Modifications of Igα and Igβ Expression as a Function of B Lineage Differentiation*

Transcription of the mb1 andB29 genes is initiated when lymphoid progenitors enter the B cell differentiation pathway, and their transmembrane Igα and Igβ products constitute essential signaling components of pre-B and B cell antigen receptors. We analyzed Igα/Igβ biosynthesis, heterogeneity, and molecular interactions as a function of human B lineage differentiation in cell lines representative of the pro-B, pre-B, and B cell stages. All B lineage representatives produced a 36-kDa Igβ form and three principal Igα forms, transient 33/40-kDa species and a mature 44-kDa glycoprotein. Deglycosylation revealed a major Igα core protein of 25 kDa and a minor 21-kDa Igα protein, apparently the product of an alternatively spliced mRNA. In pro-B cells, the Igα and Igβ molecules existed primarily in separate unassembled pools, exhibited an immature glycosylation pattern, did not associate with surrogate light chain proteins, and were retained intracellularly. Their unanticipated association with the Lyn protein-tyrosine kinase nevertheless suggests functional potential for the Igα/Igβ molecules in pro-B cells. Greater heterogeneity of the Igα and Igβ molecules in pre-B and B cell lines was attributable to increased glycosylation complexity. Finally, the Igα/Igβ heterodimers associated with fully assembled IgM molecules as a terminal event in B cell receptor assembly.

Expression of Ig␣ and Ig␤ transcripts begins very early in B lineage differentiation prior to the onset of D H -J H rearrangements in the HC locus (16,17), and Ig␤-deficient mice are unable to generate HC-producing pre-B cells (18). Surprisingly, B cell development in Ig␤ Ϫ/Ϫ mice appears to be compromised as early as the pro-B stage when V H -DJ H rearrangements are occurring, thereby suggesting an Ig␤ role in B lymphopoiesis even prior to HC synthesis. Although pro-B cell lines from humans also produce Ig␣ and Ig␤, expression of these as components of cell surface receptors has not been demonstrable (4). Correspondingly, the LC in human pro-B cells were found exclusively in the endoplasmic reticulum and early Golgi compartments, where they transiently associated with 40-, 60-, and 98-kDa proteins before undergoing intracellular degradation (7,8). In murine pro-B cells, however, Ig␣/Ig␤ heterodimers have recently been found on the cell surface, perhaps in association with calnexin (19,20).
The physiological role of Ig␣ and Ig␤ during the earliest stages in B lineage differentiation thus remains unclear, and may differ in mice and humans. In this analysis of human B lineage cells, we have compared Ig␣ and Ig␤ expression, heterogeneity, and molecular association in pro-B cells versus their more mature pre-B and B cell offspring. The results reveal a remarkable progressive complexity of the Ig␣ and Ig␤ glycoproteins during B lineage differentiation, an unanticipated intracellular association with a Src family protein-tyrosine kinase in pro-B cells, and late stage Ig␣/Ig␤ union with assembled IgM molecules to form the BCR on B cells.
Cell Surface Biotinylation and Biosynthetic Protein Labeling-Viable cells (5 ϫ 107) washed twice in PBS were incubated with 1 mg/ml biotin (NHS-sulfonyl biotin, Pierce) in PBS for 1 h at 4°C. Cells with biotinylated cell surface proteins were washed once in chilled RPMI 1640 and twice in PBS before lysis in 1% digitonin or 0.5% Triton X-100 lysis buffer. Cells (1.5 ϫ 10 8 ) were also metabolically labeled with [ 35  Immunoprecipitation-Labeled cells were harvested and lysed in 1% Nonidet P-40, 1% Triton X-100, or 1% digitonin lysis buffer. Nuclei were sedimented at 10,000 ϫ g for 20 min, and the supernatants used for immunoprecipitation. After successive incubations with bovine serum albumin and IgG-coupled Sepharose 4B beads, the precleared lysates were incubated with beads coupled with test or control antibodies. Bound materials were eluted with Laemmli's sample buffer (29) and resolved by SDS-PAGE, using 10% or 12% acrylamide. In reprecipitation experiments, digitonin-treated cell lysates were incubated with anti-Ig␤ or anti-Ig␣ mAbs, and bound materials resuspended in 500 l of 1% Nonidet P-40 lysis buffer were incubated with anti-Lyn or anti-Syk antibodies. Likewise, materials bound by anti-Lyn antibodies were eluted and immunoprecipitated with anti-Ig␤ antibodies. For two-dimensional gel electrophoretic analysis, immunoprecipitates were separated initially on nonreducing SDS-polyacrylamide (9 -10%) gels. The lanes were then excised, equilibrated for 45 min in SDS sample buffer containing ␤-mercaptoethanol, and rotated 90°before electrophoresis in the second dimension on SDS-polyacrylamide (10%) gels under reducing conditions.
Western Blots-Anti-Ig␣, anti-Ig␤, and anti-immunoprecipitates were resolved on one-dimensional SDS-PAGE and two-dimensional diagonal gels, blotted onto nitrocellulose membrane (Schleicher & Schuell), before incubation with anti-, anti-Ig␤, or anti-Ig␣ mAbs. In some experiments, the anti-Ig␣ and anti-Ig␤ precipitates were submitted to a second immunoprecipitation with an anti-phosphotyrosine mAb; the eluted material was electrophoresed, blotted, and reincubated with the anti-phosphotyrosine mAb. The blots were developed with the ECL chemiluminescence system (Amersham Pharmacia Biotech) using a horseradish peroxidase-conjugated goat anti-mouse Ig (Sigma). In cell surface biotinylation experiments, immunoprecipitates were resolved by SDS-PAGE, blotted as described above, then incubated with a horseradish peroxidase-conjugated streptavidin (Sigma) and revealed with the ECL method.
Deglycosylation-Immunoadsorbed proteins were treated with endoglycosidase H (endo H), endoglysosidase F (endo F), N-glycanase, O-glycanase, or combinations of these enzymes for 18 h at 37°C, according to the manufacturer's instructions (Roche Molecular Biochemicals), before elution with Laemmli's buffer. Fetuin protein was used as a control for optimal digestion.
In Vitro Kinase Assay-Nalm16 cells were washed twice in PBS and lysed in 1% digitonin, 25 mM Hepes, pH 7.2, 150 mM NaCl, 5 mM KCl, 5 mM EDTA, 1 mM orthovanadate, and protease inhibitors before lysate immunoprecipitation with anti-Ig␣, anti-Ig␤, or control mAbs. Bound materials were washed three times with lysis buffer and once with kinase buffer (25 mM Hepes, pH 7.2, 150 mM NaCl, 5 mM KCl, 5 mM MnCl 2 , 5 mM MgCl 2 , 1% digitonin) before resuspension in 50 l of kinase buffer containing 1 mCi of [␥-32 P]ATP (Ͼ4000 Ci/mmol, ICN Biomedicals, Orsay, France). After a 20-min incubation at room temperature, beads were washed three times with lysis buffer, then either eluted in 50 l of SDS sample buffer and boiled for 5 min or submitted to reprecipitation as follows: elution in 50 l of 0.5% SDS lysis buffer instead of digitonin, incubation for 10 min at 60°C, 10-fold dilution in 500 l of 1% Triton X-100, then immunoprecipitation. In one series of experiments, Nalm16 cells were incubated with the CB3-1 anti-Ig␤ antibody (30 g/ml) or a control mAb (␥1 isotype) for various intervals (0, 1, and 5 min) before exposure to 1% Triton X-100 lysis buffer containing 5 mM EDTA, 1 mM orthovanadate, and protease inhibitors. Cell lysates were electrophoresed, blotted, and probed with an antiphosphotyrosine mAb. Alternatively, lysates of the anti-Ig␤-stimulated pro-B cells were immunoprecipitated with an anti-phosphotyrosine mAb, and the immunoprecipitate was electrophoresed, blotted, and probed with the anti-phosphotyrosine mAb.
Densitometric Analysis-Radiolabeled protein intensities were determined relative to cell surface HC levels using a densitometer (Ultra-Scan XL) and a software program (GelScan XL; Amersham Pharmacia Biotech, Uppsala, Sweden). Background readings of the autoradiographic films were designated zero.

Characterization of Ig␣ and Ig␤ Molecules in Pro-B Cell
Lines-Four molecules with apparent molecular masses of 44, 40, 36, and 33 kDa were immunoprecipitated with anti-Ig␣ antibodies from the metabolically labeled Nalm16 and RS4;11 pro-B cell lines. In contrast, two molecules of 44 and 36 kDa were precipitated by the anti-Ig␤ mAb from both pro-B cell lines (Fig. 1A). Analysis of the anti-Ig␣ precipitates by twodimensional gel electrophoresis indicated the covalent linkage of a minor fraction of the available pools of the 44-and 36-kDa species (Fig. 1C, and data not shown), with the major portion of the 44-kDa molecules remaining on the diagonal. Similarly, two-dimensional gel analysis of anti-Ig␤ immunoprecipitates indicated that most of the 36-kDa molecules migrated on the diagonal, whereas small fractions were found in off-diagonal positions indicative of their formation of homodimers or heterodimers with the 44-kDa protein (Fig. 1D). Western blot analysis confirmed the Ig␤ identity of the 36-kDa molecules (Fig. 1B, lanes 3 and 4). It also indicated reactivity of the 44-, 40-, and 33-kDa molecules with the anti-Ig␣ antibody, thus confirming the molecular heterogeneity of Ig␣ proteins (Fig.  1B, lanes 1 and 2). Western blot analysis of anti-Ig␤ immunoprecipitates resolved on two-dimensional gels indicated that most of the Ig␤ molecules exist in a free pool, whereas minor fractions exist as covalently linked heterodimers or homodimers in the Nalm16 pro-B cell line (Fig. 1, D and F). A similar two-dimensional analysis of the anti-Ig␣ precipitates showed that only a fraction of the 44-kDa Ig␣ species ran off the diagonal (Fig. 1, C and E), thus indicating that the 40-and 33-kDa Ig␣ forms do not form covalent bonds with Ig␤.
Pulse-chase analysis of the RS4;11 and Nalm16 pro-B cells indicated that newly synthesized Ig␣ and Ig␤ proteins exist initially as completely separate pools with limited Ig␣ and Ig␤ association occurring thereafter ( Fig. 2A, and data not shown). The levels of newly synthesized 40-and 33-kDa Ig␣ variants progressively declined with estimated half-lives of less than 30 min, whereas the levels of 44-kDa Ig␣ molecules were maintained over the 3-h observation period (Fig. 2B). Notably, neither the anti-Ig␣ nor the anti-Ig␤ immunoprecipitates of pro-B cell lysates contained LC proteins, and the Ig␣ and Ig␤ molecules could not be detected on the surface of the Nalm16 pro-B cells (data not shown).
These results indicate that the Ig␣ and Ig␤ proteins produced by human pro-B cell lines exist largely in unassembled free pools, although a limited fraction of these molecules form Ig␣/Ig␤ heterodimers and trace amounts of Ig␤ homodimers. While Ig␤ appears to be expressed as a single species, Ig␣ is expressed in three sizes, the smaller two of which apparently represent immature forms that are relatively transient in nature and do not form disulfide-linked dimers.
Ig␣ Protein Heterogeneity in Pro-B Cell Lines-Ig␣ and Ig␤ heterogeneity may vary as a function of B lineage differentiation stage and the expressed immunoglobulin isotype, in part due to differential glycosylation (4, 31-40). Experiments were therefore conducted to determine whether the three Ig␣ species in pro-B cells reflect variably glycosylated forms or other modifications of protein structure. When lysates of metabolically labeled RS4;11 cells were precipitated with anti-Ig␣ or anti-Ig␤ antibodies and the bound material treated with endo F or with N-glycanase and endo F, anti-Ig␣ precipitates were resolved as two bands of 25 and 21.5 kDa, whereas anti-Ig␤ precipitates yielded a single band of 24.5 kDa (Fig. 2C, and data not shown). Western blot analysis of the anti-Ig␣ or anti-Ig␤ precipitates confirmed the Ig␣ nature of the 25-and 21.5-kDa bands, and the single band of 24.5-kDa Ig␤ forms (Fig. 2C). When the anti-Ig␣ and anti-Ig␤ immunoprecipitates of unlabeled RS4;11 and Nalm16 pro-B cell lysates were treated with endo H, immunoblot analysis again indicated 25-and 21.5-kDa Ig␣ species and a single 24.5-kDa Ig␤ band (data not shown). This endo H sensitivity pattern suggests restriction of the Ig␣ and Ig␤ glycoproteins to the endoplasmic reticulum and early Golgi in human pro-B cells.
Ig␣ and Ig␤ Transcript Heterogeneity-The fact that the molecular mass of the Ig␣ core protein predicted from complete transcription of the human mb1 gene is 24.5 kDa suggested that the 21.5-kDa deglycosylated molecule could be generated by post-transcriptional modification. Mb-1 (Ig␣) and B29 (Ig␤) transcripts in the Nalm16 and RS4;11 pro-B cell lines were therefore analyzed by RT-PCR. The Ig␣ reactions yielded a major 660-bp DNA fragment and a minor 550-bp DNA fragment (Fig. 3A). Sequence analysis indicated that the 660-bp PCR product corresponds to the full-length Ig␣ transcript, whereas the 550-bp fragment reflects the amplification of an alternatively spliced Ig␣ mRNA. This spliced form could be attributed to the use of a cryptic splice site located 186 bp after the start of the second exon, which is joined to the normal splice acceptor site of the third exon. The resulting transcript, which is devoid of 114 bp of the normal sequence, would maintain the same reading frame as the full-length transcript, and therefore could encode an Ig␣ protein lacking part of the extracellular domain. This truncated protein would not be expected to contain the cysteine residue involved in covalent association with Ig␤, but would maintain the transmembrane and cytoplasmic portions as well as four N-linked glycosylation sites.
Ig␤ reactions produced major 600-bp and minor 290-bp DNA amplification products (Fig. 3B). The sequence of the larger fragment contained all six CD79b exons, whereas the smaller product reflected a complete loss of the third exon. The 105 codon truncation maintains a correct reading frame in the 3Ј portion of the short Ig␤ transcripts that could encode for an 11-kDa protein. The predicted truncated Ig␤ protein would lack all N-linked glycosylation sites and the ability to form disulfide-bonded heterodimers with Ig␣. Antibodies to the C-terminal portion of Ig␤ are needed to determine whether this truncated Ig␤ is expressed in pro-B cells. The present RT-PCR and sequence analyses of pro-B cell lines thus agree with those previously reported for human pre-B and B cell lines (41)(42)(43)(44)(45).
Association of Ig␣ and Ig␤ Molecules with Protein-tyrosine Kinases in Pro-B Cell Lines-Antigen ligation of the BCR complex on B cells leads to interaction of the Ig␣/Ig␤ subunits with Src family kinases (Lyn, Fyn, Blk, Hck, and Lck) and Syk kinase to initiate cell activation (46 -56). Pre-BCR interaction with Src kinases has also been demonstrated (12,57). It was therefore of interest to determine whether interaction of the Ig␣/Ig␤ molecules with these protein-tyrosine kinases may occur at the pro-B cell stage. Both Lyn (53/56 kDa) and Syk (70 kDa) tyrosine kinases were identified in digitonin lysates of metabolically labeled RS4;11 pro-B cells, although the level of background radioactivity did not permit clear resolution of their association status with Ig␣ and Ig␤ molecules. When the 1 and 3) and anti-Ig␤ immunoprecipitates (lanes 2 and 4) of metabolically labeled pro-B cells. Cells were lysed in 1% digitonin lysis buffer and the immunoprecipitates separated under reducing conditions on a 12% SDS-polyacrylamide gel. Relative molecular masses in kilodaltons are indicated. Western blot analysis (see below) suggests that the higher molecular mass band seen in the anti-Ig␣ precipitates primarily represents actin. B, Western blot analysis of Ig␣ and Ig␤ molecules. Pro-B cells were lysed in a 1% Nonidet P-40 solution, and the cell lysates incubated with anti-Ig␣ or anti-Ig␤ mAbs. Immunoprecipitates resolved by SDS-PAGE under reducing conditions were transferred to nitrocellulose membranes before Western blot analysis with anti-Ig␣ (lanes 1 and 2) and anti-Ig␤ mAbs (lanes 3 and 4). Western blots were developed with horseradish peroxidase-conjugated goat antimouse Ig antibodies using a chemiluminescence system. C and D, two-dimensional gel analysis of Ig␣ and Ig␤ molecules produced by Nalm16 pro-B cells. Digitonin lysates of metabolically labeled cells were incubated with monoclonal antibodies against Ig␣ (C) or Ig␤ (D). Immunoprecipitates were resolved in SDS-PAGE under nonreducing conditions in the first dimension and under reducing conditions in the second dimension. E and F, Ig␣ and Ig␤ identification by Western blot analysis of pro-B cell lysates in two-dimensional gels. Anti-Ig␣ or anti-Ig␤ precipitates from digitonin lysates of unlabeled RS4;11 pro-B cells were separated by two-dimensional gel electrophoresis, transferred to nitrocellulose membranes, and developed with anti-Ig␣ (E) or anti-Ig␤ (F) antibodies as indicated above. Covalent association of a minor fraction of the 44-kDa Ig␣ molecules and the 36-kDa Ig␤ molecules in the pro-B cells is indicated by their off-diagonal position. The arrow indicates the trace amounts of Ig␤ components of homodimeric units. Note that the photographic magnification of E and F differs slightly from that of C and D.

FIG. 1. Analysis of Ig␣ and Ig␤ expression in pro-B cell lines. A, Anti-Ig␣ (lanes
anti-Lyn and anti-Syk eluates were submitted to a second immunoprecipitation with the anti-Ig␤ antibody, Ig␤ was detected in the anti-Lyn immunoprecipitate (Fig. 4A, lane 1) but not in the anti-Syk immunoprecipitate (data not shown). A band of 44 kDa, likely Ig␣, was also coprecipitated with anti-Lyn. Conversely, p53/56 Lyn was identified in anti-Ig␤ precipitates (Fig. 7A, lane 2), whereas Syk was not (data not shown).
An in vitro kinase assay was conducted using unstimulated and pervanadate-treated Nalm16 pro-B cells. The anti-Ig␣ antibody coprecipitated a faint 53/56-kDa doublet with kinase activity in unstimulated Nalm16 cells, and pervanadate treatment, employed to alter the kinase/phosphatase equilibrium to favor activation of the tyrosine kinases, strongly enhanced the Lyn 53/56 signal (Fig. 7B). The 53/56 doublet was also revealed by an anti-phosphotyrosine mAb in the anti-Ig␣ immunoprecipitates of Nalm16 pro-B cells preincubated with pervanadate (Fig. 4C). An additional 44-kDa band, likely Ig␣, was also seen. In contrast, Syk could not be identified in anti-Ig␣/Ig␤ precipitates of pervanadate-treated pro-B cells. Similarly, Fyn, which was also abundant in pro-B cells, was not detected in association with Ig␣/Ig␤.
The above data suggest that Lyn preferentially associates with the minor population of Ig␣/Ig␤ heterodimers within pro-B cells. In order to examine the possibility that an otherwise undetectable level of cell surface Ig␣/Ig␤ molecules accounted for the Lyn association, we examined the tyrosine phosphorylation status of proteins in pro-B cells before and after treatment with a known stimulatory antibody against an extracellular Ig␤ epitope. No differences were observed in the tyrosine phosphorylation status of Lyn or other proteins were observed within 5 min following the anti-Ig␤ treatment of the pro-B cells (data not shown), thereby mitigating against the possibility of Lyn association with pro-B cell surface Ig␣/Ig␤.

Comparison of Ig␣ and Ig␤ Expression in Pre-B and B Cell
Lines-Two-dimensional gel analysis of anti-Ig␣ and anti-Ig␤ immunoprecipitates indicated a progressive increase in heterogeneity of the Ig␣/Ig␤ heterodimers in pre-B and B cells (Figs. 5 and 6). Additional minor Ig␣ and Ig␤ forms of lower molecular weights were also revealed by this Western blot analysis. Ig␣ and Ig␤ existed in major and minor heterodimeric forms in pre-B cell lines as well as in a B cell line. Comparative twodimensional gel analysis of the Ramos B cell line also revealed prominent fan-like extensions of the major Ig␣ and Ig␤ units (Fig. 5, C-E), the identity of which was confirmed by Western blot analysis (Fig. 6, C and D). In both the pre-B and B cells, HC were coprecipitated with the Ig␣ and Ig␤ molecules. Comparative analysis of the anti-HC precipitate (Fig. 5E) with the anti-Ig␣ and anti-Ig␤ precipitates (Fig. 5, C and D) indicates that the (HC) 2 /(LC) 2 complexes could be coprecipitated with antibodies against the Ig␣ and Ig␤ molecules, while the HC/ LC subunits could not. EndoF digestion of the anti-Ig␣ precipitates from pre-B and B cells revealed core proteins similar to those observed for pro-B cells, and truncated mb1 and B29 transcripts were likewise identified in pre-B and B cells (Fig. 3). A minor population of Ig␤ homodimers was also detected in pre-B and B cells, but these Ig␤ homodimers were not seen in the anti-precipitates (Fig. 5E), anti-Ig␣ precipitates (Fig. 5, A and C), or on the cell surface (Fig. 7C). Pulse-chase analysis of anti-Ig␣ precipitates from metabolically labeled cells indicated that Ig␣/Ig␤ association is initiated immediately after their biosynthesis in pre-B and B cells (Fig. 7A). However, completion of the complex Ig␣ and Ig␤ glycosylation process required more than 2 h after biosynthesis, as illustrated in pre-B cells in Fig. 7B. The final heterogeneity of cell surface Ig␣/Ig␤ components was found to be indistinguishable in the pre-BCR on pre-B cells and BCR on B cells (Fig. 7C).

FIG. 2. Analysis of Ig␣ and Ig␤ biosynthesis and glycosylation status in pro-B cells. Pulse-chase analysis of Ig␣ and Ig␤ in RS4;11 pro-B cells (A and B).
Cells were pulse-labeled with [ 35 S]Met and [ 35 S]Cys, chased with cold amino acids for various time intervals, and then lysed before incubation with antibodies against Ig␣ or Ig␤. Immunoprecipitates were analyzed by SDS-PAGE under reducing conditions. A, Ig␣ and Ig␤ molecules are unassociated at time 0. B, Analysis of the biosynthetically labeled 44-, 40-, and 33-kDa Ig␣ molecules at later time points, by determination of relative autoradiographic intensities, indicates the relative stability of the 44-kDa Ig␣ form and the transient nature of the 33/ 40-kDa species. C, 24.5-kDa Ig␤ core protein and 21.5/25-kDa Ig␣ core proteins are revealed by deglycosylation. Nonlabeled RS4;11 cell lysates were subjected to immunoprecipitation with anti-Ig␣ or anti-Ig␤ anti-bodies, the immunoprecipitates treated with endo F, and the deglycosylated Ig␣ and Ig␤ proteins identified by immunoblots as described in Fig. 1.

DISCUSSION
In these studies, we observed a remarkable diversity of the Ig␣ and Ig␤ glycoproteins, the nature of which is altered as a function of B lineage progression. Ig␣ species of 44, 40, and 33 kDa were produced by pro-B, pre-B, and B cell lines, whereas Ig␤ was found in a single 36-kDa form. The 40-and 30-kDa Ig␣ species represented transient immature forms that did not associate with Ig␤, whereas the 44-kDa Ig␣ and 36-kDa Ig␤ were found to be relatively stable and to associate with each other to form heterodimers. The formation of Ig␣/Ig␤ heterodimers and the complexity of their glycosylation patterns were shown to increase dramatically as a function of B cell differentiation.
Two Ig␣ protein backbones of approximately 25 and 21.5 kDa were revealed in deglycosylation studies, and two types of mb1 (Ig␣) gene transcripts were identified that could account for these. The smaller, less-abundant variant, which lacked 114 bp as a consequence of alternative splicing of the Ig␣ mRNA, maintains the same reading frame as the full-length transcript. It has the potential to give rise to a 4-kDa deleted protein that likely corresponds to the truncated Ig␣ detected in B lineage cells. The predicted product of this small mb1 transcript would  1 and 2) and anti-Ig␣ precipitates (lanes 3 and 4) of pervanadate (P)-stimulated (ϩ) and unstimulated (Ϫ) Nalm16 cells were separated by SDS-PAGE before immunoblotting with an anti-phosphotyrosine antibody. lack the cysteine residue involved in forming disulfide-linked heterodimers with Ig␤, but would maintain many N-linked glycosylation sites. Similarly, two B29 (Ig␤) transcripts were identified. The smaller one has the potential to encode a 11-kDa protein that could not covalently bind Ig␣ and would lack glycosylation sites. All of the representative pro-B, pre-B, and B cell lines expressed identical patterns of Ig␣ and Ig␤ tran-scripts, and deglycosylation with N-glycanase revealed the truncated 21-kDa Ig␣ molecule in cells representative of each differentiation stage. In mature human B cells, a post-transcriptional regulation of mb1 and B29 gene expression has been suggested. In particular, activation of mature B cells with anti-IgM antibody, interleukin 4, or lipopolysaccharide was shown to induce alternative splicing of mb1 and B29 (45).
The molecular interactions and functional potential of the truncated Ig␣ and Ig␤ molecules are poorly understood. The predicted amino acid sequences of the truncated human Ig␣ and Ig␤ proteins suggest they would not covalently associate with each other, and therefore would not be incorporated into either the BCR or pre-BCR complexes. Accordingly, fibroblast co-transfection of human HC and LC genes with mb1 and B29 variants failed to reconstitute the BCR (45). In the mouse, a C-terminally truncated Ig␤ product was detectable only in activated B cells (38). A truncated murine Ig␣ product has been found to be preferentially associated with IgD, whereas the predicted full-length Ig␣ product associated primarily with IgM (39). In late stage murine pre-B cells, a truncated 23 kDa protein has been identified with a monoclonal antibody against an extracellular Ig␣ epitope (5). This suggests that truncated Ig␣ and Ig␤ proteins may be differentially expressed according to the stage of B lineage differentiation in the mouse. Truncated Ig␣ variants may also be secreted by bovine B lymphocytes (58).
A functional potential for the Ig␤ homodimers that we observed in human B lineage cells was not revealed in these studies. The Ig␤ homodimers were not found to associate with either HC or Ig␣, nor were they detected on the surface of pre-B and B cells. It is theoretically possible that Ig␤ homodimers play an undefined role inside the cell or, less likely, on the cell surface in levels below our detection threshold.
The Ig␣ and Ig␤ interactions and glycosylation heterogeneity increased progressively in representative pro-B, pre-B, and B cell lines. The Ig␣ and Ig␤ molecules in pro-B cells were found largely in separate pools, with only a minor fraction forming disulfide-bonded Ig␣/Ig␤ heterodimers. In contrast to the Ig␣/ Ig␤ status in pro-B cells, Ig␣ readily associated with Ig␤ in pre-B cells and in B cells. Variable glycosylation of the Ig␣/Ig␤ heterodimers occurred during their progression through the Golgi to reach the cell surface in association with HC to form pre-B receptors (pre-BCR) and B cell receptors (BCR), respectively. While the final Ig␣/Ig␤ glycosylation spectra in the cell surface pre-BCR and BCR were indistinguishable, a restricted fraction of mature glycosylated molecules was observed in the Ig␣/Ig␤ pool in pre-B cells relative to that seen in B cell lines. This striking feature, which is also evident in data obtained in prior studies (4,6), reflects the relative inefficiency of pre-BCR assembly in pre-B cells compared with BCR assembly in B cells.
Ig␣/Ig␤ heterodimers are essential elements in pre-B and B receptor signaling (1, 9 -15). They mediate B cell activation by interaction with Syk and Src family tyrosine kinases and also serve as pre-BCR signal-transducing components to promote pre-B cell differentiation and allelic exclusion (59 -62). Much less is known about functional Ig␣/Ig␤ potential before VDJ H gene rearrangement. One important clue, however, is provided by the demonstration that mice lacking in Ig␤ exhibit a block at the pro-B cell stage in differentiation prior to the completion of V-DJ H rearrangements (18). In contrast, V-DJ H rearrangement proceeds normally in mice that have a c mutation that prevents membrane-bound HC expression (63). These observations suggest that the Ig␣/Ig␤ molecules may play an important biological role during the pro-B cell stage in differentiation before HCs are expressed. A recent study suggests that Ig␣/ Ig␤ heterodimers may be expressed with calnexin on the surface of pro-B cells from RAG-2-deficient mice (20). Ligation of this Ig␣/Ig␤ cell surface complex induced rapid, transient phosphorylation of Ig␣ and associated tyrosine kinase to promote pro-B differentiation. However, these findings in mice may not be directly applicable to humans, given that Ig␣/Ig␤ heterodimers apparently do not reach the cell surface of human pro-B cells. Instead, our analysis of human pro-B cells indicates the intracellular association of Ig␣/Ig␤ with Lyn, a member of the Src tyrosine kinase family. The possibility that the association of Ig␣/Ig␤ with Lyn is artifactual, occurring after pro-B cell lysis, is unlikely since Ig␣/Ig␤ binding to other phosphoproteins was not observed in the pro-B cells. Specifically, phosphorylated Fyn and Syk, which are also abundant in pro-B cell lines, were not found to be associated with Ig␣ and Ig␤. Our findings in human pro-B cells thus indicate that, although Ig␣ and Ig␤ molecules exist primarily in separate pools in the ER/early Golgi compartments, do not associate with surrogate light chains, and fail to reach the cell surface, a minor population of intracellular Ig␣ and Ig␤ molecules associate with the Lyn protein-tyrosine kinase with potentially functional consequences.
The Ig␣/Ig␤ heterodimers are known to be essential for transport of the pre-BCR and BCR components to the cell surface, as well as for their subsequent mediation of signal transduction. Our observation that Ig␣/Ig␤ heterodimers in B cells were associated with the (HC) 2 /(LC) 2 units, but not with the precursor HC/LC subunits, indicates that the Ig␣/Ig␤-IgM association represents one of the terminal events in BCR assembly. In view of the possibility that the BCR complex includes two Ig␣/Ig␤ heterodimers, one Ig␣/Ig␤ heterodimer associated with each HC in the BCR complex (1), it may be important to determine whether when the Ig␣/Ig␤-HC association occurs immediately before or after assembly of the membrane-bound IgM subunits.