we studied the intracellular trafficking pathway of the Igα/Igβ. We show that in the absence of antigen binding, the Igα/Igβ constitutively traffics with mIg from the plasma membrane, through the early endosomes, to the MHC class II peptide-loading compartment. Cross-linking the BCR does not alter the trafficking pathway; however, it accelerates the transport of the Igα/Igβ to the MHC class II peptide-loading compartment. This suggests that the Igα/Igβ heterodimer is involved in BCR-mediated antigen transport through the entire antigen transport pathway.

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 degrada-tion 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)(8)(9)(10)(11)(12)(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, BCRmediated antigen processing requires 1 ⁄1000th to 1 ⁄10,000th the antigen as compared with antigen taken up by fluid phase pinocytosis (23)(24)(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, upregulate 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 mIgassociated 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)(32)(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 tyrosinebased 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.

EXPERIMENTAL PROCEDURES
Cell Lines and Antibodies-The B cell lymphoma CH27 was generated and characterized by Haughton et al. (36) and is an H-2 k , IgM ϩ , Fc␥RIIB1 Ϫ 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-E k -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 (HϩL) (anti-Ig), goat antibodies specific for mouse chain (anti-), Fab fragment of goat anti-antibody (Fab-anti-), horseradish peroxidase-conjugated goat anti-antibody (HRP-anti-), and goldlabeled goat anti-rat antibody were purchased from Jackson Immu-noReseach (West Grove, PA). Gold-labeled Fab-anti-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 Cterminal 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.
HRP-mediated Cross-linking Assay-Surface biotinylated cells were pulsed with HRP-anti-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 3Ј,3-diaminobenzidine (DAB) reaction buffer (0.5 mg/ml DAB and 0.03% H 2 O 2 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, H 2 O 2 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 ϫ 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-antifirst and incubated in 10% complete medium at 37°C for 2 h to chase the HRP-anti-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 HRPmediated cross-linking reaction and processed as detailed above.
Immunoelectron Microscopy-Cells were pulsed with gold-labeled Fab-antifor 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 antiantibody 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 antiantibody, 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-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␤. 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-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/endo-plasmic 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--treated and untreated cells was recovered from the fractions containing the early endosomes. In the anti--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--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--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.
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-as an antigen and the mIg and the Ig␣/Ig␤ on the cell surface were labeled with biotin. In the presence of DAB and H 2 O 2 , HRP catalyzes nonspecific crosslinking 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-is not membrane permeable, HRP-mediated crosslinking reactions only occur in vesicles where HRP-anti-is present. A reduction in the amount of the immunoprecipitated proteins indicates co-localization of these proteins with HRP-anti-.
Surface biotinylated CH27 cells were pulsed with HRP-anti-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-. 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, H 2 O 2 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 mIgantigen complex after internalization, the Ig␣/Ig␤ and HRPanti-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 H 2 O 2 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 H 2 O 2 , 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-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-was still in the early endosomes but also at the later chase times when HRPanti-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.
In the control experiment, CH27 cells pulsed with HRPanti-for 15 min at 37°C was chased at 37°C for 2 h. By this time, HRP-anti-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 H 2 O 2 , 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-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-(8 nm) and chased at 37°C for 1 h to label the MIIC. The ultrathin cryosections were labeled with the class II I-E k -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-antiaccumulated 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-, indicating that after 1 h of chase, the Fab-anti-, the BCR internalized anti- gen, accumulated in the MIIC. As shown in Fig. 4A, the multivesicular structures in which the Fab-anti-accumulated were also labeled by anti-Ig␣ mAb. Thus, at the steady state, the Ig␣/Ig␤s are present in the MIIC.
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-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 streptavi-din-HRP (Fig. 5A). As shown in Fig. 5A, biotinylated Ig␣/Ig␤ was recovered in the anti-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.
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-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 FIG. 3. Co-localization of the Ig␣/Ig␤ heterodimer with mIgM. A, surface biotinylated CH27 cells were pulsed with HRP-anti-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 H 2 O 2 . 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-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 (Ϯ S.E.) of the results of three independent experiments is shown. C, cells were pulsed with HRP-anti-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 H 2 O 2 . 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.
vesicles, which is consistent with the results we showed early in this report. The Ig␣ in the anti-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.
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)(33)(34)(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 demon- 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-antibody in 1% digitonin lysis buffer (B). The immunoprecipitates were analyzed by SDS-PAGE and Western blotting and blotted with anti-Ig␣ antibody. strate 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.