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The Journal of Immunology, 2000, 165: 1427-1437.
Copyright © 2000 by The American Association of Immunologists

Aberrant Trafficking of the B Cell Receptor Ig-{alpha}ß Subunit in a B Lymphoma Cell Line1

Colm Condon2, Sharon L. Hourihane2, May Dang-Lawson, Jessica Escribano and Linda Matsuuchi3

Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The B cell Ag receptor (BCR) has two important functions: first, it binds and takes up Ag for presentation to T lymphocytes; and second, it transmits signals that regulate B cell development. Normal expression of the BCR requires the association of the Ag binding subunit, membrane IgM (mIgM), with the signaling component, the Ig-{alpha}ß heterodimer. After assembly in the endoplasmic reticulum, the intact BCR travels through the secretory pathway to the cell surface. In this paper, we report two variants of the B lymphoma cell lines, WEHI 279 and WEHI 231, that have both lost the ability to express µ heavy chain and consequently do not express mIgM. However, these variants do express the Ig-{alpha}ß heterodimer. In one variant, WEHI 279*, the Ig-{alpha}ß remained trapped intracellularly in the absence of mIgM. The other variant, 303.1.5.LM, expressed an aberrantly glycosylated Ig-{alpha}ß on the cell surface that was capable of signaling after cross-linking with anti-Ig-ß Abs. Further characterization uncovered a point mutation in the 303.1.5.LM mb1 gene that would change a proline for a leucine in the extracellular domain of Ig-{alpha}. The 303.1.5.LM Ig-{alpha}ß could not associate with a wild-type mIgM after µ heavy chain was reconstituted by DNA transfection. Thus, this mutation could define a region of the Ig-{alpha} polypeptide that is important for recognition by the endoplasmic reticulum quality control system, for association with glycosylating enzymes, and for the association of Ig-{alpha}ß subunits with mIgM subunits to create a complete BCR complex.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigen receptors on the surface of B lymphocytes mediate the specific uptake of Ags for subsequent presentation to T cells and initiate intracellular signals that regulate B cell growth and differentiation (1). The B cell Ag receptor (BCR)4 is a multisubunit transmembrane protein complex composed of a membrane form of Ig (mIg) that is noncovalently associated with the accessory proteins, Ig-{alpha} and Ig-ß. While the mIg portion of the receptor serves as the Ag binding subunit, the Ig-{alpha}ß is responsible for the propagation of intracellular signals (2). Tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAM) located in the cytoplasmic tails of Ig-{alpha}ß become phosphorylated after BCR engagement and then serve as docking sites for signaling components that are recruited to the BCR complex (3). The Ig-{alpha}ß accessory proteins associated with mIgM are also necessary for the intracellular trafficking of the BCR to and from the plasma membrane (4, 5, 6, 7). Incompletely assembled forms of the BCR are retained in the endoplasmic reticulum (ER) and/or Golgi apparatus by a "quality control" system that involves a variety of chaperone proteins (4, 8, 9, 10). Indeed, we and others have shown that all four chains of the BCR (Ig heavy chain, Ig light chain, Ig-{alpha}, and Ig-ß) must assemble into a complex before any of these chains can exit the ER and proceed to the cell surface (4, 5, 11).

Surface expression of a complete, signaling-competent BCR is an important requirement for B cell development, survival, and activation. Studies in transgenic mice have shown that the failure to express any one of the BCR chains leads to a block in B cell development (12). Moreover, Rajewsky and colleagues have shown that turning off the expression of Ig heavy chain in mature B cells rapidly results in cell death (13). Thus, the proper assembly of the BCR, its release from the ER quality control system, and its subsequent trafficking to the cell surface are essential for B cells to develop and function.

Different forms of the BCR are associated with cells in different stages of differentiation. The BCR on mature cells contains either a membrane form of IgM (mIgM) or IgD (mIgD) associated with Ig-{alpha}ß (1, 2). While both mIgM and mIgD require Ig-{alpha}ß for generating intracellular signals, mIgD can travel to the cell surface in the absence of Ig-{alpha}ß while mIgM cannot leave the ER in the absence of Ig-{alpha}ß (5). Thus, Ig-{alpha}ß may interact differently with mIgM and mIgD. Pre-B cells express a pre-BCR on their surface that consists of Ig-{alpha}ß associated with an altered form of membrane IgM. The pre-B cell mIgM contains the µ heavy chain bound to the surrogate light chains, products of the VpreB and {lambda}5 loci in the mouse (14, 15, 16, 17). Signaling through the pre-BCR is required for pre-B cells to efficiently differentiate into immature B cells that express the conventional light chains (18). µ-Chain-negative pro-B cells from recombination activating gene knockout mice have been reported to express Ig-{alpha}ß subunits on the cell surface without mIg. This cell-surface Ig-{alpha}ß is associated with the chaperone protein calnexin as well as three other unidentified cell-surface proteins (19, 20). It is not clear how the Ig-{alpha}ß protein complex and an ER-resident protein (calnexin) escape the ER quality control system and traffic to the cell surface in the absence of mIgM. Nevertheless, the Ig-{alpha}ß on the surface of pro-B cells is capable of initiating tyrosine kinase-based signaling (19) and driving differentiation in vivo of pro-B cells to pre-B cells. This, together with recent data showing that loss of Ig-{alpha} or Ig-ß causes an earlier block in B cell development than does loss of the surrogate light chain (11, 21), suggests that signaling through Ig-{alpha}ß is necessary for the early steps in B cell development.

Given that the association between Ig-{alpha}ß and mIgM and the subsequent generation of a signaling-competent cell-surface form of the BCR is critical for B cell development, it is important to understand how Ig-{alpha}ß interacts with the BCR. While structural studies have suggested that both the extracellular domains and transmembrane domains of the mIgM and Ig-{alpha}ß subunits interact (22, 23, 24), the residues involved have not been mapped. In addition, the sequences on mIgM and Ig-{alpha}ß that are recognized by the ER quality control system have not been identified. Trafficking to the cell surface of incompletely assembled BCR complexes could interfere with signaling by complete BCR complexes and result in blocks in B cell development. In contrast, the ability of Ig-{alpha}ß to move to the cell surface in the absence of mIgM on pro-B cells suggests that the trafficking of this subunit may be developmentally regulated.

In this study, we have identified two variants of B lymphoma cell lines that have lost expression of µ heavy chain and fail to form mIgM. However, both variants retain the ability to express the Ig-{alpha} and Ig-ß chains. In the mIgM-negative WEHI 279* variant of the WEHI 279 B lymphoma cell line, the Ig-{alpha}ß chains were subject to the rules of quality control and remained trapped intracellularly, presumably in the ER. In contrast, in the 303.1.5.LM variant of the WEHI 231 B lymphoma cell line mutant, 303.1.5, the Ig-{alpha}ß chains were glycosylated differently than wild type and were expressed on the cell surface in the absence of mIgM. Moreover, this cell-surface Ig-{alpha}ß could initiate tyrosine kinase activation. Not only was the 303.1.5.LM Ig-{alpha}ß able to proceed to the cell surface in the absence of mIgM, but when a transfected µ-chain was expressed in these cells, the 303.1.5.LM Ig-{alpha}ß was unable to associate with the resulting mIgM. Sequence analysis of the mb1 and b29 genes expressed by the 303.1.5.LM cells revealed that there was a point mutation that changes a proline to a leucine in the extracellular domain of Ig-{alpha}. Thus, this mutation in Ig-{alpha} may provide insights into the structural requirements for the interaction of Ig-{alpha}ß with both mIgM and with components of the ER quality control system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and tissue culture

The B lymphoma cell lines WEHI 231, WEHI 279*, and BalI7 were obtained from Dr. A. L. DeFranco (University of California, San Francisco, CA). The 303.1.5 variant of the WEHI 231 B lymphoma cell line has been described previously (25, 26). WEHI 279* was discovered as a spontaneous µ-chain-negative variant of WEHI 279 (27) during a routine characterization of this cell line. Cells were grown at 37°C in RPMI 1640 supplemented with 10% FCS (Intergen, Purchase, NY), 50 µM 2-ME, 500 U/ml penicillin, 500 µg/ml streptomycin, and 2 mM L-glutamine.

Anitbodies

Polyclonal rabbit anti-mouse µ heavy chain Abs were obtained from either BioCan Scientific (Mississauga, Ontario, Canada), or ICN (Mississauga, Ontario, Canada). Polyclonal rabbit antisera against mouse {kappa} or mouse {lambda} were obtained from ICN. Affinity purified goat anti-mouse µ-chain Abs and goat anti-µ-FITC Abs were obtained from Jackson ImmunoResearch (West Grove, PA). We have previously described the rabbit polyclonal antisera that recognize murine Ig-{alpha} (28). Abs against murine Ig-ß were produced by immunizing rabbits with a peptide from the carboxyl-terminal region of Ig-ß (amino acids 76–96) (9). Other polyclonal rabbit antisera that recognize murine Ig-{alpha} were gifts from Dr. J. Cambier (National Jewish Hospital, Denver, CO) and Dr. J. Jongstra-Bilen (University of Toronto). The 4G10 anti-phosphotyrosine mAb was purchased from Upstate Biotechnology (Lake Placid, NY). Goat and sheep anti-IgG-Abs linked to HRP were obtained from ICN. Goat F(ab')2 anti-hamster IgG-FITC was obtained from Southern Biotechnology Associates (Birmingham, AL). Protein A-HRP and protein G-HRP were obtained from Amersham Life Sciences (Oakville, Ontario, Canada). Protein A coupled to Sepharose CL-4B and protein G coupled to Agarose were obtained from Sigma, (St. Louis, MO).

A hamster hybridoma that produces a mAb (HM79-16) that recognizes the extracellular domain of murine Ig-ß (20) was a gift from Dr. T. Nakamura (University of Tokyo, Tokyo, Japan). The HM79-16 mAb was purified from hybridoma culture media using a protein G-Agarose affinity column. Purified Ab was biotinylated using sulfo-NHS-biotin (Pierce, Rockford, IL). Streptavidin-FITC was obtained Molecular Probes (Eugene, OR).

Preparation of cell extracts

Cells were solubilized at 4°C in either MG lysis buffer (20 mM Tris-HCl, pH 8.0, 1% Triton X-100, 2 mM EDTA, 10% glycerol, 137 mM NaCl), NDET lysis buffer (10 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.4% deoxycholate, 66 mM EDTA), or digitonin lysis buffer (10 mM triethanolamine, pH 7.8, 1% digitonin, 150 mM NaCl, 1 mM EDTA) (29). All of the lysis buffers contained 1 µM pepstatin A, 1 µM aprotinin, 1 µM PMSF, and 1 µM leupeptin to inhibit proteases. After 10 min on ice, nuclei were removed by centrifuging at 14,000 rpm for 10 min in a cold microfuge. When cell extracts were prepared in MG lysis buffer, the extracts were adjusted to 0.3% SDS and 0.4% deoxycholate after removing the nuclei. Protein concentrations were determined using a bicinchoninic acid protein assay (Pierce).

Iodination of cell-surface proteins

Cells were labeled with the membrane-impermeant Thompson’s iodination reagent, 125I-sulfosuccinimidyl(hydroxyphenyl)propionate as described previously (30). Briefly, 125I-sulfosuccinimidyl(hydroxyphenyl)propionate was added to 5 x 106 B lymphocytes that had been washed and resuspended in 0.5 ml PBS containing 1 mg/ml glucose. The labeling reaction as quenched by adding by adding 1 ml of PBS/glucose containing 1 mg/ml L-lysine, and the cells were solubilized in detergent buffers supplemented with 2 mg/ml L-lysine as described above.

Biotinylation of cell-surface proteins

Cell-surface proteins were biotinylated according to Lisanti et al. (31). A total of 5 x 106 B lymphocytes were labeled with 1 mg/ml sulfo-NHS-biotin (Pierce). The biotinylation reaction was quenched by adding 7.5 ml ice-cold RPMI 1640 containing 2 mg/ml L-lysine, and cell extracts were prepared in detergent buffers containing 2 mg/ml L-lysine as described above.

Metabolic labeling of cellular proteins and pulse-chase experiments

Intracellular pools of L-methionine and L-cysteine were depleted by incubating B cells for 1 h at 37°C in RPMI 1640 lacking L-methionine and L-cysteine that was supplemented with 10% dialyzed FCS, 2 mM L-glutamine, penicillin, and streptomycin. The cells were then pelleted and resuspended in 37°C RPMI 1640 lacking L-methionine and L-cysteine that was supplemented with 2% FCS, L-glutamine, penicillin, streptomycin, and 1 mCi Trans [35S]-label. After the 15-min pulse period, the labeling of the cells was terminated by adding unlabeled L-methionine and L-cysteine to a final concentration of 2 mg/ml. The cell suspension was diluted to 50 ml with cold PBS containing 2 mg/ml L-methionine and L-cysteine, and after pelleting the cells they were resuspended in 37°C RPMI 1640 containing 10% FCS and 2 mg/ml L-cysteine and L-methionine. At various times (chase), 1 ml of cells was removed, and the cells were washed twice with cold PBS containing 2 mg/ml methionine and cysteine before being lysed in NDET lysis buffer containing protease inhibitors as described above.

Immunoprecipitations

Cell lysates were precleared with 100 µl of a 50% v/v slurry of protein A-Sepharose for 30 min at 4°C. The precleared lysates were then incubated for either 3 or 18 h at 4°C with Abs that had been titrated and shown to be in excess, allowing for quantitative immunoprecipitation. Immune complexes were recovered by adding 100 µl of a 50% v/v slurry of protein A-Sepharose. After 1 h, the beads were washed once by pelleting through a 0.5-ml cushion of 0.5 lysis buffer/30% sucrose, once with 1 ml of detergent lysis buffer, and once with distilled water (32). Proteins were eluted by boiling in SDS-PAGE sample buffer and analyzed by SDS-PAGE gels and Western blotting.

Endoglycosidase treatment

Carbohydrates were enzymatically removed from proteins in immunoprecipitates and in cell lysates. Samples for digestion were prepared in 100 mM sodium citrate, pH 5.5, containing 2% SDS for endoglycosidase H (Endo H) digestion or in 200 mM NaPO4, pH 7.1, 20 mM EDTA, 2% octylglucoside, 2% 2-ME, 0.2% SDS for N-glycosidase F (PNGase F) digestion. Samples were digested for 20 h at 37°C with either 1 U Endo H from Streptomyces plicatus or 1 U N-glycosidase F from Flavobacterium meningosepticum (both from Boehringer Mannheim Biochemicals, Laval, Quebec, Canada). After digestion, SDS-PAGE sample buffer was added and the proteins were analyzed on 12% SDS-PAGE gels followed by Western blotting as described below.

Gel electrophoresis and immunoblotting

Immunoprecipitates and cell extracts (5–15 µg protein) were separated by SDS-PAGE. Unless otherwise indicated, all gel samples were treated with 2% 2-ME to break disulfide bonds. For gels containing Trans [35S]-labeled proteins, the gels were stained with Coomassie blue and then soaked in 0.5 M sodium salicylate for 20 min before drying the gel onto 3-mm paper and exposure to x-ray film.

For Western blotting, SDS-PAGE gels were transferred using a Transblotter (Bio-Rad, Richmond, CA) to nitrocellulose filters or to Immobilon polyvinylidene difluoride membranes (Millipore, Nepean, Ontario, Canada) for 4.5 h at 0.5 amps. Filters were blocked overnight at 4°C in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 5% BSA. After blocking, the filters were incubated with primary Abs diluted in TBS/0.05% Tween 20 (TBST) overnight at 4°C. The Abs to Ig-{alpha}, Ig-ß, µ heavy chain, and {kappa} light chain were used at 1:1000, while the 4G10 anti-phosphotyrosine mAb was used at 1 µg/ml. The filters were washed 4 x 15 min with TBST, then bound Abs were detected using either HRP-conjugated protein A, HRP-conjugated goat anti-mouse IgG, both diluted 1:10,000 in TBST. After 30 min at room temperature, the filters were washed 4 x 15 min with TBST, and immunoreactive bands were detected using an enhanced chemiluminescence detection kit (Amersham). In some instances, filters were probed with 125I-protein A diluted 1:10,000, washed as described above, and exposed to x-ray film. To reprobe filters, bound Abs were eluted by soaking the filter for 10 min at room temperature in TBS, pH 2. After neutralizing the filter by soaking in TBS for 10 min, the filter was reblocked with TBS/5% BSA for 30 min and then reprobed with Abs.

Transfections

Approximately 107 303.1.5.LM cells were washed once and resuspended in 0.8 ml HEPES buffered saline. The cells were mixed with 24 µg of an expression plasmid containing the CMV promoter/enhancer combined with a genomic clone for the membrane form of murine µ heavy chain (4). Then, 10 µg of a plasmid containing a gene conferring neomycin resistance was also added to the cells. The cells were transfected by electroporation at 300 V/250 mF for 0.6 s. Stable, drug-resistant clones were selected in growth medium containing 0.3 mg/ml neomycin (G418). Clones were screened for expression of µ heavy chain by analyzing detergent cell lysates by immunoblotting.

Immunofluorescence

303.1.5.LM, WEHI 279*, or WEHI 231 cells were stained with fluorescinated goat anti-µ or with biotinylated hamster anti-murine Ig-ß plus streptavidin-FITC (Molecular Probes). The cells were then analyzed by flow cytometry on a Becton Dickinson FACScan (San Diego, CA).

Signaling assays

B lymphocytes were washed once in quin saline (25 mM HEPES, pH 7.2, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM MgSO4, 2 mM glutamine, 1 mM sodium pyruvate, 1 mg/ml glucose, 50 µM 2-ME) containing 1 mg/ml BSA at room temperature and resuspended to 2.5 x 106 cells/ml in 37°C quin saline plus 1 mg/ml BSA. After 5 min at 37°C, 0.5 ml of cells were stimulated by adding either anti-IgM Abs to a final concentration of 100 µg/ml or by adding biotinylated anti-Ig-ß Abs (30 µg/ml final concentration) plus streptavidin (40 µg/ml final). Reactions were stopped after 5 min by adding 1 ml ice-cold PBS containing 1 mM sodium vanadate. The cells were washed once with cold PBS/vanadate and lysed on ice for 10 min in MG lysis buffer containing 1 mM vanadate, 1 mM PMSF, 0.01 mg/ml leupeptin, and 0.01 mg/ml aprotinin. Protein concentrations were determined using the bicinchoninic acid assay. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the 4G10 anti-phosphotyrosine mAb as previously described (28).

DNA manipulations

RNA was prepared from WEHI 231 and 303.1.5.LM cells using the RNAid extraction kit (BIO/CAN, Montreal, Canada). cDNA was prepared using the first-strand cDNA synthesis kit (Pharmacia Biotech, Piscataway, NJ). The cDNA was recovered using a Geneclean II kit (Bio 101, Vista, CA). The T to C point mutation at position 377 of the mb1 genomic clone was generated using the Quickchange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The primer pair used was MB1–53 (5'-CCAGTCCCTAGGCTCTTCCTGGACATGG-3') and MB1–35 (5'-CCATGTCCAGGAAGAGCCTGAGGACTGG-3').

PCR amplification

PCR amplification was performed using "Ready To Go" PCR beads (Pharmacia Biotech). Amplification of the 303.1.5.LM Ig-{alpha} gene (mb1) was performed using 5 µl of 303.1.5.LM cDNA and 25 pmol each of the mb1 5' primer (5'-GGACATCTAGATCATGGCTTTTCCAGCT-3') and the mb1 3' primer (5'-CGGAAGCTTACGATGCCAGGGGGTCTA-3') in a final volume of 25 µl. Amplification of 303.1.5.LM Ig-ß was performed using 25 pmol each of the b29 5' primer (5'-TGGGCAAGCTTACAGAGCAGTGACCATG-3') and the b29 3' primer (5'-GAAGGTCTAGATCATTCCTGGCCTGGAT-3'). PCR were conducted in a DNA thermal cycler 480 (Perkin-Elmer Cetus, Norwalk, CT) under the following program: 94°C for 4 min (1 cycle); 95°C for 45 s, 55°C for 2 min, 72°C for 2 min (35 cycles); 72°C for 5 min (1 cycle); 4°C soak. PCR products were gel purified, blunt ended with Klenow polymerase, and ligated into EcoRV-digested pBluescript (Stratagene). Bacterial colonies containing the PCR product were identified by blue-white screening. DNA was prepared using the QiaPrep spin mini-prep kit (Qiagen, Chatsworth, CA) and analyzed by restriction enzyme digestion. DNA sequencing was performed by the Nucleic Acid and Protein Sequencing unit at the University of British Columbia.

Sequencing mb1 and b29 PCR products

Nine pBluescript clones of amplified mb1 cDNA were sequenced. The sequencing primers used were the pBluescript T3 primer (5'-ATTAACCCTCACTAAAG-3'), the pBluescript T7 primer (5'-AATACGACTCACTATAG-3'), and the internal mb1 primer (5'-TTCAGCCTTCAGTCTAACATCACA-3'). In addition, three separate PCR reactions of 303.1.5.LM cDNA amplified with the mb1 primers were sequenced directly without cloning using one of the following mb1 primers: 5'-GGACATCTAGATCATGGCTTTTCCAGCT-3', 5'-CGGAAGCTTACGATGCCAGGGGGTCTA-3' or 5'-TTCAGCCTTCAGTCTAACATCACA-3'. Two pBluescript clones of amplified b29 cDNA were sequenced. The primers used for sequencing the PCR-amplified b29 genes were 5'-TCTAGTCTTAGGATTCAGCACGTT-3' and 5'-ATGGTGAAGTTTCACTGCTACACA-3'. Primers were synthesized by BIO/CAN or by the Hormone Research Institute (University of California, San Francisco, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Variants of B lymphoma cell lines that do not express µ heavy chain but still express light chain as well as Ig-{alpha} and Ig-ß

While studying the trafficking of BCR subunits to the cell surface, we identified two variants of murine B lymphoma cell lines that did not express µ heavy chain but still expressed light chain as well as Ig-{alpha} and Ig-ß. Heavy and light chain expression were examined both by immunoprecipitation from Trans [35S]-labeled cells and by Western blotting (Fig. 1Go). The WEHI 231 and BalI7 B cell lines were used as positive controls for expression of µ heavy chain and {kappa} light chain. The first variant, 303.1.5.LM, is a clonal variant of the WEHI 231 immature B cell line called 303.1.5. This cell line (303.1.5) was established by Page and colleagues from a population of chemically mutagenized WEHI 231 cells that were selected for their resistance to anti-IgM-induced cell death (26). These cells expressed normal levels of the {kappa} light chain but made very little µ chain (26). Limiting dilution cloning of the 303.1.5 cells allowed us to isolate a subclone, 303.1.5.LM, that no longer expressed detectable amounts of µ heavy chain even though it expressed normal amounts of {kappa} light chain (Fig. 1Go, A and B). When 10 times more protein was loaded on a gel, no µ heavy chains could be detected in 303.1.5.LM cells (data not shown). The second µ chain-deficient B variant that we identified was WEHI 279*, which was initially identified as a variant of the WEHI 279 cell line that did not exhibit tyrosine kinase activation in response to anti-IgM Abs. Fig. 1GoA shows that WEHI 279* cells synthesize light chains but do not make detectable amounts of µ heavy chain.



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  		FIGURE 1. Characterization of µ chain-deficient variants of B lymphoma cell lines. A and C, Cell lines were metabolically labeled with Trans [35S]-label and solubilized in NDET lysis buffer, which dissociates the mIgM from the Ig-{alpha}ß subunit. The cell extracts were then immunoprecipitated with either anti-µ (A) or anti-Ig-{alpha} (C). Immunoprecipitated proteins were separated on 12% SDS-PAGE gels under reducing conditions. B and D, WEHI 231 and 303.1.5.LM cells were solubilized in MG lysis buffer, which also dissociates the mIgM from the Ig-{alpha}ß subunit. Cell extracts (10 µg protein) were separated on 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose. The filters were probed with Abs to the µ heavy chain, {kappa} light chain, Ig-{alpha}, or Ig-ß. Bands corresponding to the µ, {kappa}, Ig-{alpha}, and Ig-ß-chains are indicated to the right of each panel. Molecular mass markers are indicated to the left. The experiment in each panel was performed three times with similar results. E, Specificity control for the rabbit anti-Ig-ß polyclonal antisera used in these studies. Cell lysates from Ig-ß-expressing WEHI 231 cells (2.75 µg), Ig-ß-expressing transfected nonlymphoid cells (AtT20 + BCR) (15 µg), and untransfected control cells (AtT20) (15 µg) were separated on SDS-PAGE, transferred to nitrocellulose, and blotted with rabbit anti-mouse Ig-ß antisera followed by protein A-HRP. The Ab was inhibited from binding to immunoreactive bands in the presence of various amounts of Ig-ß-specific peptide that was used as the immunogen (amino acid single letter code FRKRGSQQPQELVSEEGRIVQGGC). A 4-µg/ml concentration of inhibitory peptide was used in the image shown on the right hand side of the gel.

 
We next examined the expression of the Ig-{alpha} and Ig-ß proteins in these variant B lymphoma cell lines. Trans [35S]-labeled cells were solubilized in lysis buffer containing 1% Triton X-100, which dissociates the mIgM subunit from the Ig-{alpha}ß subunit (29). The Ig-{alpha}ß complex was then immunoprecipitated with Abs directed against Ig-{alpha} (Fig. 1GoC). This analysis showed that both 303.1.5.LM cells as well as WEHI 279* cells synthesized 35S-labeled proteins that could be immunoprecipitated with anti-Ig-{alpha} Abs. Shortly after synthesis, these proteins had molecular masses similar to those expected for the various glycosylated forms of Ig-{alpha} and Ig-ß that are early in the secretory pathway. Immunoblotting 303.1.5.LM total cell extracts with Abs directed against Ig-{alpha} and Ig-ß showed that both of these proteins were present in 303.1.5.LM cells but note the differences in molecular masses of the Ig-{alpha} from wild-type and mutant 303.1.5 are compared (Fig. 1GoD). The anti-Ig-{alpha} Abs used have been characterized previously (see Ref. 28). The specificity of the anti-Ig-ß Abs is shown in Fig. 1GoE. Cell extracts prepared from WEHI 231 cells expressing Ig-ß, transfected nonlymphoid cells expressing Ig-ß, and untransfected control cells were blotted with the anti-Ig-ß Abs. The different molecular mass forms of Ig-ß between 55 and 30 kDa are clearly seen in the two Ig-ß-expressing cell lines and not in the untransfected control. The reactivity of these bands with the anti-Ig-ß Abs was inhibited by the presence of the Ig-ß-specific peptide that was used as the immunogen.

The 303.1.5.LM Ig-{alpha}ß becomes Endo H resistant while WEHI 279* Ig-{alpha}ß remains Endo H sensitive

In both 303.1.5.LM cells and WEHI 279* cells, complete Ig-{alpha}ß subunits are produced in the absence of mIgM subunits due to a defect in µ heavy chain production. Normally, incompletely assembled BCR complexes and individual BCR chains are retained in the ER by the quality control system. However, at least in pro-B cells, Ig-{alpha}ß can traffic to the cell surface in the absence of mIgM. Therefore, we investigated whether the Ig-{alpha}ß subunits in 303.1.5.LM cells and WEHI 279* cells were retained in the ER or proceeded to the cell surface. One of the best measures of whether proteins exit the ER and enter the Golgi apparatus and move on to the cell surface is their acquisition of mature, complex carbohydrate structures that render them resistant to digestion by Endo H. Endo H cleaves off high mannose carbohydrate side chains that are present on glycoproteins that are early in the secretory pathway. In the medial Golgi complex, these high-mannose carbohydrates are trimmed and new sugars are added, generating mature, complex side chains that are resistant to Endo H cleavage. Thus, treatment with Endo H can be used to determine the types of carbohydrates on the glycoprotein and the approximate location of the glycoprotein in the secretory pathway.

A pulse-chase experiment was performed in which 303.1.5.LM cells, WEHI 279* cells, and WEHI 231 cells were labeled for 15 min with Trans [35S]-labeled methionine and cysteine and chased with nonradioactive medium for varying amounts of time. The Ig-{alpha}ß complexes synthesized during the labeling period and subsequent chase were recovered by immunoprecipitation with anti-Ig-{alpha} Abs. Half of the sample was treated with Endo H, the other half sample left untreated, and both samples were analyzed by SDS-PAGE (Fig. 2Go). We found that the WEHI 279* Ig-{alpha}ß was Endo H sensitive immediately after the pulse (time 0) and remained completely Endo H sensitive for at least 4 h (Fig. 2GoB). This indicated that the carbohydrates on the WEHI 279* Ig-{alpha}ß were of the high-mannose type, consistent with these proteins being retained in the ER. In contrast, while the 303.1.5.LM Ig-{alpha}ß was initially Endo H sensitive, Endo H-resistant forms began to appear after 0.5–1 h of chase, with the Endo H-resistant forms becoming more predominant at 3–6 h of chase (Fig. 2GoA). This indicated that a significant portion of the carbohydrates on the 303.1.5.LM Ig-{alpha}ß were of the mature, modified type and suggested that some of these Ig-{alpha}ß complexes had exited the ER and were possibly on their way to the cell surface. The acquisition of Endo H resistance by the 303.1.5.LM Ig-{alpha}ß exhibited the same kinetics as that for Ig-{alpha}ß in the wild-type parental WEHI 231 cells in which large amounts of Ig-{alpha}ß traffic to the cell surface as part of BCR complexes (compare Fig. 2Go, A and C). This suggested that despite the lack of mIgM, the Ig-{alpha}ß in 303.1.5.LM cells was able to exit the ER and progress through the Golgi with normal kinetics. In contrast, the Ig-{alpha}ß in WEHI 279* cells appeared to be retained in the ER by the quality control system as would have been expected in cells lacking mIgM (Fig. 2GoB). However, it was noted that there were differences in the molecular masses of the Ig-{alpha}ß from 303.1.5.LM when compared with wild-type WEHI 231 cells that are most likely due to differences in glycosylation (Fig. 2Go, A and C).



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  		FIGURE 2. Endo H sensitivity of Ig-{alpha}ß chains in WEHI 279* cells, 303.1.5. LM cells, and WEHI 231 cells. The cells were pulse labeled for 15 min with Trans [35S]-label and then "chased" with medium containing unlabeled methionine and cysteine for the indicated times. Cells were solubilized in NDET lysis buffer, which dissociates the mIgM from the Ig-{alpha}ß subunit. 303.1.5. LM cell extracts (A) and WEHI 279* cell extracts (B) were immunoprecipitated with anti-Ig-{alpha} Abs. The {kappa} light chains expressed by both of these cell lines were not immunoprecipitated in this experiment. WEHI 231 cell extracts (C) were immunoprecipitated with a mixture of anti-µ and anti-Ig-{alpha} Abs. The anti-µ immunoprecipitate also contains the {kappa} light chain associated with µ heavy chain. Only the region of the gel corresponding to Ig-{alpha}ß and light chain is shown. Immunoprecipitates were incubated with or without Endo H, which removes high mannose-containing sugars that are found primarily on proteins that are located early in the secretory pathway, for example, in the ER. Proteins were separated on 12% SDS-PAGE gels under reducing conditions. Note that immunoprecipitation with anti-Ig-{alpha} Abs should coimmunoprecipitate covalently associated Ig-ß. Thus some of the bands seen in anti-Ig-{alpha} immunoprecipitates may be Ig-ß bands. Similarly, immunoprecipitation with anti-µ heavy chain Abs will coimmunoprecipitate covalently associated {kappa} light chain (C). {kappa} light chains migrate on these gels at the same position as Endo H-digested Ig-{alpha} but does not interfere with the detection of mature Endo H-resistant forms of Ig-{alpha}. Ig-{alpha}U, Ig-{alpha} that was not digested by Endo H; Ig-{alpha}S, Endo H-sensitive forms of Ig-{alpha}; Ig-{alpha}R, Endo H-resistant forms of Ig-{alpha}; {kappa}, {kappa} light chain. The experiments shown in A and C were performed three times with similar results, and the experiment shown in B was performed twice with similar results.

 
The 303.1.5.LM Ig-{alpha}ß is expressed on the cell surface in the absence of mIgM

The Endo H data shown in Fig. 2Go suggested that in 303.1.5.LM cells, but not in WEHI 279* cells, Ig-{alpha}ß was able to progress through the secretory pathway in the absence of mIgM. Therefore, several types of experiments were conducted to determine whether the Ig-{alpha}ß in 303.1.5.LM cells could in fact traffic to the cell surface in the absence of mIgM. First, 303.1.5.LM cells and WEHI 279* cells, as well as normal B lymphoma cells as controls, were surface iodinated with the membrane-impermeant Thompson’s iodination reagent (Fig. 3Go). The labeled cell-surface proteins were immunoprecipitated with either anti-µ heavy chain Abs or with anti-Ig-{alpha} Abs to determine whether or not the cells express mIgM or Ig-{alpha}ß on the cell surface. Fig. 3GoA shows that 125I-labeled µ heavy chain and {kappa} light chain were present on the surface of WEHI 231 and BalI7 cells but not on the surface of 303.1.5.LM cells or WEHI 279* cells. Moreover, no iodinated Ig-{alpha}ß could be immunoprecipitated from WEHI 279* cells (Fig. 3GoB), consistent with the fact that Ig-{alpha}ß in these cells were retained inside the cell by the ER quality control system. In contrast, substantial amounts of 125I-labeled Ig-{alpha}ß were present in 303.1.5.LM cells (Fig. 3GoB), indicating that the 303.1.5.LM Ig-{alpha}ß was present on the cell surface in the absence of mIgM. All of the iodinated Ig-{alpha}ß present in extracts of 303.1.5.LM cells was resistant to Endo H (Fig. 3GoC), consistent with it having traversed the ER and Golgi and transported to the cell surface. Note that in the WEHI 231 cells, some of the cell surface Ig-{alpha}ß was partially Endo H sensitive. Thus, in the wild-type WEHI 231 cells there may be some heterogeneity in the carbohydrate side chains attached to Ig-{alpha}ß and that this heterogeneity was different in 303.1.5.LM carbohydrate side chains. However, the molecular masses of these WEHI 231 wild-type Endo H-sensitive forms are not equivalent to the Endo H-sensitive ER forms, which run at a molecular mass similar to {kappa} light chain (~25 kDa) (Fig. 2GoC). Nevertheless these data, using the membrane-impermeant iodination reagent strongly suggest that the 303.1.5.LM Ig-{alpha}ß can be expressed on the surface of these cells.



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  		FIGURE 3. Expression of Ig-{alpha}ß on the surface of 303.1.5.LM cells. Cell-surface proteins were labeled with 125I using the membrane-impermeant Thompson’s reagent. Cells were solubilized in NDET lysis buffer, which dissociates the mIgM from the Ig-{alpha}ß subunit. Iodinated proteins were immunoprecipitated with either anti-µ heavy chain Abs (A) or anti-Ig-{alpha} Abs (B) and separated on 12% SDS-PAGE gels under reducing conditions. In C, the immunoprecipitated proteins were subjected to Endo H digestion (+) or left untreated (-) before being analyzed by SDS-PAGE. Migration of µ, {kappa}, and Ig-{alpha}ß are indicated to the right of each panel. Molecular mass markers are indicated to the left of each panel. The experiment in each panel was performed four times with similar results.

 
The availability of an Ab that recognized the extracellular domain of Ig-ß allowed us to perform flow cytometry to provide additional evidence that Ig-ß was present on the surface of 303.1.5.LM cells. Staining with a biotinylated anti-Ig-ß mAb (HM79-16) followed by streptavidin-FITC revealed that Ig-ß was present on the surface of 303.1.5.LM cells (Fig. 4Go). Indeed, the amount of accessible Ig-ß on the surface of 303.1.5.LM cells was equal to or greater than that on the surface of the mIgM+ parental WEHI 231 cell line. In contrast, there was little or no Ig-ß on the surface of WEHI 279* cells, consistent with the iodination experiments which indicated that Ig-{alpha}ß is not found on the surface of WEHI 279* cells (see Fig. 3Go). The difference in staining between wild-type WEHI 231 cells and 303.1.5.LM cells with the mAb anti-Ig-ß could be due to differences in accessibility of the Ig-ß when it forms part of a complete BCR complex with mIgM as opposed to expression only with Ig-{alpha}.



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  		FIGURE 4. Expression of Ig-ß on the surface of 303.1.5.LM cells but not WEHI 279* cells. WEHI 231, 303.1.5.LM, and WEHI 279* cells were incubated with or without a biotinylated hamster anti-mouse Ig-ß mAb (HM79-16) that recognizes the extracellular domain of Ig-ß (19 ) followed by staining with streptavidin-FITC and analysis by FACScan. Representative data from one of three similar experiments are shown. Similar results were obtained when the cells were stained with nonbiotinylated HM79-16 followed by goat anti-hamster-FITC.

 
While the flow cytometry data indicated that Ig-ß is on the surface of 303.1.5.LM cells, we could not use flow cytometry to confirm the presence of Ig-{alpha} on the surface of 303.1.5.LM cells because the available anti-Ig-{alpha} Abs are directed against the cytoplasmic domain of Ig-{alpha}. However, we were able to show, by quantitative immunodepletion experiments (Fig. 5Go), that virtually all of the Ig-ß in 303.1.5.LM cells was associated with Ig-{alpha}. Fig. 5GoA shows that four to five successive immunoprecipitations with anti-Ig-{alpha} Abs removed all of the Ig-{alpha} from 303.1.5.LM cell extracts. When the cell extracts that were depleted of Ig-{alpha} were examined for the presence of Ig-ß (Fig. 5GoB), we found that nearly all of the Ig-ß had been removed by coimmunoprecipitation with anti-Ig-{alpha}. Thus, the vast majority of the Ig-ß in 303.1.5.LM cells is associated with Ig-{alpha}, making it likely that the Ig-ß on the surface of these cells is associated with Ig-{alpha}.



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  		FIGURE 5. Coimmunoprecipitation of Ig-{alpha} with Ig-ß in 303.1.5.LM cells. 303.1.5.LM cells were solubilized in MG lysis buffer containing 1% Triton X-100 and 0.3% SDS to dissociate mIgM from the Ig-{alpha}ß subunit. Cell extracts were sequentially immunoprecipitated five times with anti-Ig-{alpha} Abs. The proteins precipitated from the first through fifth immunoprecipitation of the same cell extract were run in lanes 1–5, a portion of the initial cell extract before immunoprecipitation was run in lane 6, and a similar portion of cell extract remaining after each sequential immunoprecipitation was run in lanes 7–11 (i.e., lane 7 is what was left after the first anti-Ig-{alpha} immunoprecipitation while lane 11 is what was left after the fifth anti-Ig-{alpha} immunoprecipitation). Proteins were separated on 12% SDS-PAGE and analyzed by immunoblotting with anti-Ig-{alpha} (A) or anti-Ig-ß (B) Abs. Note that the anti-Ig-ß blot was performed first before stripping the filter and reprobing it with anti-Ig-{alpha} Abs. The migration of the forms of Ig-{alpha} and Ig-ß are marked by the brackets on the right side of the panels. The asterisk on the left of each panel indicates the migration of the IgG heavy chain of the rabbit anti-Ig-{alpha} Ab used for immunoprecipitation, which was detected by the protein A-HRP used to develop the blots. Molecular mass markers are located to the left of each panel.

 
Another test of whether the 303.1.5.LM Ig-{alpha}ß is in fact on the cell surface was to determine whether the addition of anti-Ig-ß Abs to the medium can initiate intracellular signaling reactions. Nagata et al. have previously shown that the Ig-{alpha}ß expressed on the surface of mIgM-negative pro-B cells is capable of initiating protein tyrosine phosphorylation in response to BCR cross-linking with the anti-Ig-ß Abs, HM79-16 (19). Using HM79-16, which recognizes the extracellular domain of Ig-ß, we asked whether cross-linking of the Ig-{alpha}ß that is presumably on the surface of 303.1.5.LM cells could stimulate protein tyrosine phosphorylation. Immunoblotting with the 4G10 anti-phosphotyrosine mAb showed that incubating 303.1.5.LM cells with the anti-Ig-ß Ab stimulated tyrosine phosphorylation of several proteins whereas anti-IgM Abs did not (Fig. 6Go). These results support the flow cytometry and cell-surface iodination data that indicated that 303.1.5.LM cells express Ig-{alpha}ß on the cell surface in the absence of mIgM. In contrast to 303.1.5.LM cells, both the anti-Ig-ß Ab and the anti-IgM Abs stimulated tyrosine phosphorylation in the parental mIgM+ WEHI 231 cells. Note that the proteins that were phosphorylated on tyrosine residues in response to anti-Ig-ß in the 303.1.5.LM cells had very similar molecular masses to those that were phosphorylated in response to anti-IgM in the WEHI 231 cells. Thus, in addition to being on the cell surface, the 303.1.5.LM Ig-{alpha}ß is able to initiate signals that are characteristic of the intact BCR.



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  		FIGURE 6. Signaling by cell-surface Ig-{alpha}ß on 303.1.5.LM cells. WEHI 231 cells and 303.1.5.LM cells were incubated for 5 min with anti-IgM, biotinylated anti-Ig-ß mAb (HM79-16), or biotinylated HM79-16 plus streptavidin. Cell extracts (10 µg protein) were separated on 10% SDS-PAGE gels and analyzed by immunoblotting with the 4G10 anti-phosphotyrosine mAb. The filter was then stripped and reprobed with anti-Ig-{alpha} Abs to ensure equal loading of protein in each lane (bottom panel). The migration of Ig-{alpha} as well as the molecular mass standards is indicated to the left. Representative data from one of three similar experiments are shown.

 
In summary, we have shown by a variety of techniques that a signaling-competent Ig-{alpha}ß is present on the surface of 303.1.5.LM cells in the absence of mIgM. Therefore, the Ig-{alpha}ß in these cells is able to escape the ER quality control machinery, which would normally retain the incompletely assembled portions of the BCR, Ig-{alpha}ß subunits, in the ER, as is the case in WEHI 279* cells. A priori, the ability of Ig-{alpha}ß to escape the quality control system in 303.1.5.LM cells could reflect either a mutation in Ig-{alpha} or Ig-ß that interferes with its interactions with the quality control system. These mutations could result in alterations in the protein structure of the Ig-{alpha}ß or they could alter the ability of the protein to be glycosylated. Glycosylation changes could interfere with or enhance the interaction of proteins with chaperones that regulate intracellular trafficking. Alternatively, a defect in the ER quality control system in 303.1.5.LM cells might allow all proteins in the ER to escape regardless of their folding or assembly state. To distinguish between these possibilities, we initiated a more detailed analysis of the 303.1.5.LM Ig-{alpha} and Ig-ß proteins.

Analysis of the 303.1.5.LM Ig-{alpha} and Ig-ß proteins

To determine whether there were mutations that resulted in significant changes (e.g., a truncation or deletion) in the 303.1.5.LM Ig-{alpha} or Ig-ß proteins, we removed all of the N-linked carbohydrates from these proteins using PNGase F and then compared the molecular masses of the deglycosylated Ig-{alpha} and Ig-ß from 303.1.5.LM cells to those for PNGase F-treated Ig-{alpha} and Ig-ß from WEHI 231 cells. Immunoblotting PNGase F-treated cell extracts with Abs to Ig-{alpha} or Ig-ß showed that deglycosylated Ig-{alpha} and Ig-ß from 303.1.5.LM cells had the same molecular masses as deglycosylated Ig-{alpha} and Ig-ß from WEHI 231 cells (Fig. 7Go, A and B). Moreover, when we cell-surface biotinylated 303.1.5.LM cells and WEHI 231 cells and examined the masses of cell surface Ig-{alpha} and Ig-ß after PNGase F treatment, we again found no difference between the Ig-{alpha} and Ig-ß from 303.1.5.LM cells and WEHI 231 cells (Fig. 7GoC). Fig. 7GoD shows that the molecular masses of PNGase F-treated 303.1.5.LM Ig-{alpha} and Ig-ß were also identical with those for PNGase F-treated Ig-{alpha} and Ig-ß from WEHI 279* cells. Because the masses of the 303.1.5.LM Ig-{alpha} and Ig-ß peptide backbones appear to be very similar to those for Ig-{alpha} and Ig-ß from the parental WEHI 231 cells and from the µ-negative WEHI 279* cells in which the Ig-{alpha}ß is retained in the ER, there does not appear to be a significantly large truncation, deletion, or insertion in the 303.1.5.LM Ig-{alpha}ß.



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  		FIGURE 7. PNGase F-treated Ig-{alpha} and Ig-ß from 303.1.5.LM cells, WEHI 231 cells, and WEHI 279* cells have similar molecular masses. A and B, WEHI 231 and 303.1.5.LM cell extracts were incubated with (+) or without (-) PNGase F. Cell extracts were then separated on 12% SDS-PAGE gels and analyzed by immunoblotting with Abs to (A) Ig-{alpha} or (B) Ig-ß. C, WEHI 231 and 303.1.5.LM cells were surface biotinylated, and the Ig-{alpha}ß complex was immunoprecipitated with anti-Ig-{alpha} Abs. Immunoprecipitates were incubated with (+) or without (-) PNGase F, and the immunoprecipitated proteins were separated on 12% SDS-PAGE gels. The biotinylated cell-surface forms of Ig-{alpha} and Ig-ß were detected by immunoblotting with streptavidin which binds to biotin. D, WEHI 231, WEHI 279*, and 303.1.5.LM cell extracts were incubated with (+) or without (-) PNGase F. Cell extracts were separated on 12% SDS-PAGE gels and analyzed by immunoblotting with Abs to Ig-{alpha} (upper panel) or Ig-ß (lower panel). The migration of deglycosylated (CHO-) forms of Ig-{alpha} and Ig-ß are indicated to the right of each panel. Molecular mass markers are indicated to the left.

 
The sequence of the 303.1.5.LM mb1 and b29 genes

Because there appeared to be no major alterations in the 303.1.5.LM Ig-{alpha} and Ig-ß proteins, we used PCR techniques to clone and sequence the 303.1.5.LM mb1 gene that encodes Ig-{alpha} and the 303.1.5.LM b29 gene that encodes Ig-ß. Each gene was sequenced by PCR directly from different total cDNA pools. In addition, individual PCR-amplified mb1 and b29 cDNAs were cloned and sequenced.

Sequencing of multiple PCR amplifications of 303.1.5.LM cDNA showed that the sequence of the 303.1.5.LM b29 cDNA was identical with that of the published wild-type b29 gene sequence (33). In addition, when we expressed one of these cloned 303.1.5.LM Ig-ß cDNAs in the AtT20 endocrine cell line, it yielded a 29-kDa protein that reacted with our anti-Ig-ß Ab (data not shown). Moreover, the 303.1.5.LM Ig-ß that was expressed in AtT20 cells remained Endo H sensitive, indicating that it could be recognized by the ER quality control system and retained in the ER. Thus, there appear to be no alterations in Ig-ß in 303.1.5.LM cells.

In contrast, sequence analysis of four independent clones of the 303.1.5.LM mb1 cDNA as well as three pools of PCR-amplified 303.1.5.LM cDNA revealed a C to T mutation at base pair number 377 (numbering of the cDNA). This sequence alteration was obtained using five different primers and in both sequencing directions. This point mutation is located 16 nt from the 5' end of exon III of the mb1 genomic sequence. No other changes from the WEHI 231 mb1 sequence were detected. This point mutation would result in the replacement of a proline residue in the extracellular domain of Ig-{alpha} with a leucine residue. This change occurs at amino acid 126 of Ig-{alpha}, a position that is located 12 residues from the putative start of the transmembrane region and is part of a sequence containing three prolines. Moreover, this mutation at amino acid 126 occurs at a position that is in the middle of a 26-aa juxtamembrane region of Ig-{alpha} that is highly conserved in mouse, human, and bovine Ig-{alpha}. The conservation of this juxtamembrane amino acid sequence suggests that it could be important for Ig-{alpha} structure and/or function. Therefore, a proline to leucine change at this position could cause a serious disruption of the three-dimensional structure of Ig-{alpha} and potentially affect its interactions with other BCR chain chaperone proteins or glycosylating enzymes.

Our sequencing analysis also revealed that compared with the Ig-{alpha} sequence published by Sakaguchi et al. (34), both the wild-type WEHI 231 mb1 gene and the 303.1.5.LM mb1 gene contained an insertion of a cytosine at position 283 of the cDNA and a corresponding deletion of a guanosine at position 300. However, our sequence agreed with the mb1 sequence published by Flaswinkel and Reth (35). The resulting frameshift mutation affects amino acids 95–100 and changes a Thr-Gly-Ala-Cys-Thr-Gly sequence to His-Arg-Gly-Leu-Tyr-Trp. This sequence change was found in 303.1.5.LM cells and in parental WEHI 231 cells and it most likely reflects an mb1 allele that is expressed in WEHI 231 cells but that differs from the mb1 allele sequenced by Sakaguchi et al. (from the C57BL/6 x DBA/2J strain of mice). WEHI 231 is derived from the NZB x BALB/c strain of mice.

In previous studies, we have had difficulties expressing an mb1 cDNA, thus the putative 303.1.5 LM mutation was introduced into an mb1 genomic clone (4). We attempted to stably express the mutated and wild-type mb1 in AtT20 cells already expressing Ig-ß, and we attempted to stably express both mb1 and b29 in nontransfected cells. In both cases, we successfully obtained stable clones expressing either Ig-{alpha} or Ig-ß but no stable clones expressing both. Additional attempts to get both mutant and wild-type Ig-{alpha} and Ig-ß coexpressed using transient expression in BOSC 293 cells were also unsuccessful.

The 303.1.5.LM Ig-{alpha}ß complex does not associate with wild-type mIgM subunits

The 303.1.5.LM Ig-{alpha}ß subunit is able to escape the ER quality control system and can move to the cell surface in the absence of mIgM in 303.1.5.LM cells. This phenomenon may be due to a mutation that we discovered in the extracellular domain of the 303.1.5.LM Ig-{alpha} protein. Because this mutation might alter the structure of Ig-{alpha}, we asked whether the 303.1.5.LM Ig-{alpha}ß complex containing the mutant Ig-{alpha} protein could associate with mIgM. To test this, we used DNA transfection to express a µ heavy chain gene in 303.1.5.LM cells. Fig. 8Go, A and B show that µ heavy chain and {kappa} light chain are expressed in 303.1.5.LM transfectants (303.1.5.µ1 and 303.1.5.µ2) and can be detected by Western blotting. Fig. 8Go, C and D show that this transfected µ-chain can associate with the endogenous {kappa} light chain in 303.1.5.LM cells and form a complete µ2{kappa}2 mIgM subunit as well as other normal mIgM assembly intermediates (µ{kappa} and {kappa}2). To determine whether this reconstituted mIgM could associate with the 303.1.5.LM Ig-{alpha}ß and traffic to the cell surface along with Ig-{alpha}ß as part of a complete BCR complex, one of the µ-chain-expressing transfectants of the 303.1.5.LM cells (303.1.5.µ1) was cell-surface biotinylated and solubilized in digitonin lysis buffer, which preserves the association between the mIgM and Ig-{alpha}ß subunits of the BCR. After immunoprecipitation with either anti-µ-chain Abs or anti-Ig-{alpha} Abs, immunoblotting with streptavidin was used to detect associated surface proteins (Fig. 9GoA). In addition, the blots were reprobed with anti-µ and anti-Ig-{alpha} Abs to detect intracellular proteins as well as cell-surface proteins. In WEHI 231 cells, anti-IgM Abs precipitated the µ- and {kappa}-chains as well as a series of 30- to 45-kDa biotinylated proteins that are the different glycosylated forms of Ig-{alpha} and Ig-ß (Fig. 9GoA). The same set of bands were immunoprecipitated with anti-Ig-{alpha} Abs, showing that the mIgM and Ig-{alpha}ß subunits of the BCR remained associated with each other under these conditions. In contrast, in 303.1.5.µ1 cells, although they express all four chains of the BCR, no Ig-{alpha} was immunoprecipitated with anti-µ-chain Abs and no µ-chain was coimmunoprecipitated with anti-Ig-{alpha} Abs. Thus, the mIgM did not associate with Ig-{alpha}ß in 303.1.5.µ1 cells. Moreover, the surface biotinylation experiments showed that while the Ig-{alpha}ß in the 303.1.5.µ1 cells was able to traffic to the cell surface, the mIgM did not. Although biotinylated Ig-{alpha}ß was found in the 303.1.5.µ1 cells, no biotinylated µ heavy chain was detected. In addition, Endo H digestion showed that all of the µ heavy chain in the 303.1.5.µ1 cells remained Endo H sensitive (Fig. 9GoB), confirming that the µ heavy chain did not progress beyond the ER in these cells. Hence, even though there were complete mIgM subunits in 303.1.5.µ1 cells (Fig. 8GoC), the Ig-{alpha}ß moved to the cell surface while the mIgM did not. There were no complete BCR complexes on the surface of these cells. Therefore, the 303.1.5.LM Ig-{alpha}ß appears to be unable to associate with mIgM and assemble the mIgM into complete BCR complexes that can exit the ER. Instead, the 303.1.5.LM Ig-{alpha}ß evades the ER quality control mechanism and traffics to the cell surface by itself.



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  		FIGURE 8. µ heavy chain expression can be reconstituted in 303.1.5.LM cells and µ heavy chain: {kappa} light chain heterodimers formed. DNA transfection using electroporation was used to re-express a murine µ heavy chain in 303.1.5.LM cells. A and B, Immunoblots of cell extracts prepared in MG lysis buffer showing expression of µ heavy chain in two independent transfected clonal cell lines, 303.1.5.µ1 and 303.1.5.µ2. Ten micrograms of cellular protein was loaded in each lane of an SDS-PAGE gel containing 10% polyacrylamide. Proteins were transferred to nitrocellulose and probed with anti-µ Abs (A) and anti-{kappa} Abs (B) as described previously. C and D, WEHI 231, 303.1.5.LM, and 303.1.5.µ1 cells were metabolically labeled with Trans [35S]-label and cell extracts immunoprecipitated with either anti-µ Abs or anti-{kappa} Abs and protein A-Sepharose. Immunoprecipitated proteins were left unreduced (C) or reduced (D) with 2-ME and were separated on SDS-PAGE gels containing 7% (C) or 10% (D) polyacrylamide. Gels were fixed, stained, and dried and exposed to x-ray film. The migration of membrane IgM (µ2{kappa}2), heavy and light chain dimer (µ{kappa}), heavy chain alone (µ), light chain dimer ({kappa}2), and light chain alone ({kappa}) are marked on the right of each panel. Molecular mass markers are shown on the left of each panel.

 


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  		FIGURE 9. The reconstituted µ heavy chain in 303.1.5.µ1 cells was not associated with Ig-{alpha}ß on the cell surface and was trapped early in the secretory pathway. A, WEHI 231, 303.1.5.LM, and 303.1.5.µ1 cell-surface proteins were labeled with sulfo-NHS-biotin as described in Materials and Methods. Cell extracts were then prepared using digitonin lysis buffer to preserve noncovalent protein interactions between the BCR chains. Membrane IgM and Ig-{alpha}ß were coimmunoprecipitated with either anti-µ Abs or anti-Ig-{alpha} Abs. Immunoprecipitates were separated on SDS-PAGE gels containing 10% polyacrylamide, proteins transferred to Immobilon membranes, and the filters probed with streptavidin-HRP to detect the biotinylated proteins. Filters were stripped and reprobed with anti-µ Abs or anti-Ig-{alpha} Abs to detect intracellular µ heavy chain and Ig-{alpha}ß. The migration of µ, {kappa}, and Ig-{alpha}ß are indicated on the right of the panel. Molecular mass markers are indicated on the left of all panels. B, The determination of Endo H sensitivity or resistance of mIgM expressed in reconstituted 303.1.5.µ1 cells was used as an indication of the intracellular location of the protein complex. WEHI 231 and 303.1.5.µ1 cells were labeled with Trans [35S]-label, cell extracts were prepared, and mIgM was immunoprecipitated with anti-µ Abs and protein A-Sepharose. Immunoprecipitates were digested with Endo H, and the samples were analyzed on SDS-PAGE gels containing 12% polyacrylamide. Radioactively labeled proteins were detected by exposure of fixed, stained, and dried gels to x-ray film. µU and µR, migration of undigested µ heavy chain and Endo H-resistant µ heavy chain; µS, migration of Endo H-sensitive µ heavy chain; {kappa}, migration of {kappa} light chain. Molecular mass markers are shown on the left of each panel.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have described two variants of B lymphoma cell lines, WEHI 279* and 303.1.5. LM, both of which have lost the expression of µ heavy chain but still express Ig {kappa} light chain, Ig-{alpha}, and Ig-ß. In both of these variant cell lines, Ig-{alpha}ß heterodimers form but do not have mIgM subunits that they can associate with. Previous work has shown that incomplete BCR complexes, as well as individual BCR chains that are not assembled into complete BCR complexes consisting of a mIgM subunit and associated Ig-{alpha}ß subunit(s), are retained in the ER by a quality control system that involves multiple chaperone proteins including BiP, calnexin, and GRP94 (8, 36). Consistent with this model, the WEHI 279* Ig-{alpha}ß subunit was never expressed on the cell surface, suggesting that it cannot escape from the quality control system in the ER. Moreover, it remained Endo H sensitive for at least 4 h, indicating that the Ig-{alpha}ß had high mannose-containing carbohydrate side chains that are characteristic of glycoproteins that are early in the secretory pathway and that have not been processed by glycosidases and glycosyltransferases that are found in the late-medial and trans-Golgi apparatus. While the WEHI 279* Ig-{alpha}ß behaved as expected for an unpaired (with mIgM) Ig-{alpha}ß subunit and was retained in the ER, surprisingly the Ig-{alpha}ß in 303.1.5.LM cells was able to escape the ER quality control system and traffic to the cell surface in the absence of mIgM. The cell-surface localization of the 303.1.5.LM Ig-{alpha}ß was confirmed by several different approaches. Moreover, the cell-surface Ig-{alpha}ß in 303.1.5.LM cells could initiate tyrosine kinase activation when anti-Ig-ß Abs were added to the cells to cross-link Ig-{alpha}ß. Sequence analysis revealed a point mutation in the 303.1.5. LM mb1 gene that resulted in a proline to leucine amino acid change at position 126 in the extracellular domain of the corresponding Ig-{alpha} polypeptide. Because this proline residue is in a highly conserved proline-rich region of Ig-{alpha}, its replacement by a leucine residue could cause significant structural alterations that allow the 303.1.5.LM Ig-{alpha}ß to escape detection by the ER quality control system and traffic to the cell surface. The structural changes caused by this mutation also correlated with aberrant glycosylation of the 303.1.5.LM Ig-{alpha}ß compared with wild-type Ig-{alpha} (see Figs. 1GoD, 2A vs 2C, 3B, 3C, 7A, 7C) and the inability of the 303.1.5.LM Ig-{alpha}ß to associate with mIgM subunits. Thus, this mutation could define a region of the Ig-{alpha} polypeptide that is important for recognition by the ER quality control system, for glycosylation, and for the association of Ig-{alpha}ß subunits with mIgM subunits to create a complete BCR complex.

The 303.1.5.LM cells were derived from a variant of the WEHI 231 B lymphoma cell line that was originally obtained after chemical mutagenesis (303.1.5) (26). There are likely to be multiple genetic defects in these cells (303.1.5), and Page et al. have previously reported that the phospholipase-C in these cells behaves abnormally (26). Thus, it is theoretically possible that some of the defects in Ig-{alpha}ß trafficking and glycosylation could be due to cellular defects in the 303.1.5.LM cells in addition to the potential role that a structural mutation in Ig-{alpha} could cause. For example, there could be mutations that depress the ER quality control system perhaps by ablating or down-regulating expression of one or more chaperone proteins. There could also be mutations in glycosidases and/or glycosyltransferases that could account for the differences in glycosylation of the 303.1.5.LM Ig-{alpha}ß carbohydrate side chains. While we cannot completely rule out that there are global defects in the 303.1.5.LM secretory pathway, we showed that the 303.1.5.LM ER quality control system can properly deal with unpaired mIgM subunits. Unpaired mIgM subunits (not paired to Ig-{alpha}ß) in µ heavy chain-transfected 303.1.5.µ1 cells did not traffic to the cell surface and remained Endo H sensitive, consistent with their retention in the ER (Fig. 9GoB). Thus, it is likely that the ability of the 303.1.5.LM Ig-{alpha}ß subunit to traffic to the cell surface is an intrinsic property of the 303.1.5.LM Ig-{alpha} and Ig-ß polypeptides and is due to the mutation in the Ig-{alpha} polypeptide, perhaps affecting its glycosylation pattern. Nevertheless, the proper retention of the mIgM subunit in the 303.1.5.LM cells strongly suggests that it is the mutation in the 303.1.5.LM Ig-{alpha} protein that alters its ability to be recognized by the ER quality control system rather than a defect in the cellular machinery. We are currently comparing the interactions of Ig-{alpha} with chaperone proteins in 303.1.5.LM cells vs those in the parental WEHI 231 cells and those in WEHI 279* cells where the unpaired Ig-{alpha}ß is retained in the ER. Such analysis may reveal which of the chaperone proteins normally interact with the extracellular domains of the Ig-{alpha}ß subunit and retain unpaired Ig-{alpha}ß subunits in the ER. Preliminary data has not detected any differences in the association and coimmunoprecipitation of 303.1.5.LM and wild-type Ig-{alpha} with the protein chaperone GRP-94 (C. Condon and L. Matsuuchi, unpublished observations).

It will also be of interest to determine how the 303.1.5. LM Ig-{alpha}ß leaves the ER and traffics to the cell surface. The simplest possibility is that Ig-{alpha}ß fails to interact with the chaperone proteins of the ER quality control system and by default progresses to the cell surface. However, it is also possible that chaperone proteins play an active role in escorting the 303.1.5.LM Ig-{alpha}ß to traffic to the cell surface. Both retention and escorting may be normal chaperone protein functions. Evidence supporting a role for chaperone proteins in actively mediating cell-surface expression of proteins comes from studies on the sperm protein calmegin, which is important for spermatogenesis. Sperm from homozygous calmegin knockout mice are not fertile. It has been suggested that calmegin acts as a chaperone protein that is required for escorting one or more sperm membrane proteins to the plasma membrane. Thus, the knockout mice lacking the appropriate chaperone have sperm that lack key surface receptors that promote the interaction and fusion with the egg. This results in infertility (37). Similarly, in pro-B cells that do not make mIgM, the Ig-{alpha} and Ig-ß appear on the cell surface in association with four proteins, one of which is calnexin, an ER chaperone protein (19, 20). It is not clear whether the ability of calnexin and the associated Ig-{alpha}ß to traffic to the cell surface in these pro-B cells is developmentally regulated or whether it is controlled by other proteins that have the ability to move to the cell surface. The discovery of protein chaperones on the cell surface associated with isolated components of lymphoid signaling receptors is not confined to cells of the B cell lineage. It has also been observed that immature thymocytes contain ER-resident protein chaperones on their cell surfaces and that cell-surface expression is eliminated upon thymocyte differentiation (38). Moreover, the protein chaperone calnexin has been found on thymocyte cell surfaces associated with the signaling component of the TCR, CD3 (39). A unifying theme seems to be the use of the signaling component of the BCR and TCR as a receptor to drive the differentiation of early lymphocyte development. One would expect calnexin to remain trapped in the ER, and it is unclear how it escapes retention. One possibility is that the 303.1.5.LM Ig-{alpha}ß or the TCR CD3 have acquired the ability to bind a yet undefined chaperone protein that can then actively escort it and calnexin to the plasma membrane.

Although the mutation in the 303.1.5.LM Ig-{alpha} protein did not prevent it from associating with Ig-ß, the 303.1.5.LM Ig-{alpha}ß was unable to interact with membrane IgM in the µ-chain-reconstituted 303.1.5.µ1 cells. While there were clearly complete µ2{kappa}2 mIgM subunits in these cells (Fig. 8Go), the 303.1.5.LM Ig-{alpha}ß subunits did not associate with them (Fig. 9Go). Instead, the 303.1.5.LM Ig-{alpha}ß subunits trafficked to the cell surface while the mIgM was retained in the ER (Figs. 3Go, 4Go, and 6Go). The simplest interpretation of these data is that the proline to leucine mutation in the 303.1.5.LM Ig-{alpha} polypeptide altered its conformation in some way such that the 303.1.5.LM subunit could not associate with the mIgM. This is consistent with previous observations that the extracellular domain of mIgM contributes to the interaction of mIgM with presumably the extracellular domain of the Ig-{alpha}ß subunit (22). An alternative explanation could be that the mutation affects the glycosylation of the Ig-{alpha}ß, and this difference in glycosylation influences the ability of the Ig-{alpha}ß to associate with mIgM. Further deletion analysis would be required to determine whether the proline-rich sequence in Ig-{alpha}, in which the proline to leucine replacement occurs in the 303.1.5.LM cells, is directly involved in binding to mIgM. A third possibility is that this proline to leucine replacement in Ig-{alpha} alters the conformation of another region of the Ig-{alpha} polypeptide that interacts with mIgM.

In summary, we have identified a mutation in the extracellular domain of the Ig-{alpha} protein that correlates with the inability of the resulting Ig-{alpha}ß subunit to bind to mIgM and with its ability to traffic to the cell surface in the absence of mIgM. Further analysis of this mutant Ig-{alpha} protein may provide structural insights into how Ig-{alpha}ß interacts with both the ER quality control system and mIgM.


   Acknowledgments
 
The WEHI 231 mutant 303.1.5 was a generous gift from Dr. A. L. DeFranco. The monoclonal anti-Ig-ß (HM79-16) was a gift from Dr. T. Nakamura. We acknowledge the expert technical assistance of Cathy Brown, Tally Vertinsky, and Lorie Joyce. We thank Dr. Michael R. Gold for reading the manuscript and Dr. A. L. DeFranco and Shaun P. Foy for helpful discussions during the course of this work. This paper is dedicated to the memory of Dr. Marian E. Koshland, who taught L. M. the importance of perseverance.


   Footnotes
 
1 This work was supported by grants (to L.M.) from the Medical Research Council of Canada and the National Science and Engineering Research Council of Canada and with funds from the University of British Columbia. Back

2 C.C. and S.L.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Linda Matsuuchi, Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia V6T 1Z4, Canada. Back

4 Abbreviations used in this paper: BCR, B cell antigen receptor; Endo H, endogylcosidase H; ER, endoplasmic reticulum; mIg, membrane Ig; PNGase F, N-glycosidase F. Back

Received for publication July 8, 1999. Accepted for publication May 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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