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Originally published In Press as doi:10.1074/jbc.M312169-A200 on December 11, 2003

J. Biol. Chem., Vol. 279, Issue 11, 10228-10236, March 12, 2004
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Binding of Free Immunoglobulin Light Chains to VpreB3 Inhibits Their Maturation and Secretion in Chicken B Cells*

Olivier Rosnet{ddagger}, Carla Blanco-Betancourt§, Karine Grivel, Kirsten Richter, and Claudine Schiff{ddagger}

From the Centre d'Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale-Université de la Méditerranée, Campus de Luminy, 13288 Marseille Cedex 09, France

Received for publication, November 6, 2003 , and in revised form, December 5, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The VpreB3 gene product was first characterized as an immunoglobulin (Ig) µ heavy chain-binding protein in mouse precursor B (pre-B) cells. Although its function is unknown, it has been proposed to participate in the assembly and transport of the pre-B cell receptor. We have identified a VpreB3 orthologous gene in chicken that is located close to the immunoglobulin light chain (LC) gene cluster and specifically expressed in the bursa of Fabricius. By overexpressing VpreB3 in the DT40 IgM+ immature chicken B cell line, we have characterized VpreB3 as an endoplasmic reticulum-resident glycoprotein that binds preferentially to free IgLC. However, binding to IgHC is observed in IgLC-deficient DT40 cells. Interaction of VpreB3 with free IgLC is partly covalent and induces retention of free IgLC in the endoplasmic reticulum, preventing their secretion without affecting IgM surface expression. Our results demonstrate that this evolutionarily conserved molecule may play a role in the regulation of the maturation and secretion of free IgLC in B cells. We discuss possible implications in the regulation of the immune response.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The B cell receptor (BCR)1 at the surface of immature and mature B cells and the pre-B cell receptor (pre-BCR) at the surface of developing B cell precursors control cell fates at different levels. Depending on the B cell maturation stage, recognition of self-antigens by the BCR can induce cell death, anergy, or receptor editing (1–4). BCR signal output also contributes to the developmental fate of peripheral B cells (2, 5). In addition, recognition of self-antigens by the BCR has also been shown to induce positive selection in transgenic models (6, 7). In precursor B (pre-B) lymphocytes, surface expression of the pre-BCR is necessary for cell proliferation, repertoire selection, and allelic exclusion (8).

The BCR consists of an antigen-binding subunit made of two covalently associated µ or {delta} immunoglobulin (Ig) heavy chains (HC), each covalently bound to an Ig light chain (LC). This dimer is bound to one Ig{alpha}/Ig{beta} heterodimer that makes up the signaling unit of the BCR. In pre-B cells, a surrogate light chain made of the association of the VpreB and {lambda}5 proteins substitutes for the conventional Ig light chain, thus making a pre-BCR. For proper expression of these protein complexes at the cell surface, it is assumed that their assembly and transport are regulated by a "quality control" mechanism inside the endoplasmic reticulum (ER). This involves interactions with ER-resident chaperones that ensure that incorrectly assembled protein complexes and assembly intermediates are retained in the ER and/or are ultimately degraded following retrotranslocation into the cytoplasm (9–12). In the case of both the BCR and pre-BCR, interactions with the Bip/GRP78/HSPA5, GRP94, and calnexin chaperones have been described (13). It has been demonstrated that Bip binds to the CH1 domain of free Ig heavy chains to retain them in the ER and that the binding of the light chains to the complex releases Bip in an ATPase-dependent manner, allowing the complete folding of Ig protein subunits (14).

Retention of assembled pre-BCR complexes in the ER has been attributed to pre-B cell intrinsic factor(s) (15) and to the non-Ig region of {lambda}5 (16). This retention leads to low pre-BCR surface expression compared with BCR expression on immature and mature B cells. This low expression is presumably necessary for the tight regulation of cell proliferation at the large pre-B cell stage in a ligand-independent (17) or ligand-dependent manner (18). Among B cell-specific proteins that bind the IgµHC in pre-B cells is the VpreB3 gene product. This small polypeptide resembles an IgLC variable domain and also shows similarities with the VpreB protein (19, 20). VpreB3 in murine pre-B cells seems to bind transiently to IgµHC chain during the course of pre-BCR assembly in the ER (21), but its function is unknown. VpreB3 mRNA is expressed at high level from the pro-B to the immature B cell stage (22), thereby indicating possible participation of VpreB3 in the biosynthesis of the BCR and pre-BCR.

In this report we describe the identification of the chicken VpreB3 gene. Using the chicken DT40 immature B cell line, we have demonstrated that binding of free IgLC to VpreB3 negatively regulates their maturation and secretion. This work reveals biochemical and functional characteristics of VpreB3 that may have appeared early in the course of the evolution of the adaptative immune system.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, Antibodies, and Reagents—DT40 cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 1% chicken serum (both heat-inactivated for 30 min at 56 °C), 50 µM 2-ME, 2 mM L-glutamine, 10 units/ml penicillin, 10 µg/ml streptomycin. DT40 VJL–/– cells (a kind gift from J. M. Buerstedde and H. Arakawa, Institute for Molecular Radiobiology, Neuherberg, Germany) were grown in the presence of 0.5 mg/ml histidinol and 1.5 mg/ml hygromycin. Polyclonal goat anti-chicken IgM-µ chain-specific Abs and anti-chicken light chain were from Bethyl Laboratories (Montgomery, TX). Anti-MYC mouse monoclonal Abs 9E10 and 9B11 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling (Beverly, MA), respectively. M1 mouse monoclonal anti-chicken µ chain was from Southern Biotech Associates (Birmingham, AL). Fluorophore-conjugated reagents for confocal microscopy were Texas Red-labeled goat anti-mouse IgG (Beckman-Coulter, Fullerton, CA), fluorescein isothiocyanate-conjugated concavalin A, BODIPY FLC5-ceramide (Molecular Probes, Eugene, OR), and rhodamine-conjugated anti-goat IgG (Sigma).

Flow Cytometry—0.2 x 106 cells were suspended in PBS 0.2% bovine serum albumin, 0.1% sodium azide containing 0.5 µg/ml of M1 Ab. After washings, staining was obtained with phycoerythrin-labeled goat F(ab')2 anti-mouse IgG (Beckman-Coulter).

Confocal Microscopy—5 x 105 DT40 cells were seeded per poly(L)lysine (Sigma)-treated glass coverslip (Marienfeld, Lauda-Koenigshofen, Germany) and allowed to adhere for 30 min at 37 °C. After fixation using a 4% paraformaldehyde solution, cells were preincubated for 20 min at room temperature with PBS, 0.2% gelatin (Sigma), 0.1% saponin (Sigma), washed in PBS, incubated for 45 min with appropriate primary Ab dilutions, washed in PBS, and incubated for 30 min with a secondary Ab dilution in the case of unconjugated primary Abs. Slides were examined using an LSM-510 Carl Zeiss (Jena, Germany) confocal microscope using the x63 objective.

Metabolic Labeling—For metabolic labeling, cells were washed once in PBS and suspended in labeling medium (RPMI 1640 minus methionine and cysteine plus 3% dialyzed fetal calf serum and 0.3% dialyzed chicken serum) at a concentration of 5 x 106 cells/ml for 45 min. For long-term labeling, the cells were subsequently suspended at a concentration of 5 x 106 in fresh labeling medium containing 50 µCi/ml L-[35S]Cys and 50 µCi/ml Expre35S35S protein labeling mix (Met 77%, Cys 17%; PerkinElmer Life Sciences) for 4 h.

For pulse-chase metabolic labeling, cells were suspended at a concentration of 40 x 106 cells/ml in fresh labeling medium containing 200 µCi/ml L-[35S]Cys and 200 µCi/ml Expre35S35S protein labeling mix for 15 min. Next, 10 volumes of normal culture medium containing an excess (5 mM) of cysteine and methionine was added. After centrifugation, cells were suspended in the same medium for the indicated chase times. Before lysis, cells were washed twice in ice-cold PBS 2% bovine serum albumin and once in ice-cold PBS.

Cell Lysis and Immunoprecipitation—Cells were lysed at a concentration of 15 to 30 x 106 cells/ml in 20 mM Tris-Hcl, pH 8, 2 mM EDTA, pH 8, 150 mM NaCl, 1% Igepal CA-630, 2.5 mM iodoacetamide, 7 µg/ml antipain, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride.

For immunoprecipitation, 10 µg of Ab were conjugated to protein G-Sepharose beads (Amersham Biosciences) for 4–12 h in lysis buffer and then saturated for 2 h in PBS, 5% bovine serum albumin. After two washes in lysis buffer, beads were incubated with cell lysates for 3–4 h. When non-radiolabeled lysates were used, immunoprecipitates were collected and washed three times in lysis buffer. When radiolabeled lysates were used, a preclearing was performed by incubating the lysates overnight in the presence of control Ig-conjugated protein G-Sepharose beads. After immunoprecipitation with specific Abs, immunoprecipitates were resuspended in 100 µl of Tris-Hcl, pH 8, 150 mM NaCl, 0.5% Igepal CA-630 (Sigma), 0.5% SDS, 0.5% sodium deoxycholate and deposited at the top of a sucrose cushion consisting of 400 µl of 10 mM Tris-HCl, pH 8, 20% sucrose overlaid by 400 µl of Tris-Hcl, pH 8, 150 mM NaCl, 0.5% Igepal CA-630, 0.5% SDS, 0.5% sodium deoxycholate, 10% sucrose. After 5 min of centrifugation, immunoprecipitates were washed once in 10 mM Tris-HCl, pH 8, 500 mM NaCl, 0.5% Igepal, 0.05% sodium deoxycholate and once in 10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% Igepal. Immune complexes were then heated in SDS/2-ME-containing loading buffer and separated on polyacrylamide gels.

Radioactive gels were fixed in 15% methanol, 7.5% acetic acid and treated with Amplify reagent (Amersham Biosciences) before drying and exposure on autoradiograhic film and/or using a Bas-1500 imager (Fujifilm, Stamford, CT). Image analysis and quantification were performed using the Tina software (Raytest, Straubenhardt, Germany).

EndoH Treatment—Washed protein G-Sepharose beads bound to immune complexes were resuspended and incubated at 95 °C for 7 min two successive times in 70 µl of 20 mM Tris-HCl, pH 6.8, 1% SDS, 5 mM dithiothreitol. Then 13 µl of trisodium citrate, pH 5.5, was added to 70 µl in the presence of protease inhibitors with or without 5 milliunits of endoH (Sigma) for 18 h.

Western Immunoblotting—Polyacrylamide gels were transferred onto Immobilon membrane (Millipore, St.-Quentin-Yvelines, France), and the membranes were probed following the recommendations of the manufacturer with the indicated Abs at a concentration of 1 µg/ml in PBS, 0.05% Tween 20, 5% nonfat dry milk. Horseradish peroxidase-coupled anti-mouse (Sigma) or anti-goat (Chemicon International, Temecula, CA) Ig Abs or protein G (Sigma) were used for detection by the enhanced chemiluminescence detection system (Amersham Bioscience).

Two-dimension Gel Analysis—Immunoprecipitates were resuspended in non-reducing sample buffer (Tris-HCl 125 mM, 20% glycerol, 2% SDS, 0.02% bromphenol blue) and run in the first dimension by 12.5 SDS-PAGE. The relevant strips were then cut out, incubated in sample buffer with 4% 2-ME at 95 °C for 10 min, and run in the second dimension using 12.5% SDS-PAGE.

VpreB3 Expression Plasmid—A full-length chicken VpreB3 EST (GenBankTM accession no. AJ395785 [GenBank] ) from the bursal dkfz426 cDNA library was obtained from J. M. Buerstedde. A Myc tag epitope-coding sequence was added at the 3'-end by PCR using the following oligonucleotides: sense primer, 5'-GTGAATTCTGCAGCCTACCATGGTCCTGGGCTT-3', and antisense primer, 5'-CGAATTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCCAGCCAGTTGATGGTGAGG-3'. The amplified product was first cloned in the pGEM-T Easy vector (Promega, Madison, WI) and subsequently in the pApuro expression vector using EcoRI restriction sites. For this latter purpose, a pApuro vector containing a Btk insert cloned at the EcoRI site was obtained from R. Guinamard (Centre d'Immunologie de Marseille-Luminy).

Transfections—All transfections were performed by electroporation at 550 V and 25 µF with 50 µg of linearized plasmid and 10 x 106 cells in 800 µl of PBS using a Genepulser II with a capacitance expander (Bio-Rad, Hercules, CA). The pApuro-derived vectors were linearized by NotI.

Reverse Transcriptase-PCR Analysis—cDNA was prepared from 2 µg of total RNA, using random hexamers and the Superscript II reverse transcriptase (Invitrogen) in a volume of 20 µl. PCR was performed in a PTC-200 apparatus (MJ Research Inc., Waltham, MA) using 1 µl of reverse-transcribed RNA for VpreB3 (or 1 µl of a 1/100 dilution for Gapdh) and 50 pmol of each primer. The first cycle was run as follows: denaturation at 94 °C for 3 min 30 s, annealing at 60 °C for 30 s, and synthesis at 72 °C for 30 s. The next cycles were run using the same conditions except that the denaturation time was 30 s and the synthesis time of the last cycle was 7 min. Oligonucleotides for VpreB3 expression analysis were: sense primer, 5'-CTCTTCCTCTGCTCCTGGATCCTAC-3', and antisense primer, 5'-AGAAATAGCGACCGTTGTCTTCCTC-3'. Sense primer, 5'-TGACGTGCAGCAGGAACACTATAAA-3', and anti-sense primer, 5'-TGAGATGATAACACGCTTAGCACCA-3', were used for the Gapdh control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a VpreB3 Orthologous Gene in Chicken—A chicken cDNA coding for a protein product with strong similarities to human and mouse VpreB3 was found by performing a Blast search on EST sequence databases using the VpreB3 protein as a query sequence. This 0.6-kb-long chicken cDNA (GenBankTM accession no. AJ395785 [GenBank] ) is likely to be full-length because it grossly corresponds to the size of the corresponding mRNA detected by Northern blot hybridization (data not shown). The 125-amino acids-long chicken VpreB3 protein shows 50 and 55% amino acid identity with the mouse and human Vpre3 proteins, respectively (Fig. 1A). It has only 40% identity with chicken IgLC variable domains (not shown). All three known VpreB3 proteins have a characteristic sequence feature ("signature") made of three invariant residues (His-96, Ala-98, Cys-99) in the FR3-like region that are not found in any other Ig-related polypeptides (Fig. 1A).



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  		FIG. 1.
Characterization of the chicken VpreB3 gene. A, sequence alignment of the human (Hsa), mouse (Mmu), and chicken (Gga) VpreB3 proteins. Boxed residues are conserved in at least two of the three proteins. Black boxes correspond to conserved amino acids figuring the "signature" of VpreB3. Cysteines involved in the stabilization of the Ig fold are indicated by black dots. An extra cysteine present in the signature is indicated by a black square. Protein subdomains (FR, framework region; CDR, complementary determining regions) that characterize Ig domains are indicated above the sequences. B, genomic organization of the VpreB3 gene and the light chain genes locus showing the unique L, VL, JL, and CL segments and the 25 VL pseudogenes ({psi}VL). C, VpreB3 mRNA expression in DT40 cells and in different tissues from chicks at the time of hatching. The results from 30, 34, and 38 cycles of amplification from reverse-transcribed RNA are shown. The larger amplification product in liver corresponds to amplified contaminating genomic DNA.

 
Another Blast search using the chicken cDNA as the query sequence found the sequence of the chicken VpreB3 gene and surrounding locus (GenBankTM accession no. AF079888 [GenBank] ). This gene has an evolutionarily conserved two-exon structure that codes for the signal peptide and V-like region, respectively, and lies 3.4 kb upstream of the VL pseudogene cluster that contributes to light chain diversity through a gene conversion mechanism (Fig. 1B). The VpreB3 gene is in the same transcriptional orientation as the unique functional VL-JL segment.

We examined VpreB3 mRNA expression by PCR amplification in the chicken DT40 B cells and in various chick tissues at the time of hatching. Expression was detected in DT40 cells and in the bursa of Fabricius but not in other tissues (Fig. 1C). Note that the larger band seen in liver was because of the amplification of contaminating genomic DNA. Because the bursa of Fabricius is a site of diversification and expansion of B cells expressing IgM at their surface, we further used the IgM+ DT40 cells to gain insight into the functional role of the VpreB3 protein.

VpreB3 Glycoprotein Binds IgLC in DT40 Cells—To characterize the chicken VpreB3 protein and interacting partners, we generated DT40 clones that express a Myc epitope-tagged version of the VpreB3 protein as well as control clones transfected with an empty expression vector. There was no evidence that VpreB3 overexpression affects IgM surface expression. Rather, DT40 clones showed low scale heterogeneity in their IgM surface expression, likely because of ongoing immunoglobulin gene diversification by gene conversion (23). Therefore, in subsequent experiments we compared VpreB3-overexpressing cells (Vp) to control cells (C) showing similar IgM staining patterns, e.g. Vp7 versus C2 and Vp21 versus C3 (Fig. 2A).



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  		FIG. 2.
VpreB3 interacts preferentially with IgLC in DT40 cells. A, analysis of IgM cell surface expression in parental DT40 and transfected DT40 clones. C2 and C3 clones were transfected with an empty pApuro expression vector and Vp7 and Vp21 clones with a Myc-tagged VpreB3 coding pApuro vector. Viable cells were examined by immunofluorescence using the M1 anti-µ Ig heavy chain Ab. B and C, indicated cells were labeled for 4 h with [35S]Met and [35S]Cys, lysed in 1% Igepal CA-630 buffer, and immunoprecipitated with the indicated Abs. Immune complexes were treated (+) or not (–) with endoH and resolved by 12.5% SDS-PAGE under reducing conditions. The bands resulting from endoH digestion are indicated by asterisks. Note that endoH-resistant IgLC and IgµHC bands are reduced in VpreB3-overexpressing cells compared with control cells.

 
After radioactive metabolic labeling of transfected cells and immunoprecipitation using anti-Myc Ab, the 16.5-kDa VpreB3 product coprecipitated with a 28-kDa protein with the same electrophoretic mobility and endoH sensitivity as IgLC (Fig. 2B). In addition, a band at the same 16.5-kDa molecular mass as VpreB3 was found in anti-IgLC immunoprecipitates and, to a much lower extent, in anti-IgµHC immunoprecipitates (Fig. 2, B and C). A major fraction of VpreB3 showed a 3-kDa decrease in size after treatment with endoH (Fig. 2, B and C), thereby demonstrating that it was mainly a high mannose-containing glycoprotein located in the ER. However, a significant VpreB3 fraction showed resistance to endoH digestion, suggesting that it could be transported into and mature in the Golgi apparatus (see below).

As shown in Fig. 2, B and C, Golgi-processed endoH-resistant mature forms of IgLC and IgµHC decreased in VpreB3-overexpressing cells compared with control transfectants. VpreB3 is thus an IgLC-binding protein that may negatively regulate maturation of BCR chains.

VpreB3 Weakly Associates with IgµHC—Our experiments indicated that coimmunoprecipitation of IgµHC with VpreB3 was not detected in anti-Myc immunoprecipitate (Fig. 2B) and detected at a very low level in anti-IgµHC immunoprecipitates (Fig. 2, B and C). The lack of detection of IgµHC in anti-Myc immunoprecipitate was also observed in Vp21 cells and in two additional independent experiments in Vp7 cells (data not shown). To unambiguously determine the capacity of VpreB3 to associate with IgµHC, similar experiments were conducted in IgLC-deficient (VJL–/–) DT40 cells expressing Myc-tagged VpreB3. Immunoprecipitations on lysates from metabolically labeled cells showed that IgµHC was detected in anti-Myc immunoprecipitates, albeit at a low level (Fig. 3). In addition, VpreB3 was still detected in anti-IgµHC immunoprecipitates from VJL–/– DT40 cells overexpressing VpreB3. VpreB3 had thus a low capacity to bind IgµHC in the absence of competing IgLC.



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  		FIG. 3.
VpreB3 interacts with IgµHC in the absence of IgLC. Indicated cells were labeled for 4 h with [35S]Met and [35S]Cys, lysed in 1% Igepal CA-630 buffer, and immunoprecipitated with the indicated Abs. Immune complexes were treated (+) or not (–) with endoH and resolved by 12.5% SDS-PAGE under reducing conditions. The bands resulting from endoH digestion are indicated by asterisks.

 
VpreB3 Forms Covalent Heterodimers with IgLC—The presence of an evolutionarily conserved extra cysteine (Cys-99) in the VpreB3 signature (Fig. 1A) prompted us to look for a possible covalent association between VpreB3 and IgLC. A two-dimension gel analysis of anti-IgLC and anti-Myc immunoprecipitates from DT40 cells expressing VpreB3-Myc (Vp7 cells) showed a fraction of VpreB3 at 16.5 kDa and IgLC proteins at 30 kDa migrating off the diagonal (Fig. 4). Their identical electrophoretic mobility (corresponding to a 46-kDa complex) in the first dimension is consistent with the existence of covalent VpreB3-IgLC heterodimers. Unexpectedly, mutation of Cys-99 to a glycine residue did not inhibit the formation of this covalent complex (data not shown). The latter observation raises the possibility that the cysteine residues usually involved in the stabilization of Ig-like domains (Cys-42 and -116 in Vpreb3) may participate in interchain disulfide bonds in VpreB3-IgLC heterodimers.



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  		FIG. 4.
VpreB3 and IgLC form covalent heterodimers. DT40 cells expressing Myc-tagged VpreB3 (Vp7 cells) were labeled for 4 h and lysed in 1% Igepal CA-630 lysis buffer. Cell lysates were immunoprecipitated with anti-Myc (left panel) or anti-IgLC (right panel) Abs. Immunoprecipitates were resolved by 12.5% SDS-PAGE under nonreducing condition in the first dimension and under reducing conditions in the second dimension. Open and black arrowheads indicate VpreB3 and IgLC, respectively.

 
Prominent Localization of VpreB3 in the ER—The localization of the VpreB3-Myc protein was delineated by immunofluorescence and confocal microscopy using anti-Myc monoclonal Ab together with fluorophore-conjugated concavalin A or ceramide to label the ER or Golgi compartment, respectively. Fig. 5 shows that VpreB3 was predominantly localized in the ER. However, small areas of superimposed anti-Myc and Golgi-specific staining confirmed that a small fraction of VpreB3 was present in the Golgi compartment. These results are in agreement with the observation that most but not all of the VpreB3 proteins are sensitive to endoH digestion (Fig. 2, B and C), thus confirming that a fraction of the VpreB3 protein pool can exit the ER to mature in the Golgi apparatus.



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  		FIG. 5.
VpreB3 is predominantly localized in the ER. DT40 cells overexpressing Myc-tagged VpreB3 (Vp7 cells) were fixed, permeabilized, and stained with fluorescein isothiocyanate-conjugated concavalin A (green, upper panel) for ER staining or Bodipy FL C5-ceramide (green, lower panel) for Golgi staining in combination with the 9B11 anti-Myc mAb revealed with Texas Red-conjugated goat anti-mouse Abs (red). Yellow in merged images indicates colocalization of the red and green signals. Differential interference contrast (DIC) images are also shown on the right.

 
Maturation and Secretion of VpreB3-containing Complexes— Pulse-chase analysis of VpreB3-containing anti-Myc immunoprecipitates from Vp7 cells showed no evidence of VpreB3 binding partners in addition to IgLC (Fig. 6A). Clearly, the half-lives of IgLC and VpreB3 in anti-Myc (Fig. 6A) immunoprecipitates were quite similar (~1 h 20 min), indicating that a pool of IgLC was stably associated to VpreB3 and degraded together at the same rate. IgLC associated with VpreB3 were totally sensitive to endoH digestion, demonstrating that VpreB3·IgLC complexes are confined to the ER. In addition to the endoH-sensitive form of VpreB3 at 16.5 kDa reported above (see Fig. 2), an additional digestion-resistant VpreB3 product was observed at 18 kDa at 30 and 60 min of chase. A similar VpreB3 product was also evident in VpreB3-Myc-expressing VJL–/– DT40 mutant cells (Fig. 6B).



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  		FIG. 6.
Maturation and secretion of VpreB3-containing complexes. DT40 Vp7 cells (A) and DT40 VJL–/– cells expressing Myc-tagged VpreB3 (B) were pulse-labeled for 15 min and lysed after variable chase periods. VpreB3 and its associated proteins were precipitated using the 9E10 anti-Myc mAb, and immune complexes were mock-treated or digested with endoH. Samples were analyzed by 12.5% SDS-PAGE. In panels A and B, the endoH-deglycosylated proteins are indicated by asterisks and the 18-kDa endoH-resistant VpreB3 isoform is indicated by arrows. C, indicated cells were similarly grown to confluence for 3 days. The supernatants were collected and immunoprecipitated with the anti-Myc 9E10 mAb. Immune complexes were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and immunoblotted with an anti-Myc mAb. D, lysates and supernatants of the indicated cells were immunoprecipitated with the 9E10 anti-Myc mAb, and immune complexes were mock-treated or digested with endoH. After separation and transfer onto polyvinylidene difluoride membrane, VpreB3 proteins were detected by immunoblotting using the anti-Myc Ab. Note the presence of an 18-kDa endoH-resistant VpreB3 protein in Vp7 culture supernatants, in contrast to the 16.5-kDa endoH-sensitive band found in the cell lysate. C and D, black circles indicate the position of nonspecific bands cross-reactive with the anti-Myc Ab. Deglycosylated VpreB3 band at 13.5 kDa is denoted VpreB3 degly.

 
An anti-Myc reactive VpreB3 product of 18 but not 16.5 kDa was detected in the culture medium of VpreB3-Myc expressing cells but not of the corresponding control cells (Fig. 6C). In contrast to the major intracellular 16.5-kDa VpreB3 isoform, this 18-kDa VpreB3 protein was fully resistant to endoH, confirming that it had been processed through the Golgi apparatus to be secreted (Fig. 6D). Secreted VpreB3 proteins were not associated with IgLC (data not shown). Therefore, a fraction of VpreB3 with unique post-translational modification(s) is able to mature through the Golgi and exit the cell in the absence of companion IgLC.

VpreB3 Overexpression Affects the Kinetics of IgLC·IgµHC Complex Maturation at the ER to Golgi Transition—Experiments shown in Fig. 2 have pointed out a possible negative effect of VpreB3 overexpression on the maturation of immunoglobulin chains. Indeed, the amount of mature IgLC was reduced 60% in Vp7 compared with C2 cells and reduced 90% in Vp21 compared with C3 cells. Therefore, the fate of newly synthesized Ig chains was investigated by anti-IgLC immunoprecipitation after pulse-chase labeling. It clearly showed that acquisition of endoH resistance by Ig chains was decreased in VpreB3-overexpressing Vp7 cells compared with C2 control cells (Fig. 7A). This was more evident in the case of IgLC for which maturation was 80–85% decreased at the first three time points analyzed (see quantification in Fig. 7B). The maturation of the IgµHC was delayed rather than quantitatively affected, as shown by the appearance of endoH-resistant form at 30 and 60 min in control and VpreB3-overexpressing cells, respectively.



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  		FIG. 7.
VpreB3 overexpression affects IgM chain maturation. A, C2 control DT40 cells (left panel) and Vp7 DT40 cells expressing VpreB3-Myc (right panel) were pulse-labeled with a [35S]Met/Cys mixture for 15 min, followed by chase periods up to 8 h, and lysed in Igepal CA-630-containing buffer. Lysates were immunoprecipitated with polyclonal goat anti-chicken IgLC Abs and mock-treated or digested with endoH as indicated. Samples were analyzed by 12.5% SDS-PAGE. B, IgLC endoH-resistant bands of C2 and Vp7 cells shown in panel A were quantified using the Tina software. Results are expressed as a percentage of the maximum value. C, indicated cells were colabeled with goat anti-chicken IgLC polyclonal Abs and Bodipy FL C5-ceramide for Golgi staining. Anti-IgLC Abs were detected with a Rhodamine-conjugated anti-goat IgG Ab. Note the reduced yellow signal in merged images in VpreB3-overexpressing cells compared with controls, indicating a reduction of Golgi-localized IgLC.

 
The effect of VpreB3 on IgLC intracellular transport and maturation was further confirmed by confocal microscopy. Fig. 7C shows that a large amount of IgLC was localized in the Golgi in C2 control cells. In contrast, Golgi-localized IgLC were barely detected in VpreB3-overexpressing Vp7 cells. Thus, overexpression of VpreB3 in DT40 cells negatively affects the transit of IgM components, mostly IgLC, from the ER to the Golgi.

VpreB3 Overexpression Inhibits the Secretion of Free IgLC— Excess free IgLC are rapidly secreted in the culture medium of DT40 cells (not shown). Because VpreB3 overexpression affects IgLC transit in the ER/Golgi secretory pathway, an effect of VpreB3 on IgLC secretion may be expected. Indeed, VpreB3 overexpression affected free IgLC secretion because we observed a reduction or absence of IgLC in culture supernatants from Vp7 and Vp21 compared with C2 and C3 cells, respectively (Fig. 8). These results demonstrate that VpreB3 overexpression can retain in the ER some free IgLC that are prone to be secreted.



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  		FIG. 8.
VpreB3 overexpression reduces IgLC secretion. Control cells (C2 and C3) and VpreB3-overexpressing DT40 cells (Vp7 and Vp21) were labeled for 2 h with a [35S]Met/Cys mixture. After centrifugation, supernatants and cells were harvested. Cells were washed and lysed in Igepal CA-630-containing buffer. Supernatants and cell lysates were precleared with control Abs and then immunoprecipitated with anti-IgLC polyclonal Abs. Immunoprecipitates were analyzed by 12.5% SDS-PAGE.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The characterization of an orthologous chicken gene suggests a role for VpreB3 in B cell development throughout evolution. Conserved linkage of VpreB3 to the light chain gene locus in chicken and human (20) indicates that it may have evolved from an ancestral, possibly germ line-joined, IgLC gene. Our failure to find sequences homologous to VpreB3 in the nearly fully sequenced genome of the bony fish Fugu rubripes argues for an emergence of an ancestral VpreB3 gene between 300 and 400 million years ago, between the radiation of the ray-finned fishes and the reptiles/birds phyla. At this time, junctional and combinatorial diversity in Ig genes was already established (24), suggesting that VpreB3 emergence did not depend on a striking change during evolution of the adaptative immune system.

When VpreB3 was first observed in mouse pre-B cells as a partner of IgµHC, it was proposed to play a role in IgµHC transport in the context of pre-BCR formation (19, 21). In mouse and human, the IgH locus is usually rearranged before the {kappa} and {lambda} LC loci, and B cell development depends on the expression of a pre-BCR at the pre-B cell stage (8). In contrast, immunoglobulin heavy and light chain gene rearrangements occur simultaneously during a 10-day window during embryonic development in chicken (25, 26); therefore, there is no need for a pre-BCR-dependent developmental checkpoint. In accordance with this view, we did not find sequences homologous to the VpreB or {lambda}5 pre-BCR-coding genes in chicken genomic and EST databases.

Because VpreB3 is expressed in the bursa of Fabricius by the time of hatching (Fig. 1), in which nearly all lymphocytes are surface IgM-positive (25, 27), it is reasonable to postulate a role for VpreB3 in immature and/or mature IgM-positive cells. This assumption is corroborated by recent reports of large-scale expression analysis showing VpreB3 expression in mouse immature B cells (22) and in human mature B cells (28). Moreover, the latter study has included VpreB3 mRNA expression in the germinal center developmental expression signature.

Despite strong mRNA expression and the capacity of Myc-tagged VpreB3 to bind IgLC in transfected DT40 cells, we were unable to detect endogenous VpreB3 in anti-IgLC immunoprecipitates of empty vector-transfected and parental DT40 cells (Fig. 2 and data not shown). Thus, the endogenous VpreB3 protein level should be low and difficult to detect. This provides a possible explanation for the lack of a detectable effect of targeted gene deletion of VpreB3 in DT40 cells (data not shown). Difficulties were also encountered in detecting endogenous VpreB3 in human pre-B cells expressing high levels of mRNA (data not shown). We speculate that VpreB3 may be subjected to a translational block that may be relieved for rapid synthesis of the protein upon requirement. A number of translational control mechanisms mediated by the 5' or 3' untranslated regions (UTR) of mRNA have been described (29), including remarkably transcript-specific inhibition (30). Interestingly, mouse, human, and chicken VpreB3 mRNAs have conserved a very short (140–180 base pairs long) 3'-UTR.

In transfected DT40 cells, VpreB3 preferentially binds IgLC. The poor capacity of VpreB3 to bind IgµHC in chicken cells contrasts with the easily detectable ability of human VpreB3 to bind IgµHC and also to {lambda}5 in pre-B cells (data not shown). This is possibly because of the formation of a high affinity ternary complex made of VpreB3, {lambda}5, and IgµHC in human pre-B cells. Therefore, the inefficiency of VpreB3 to bind IgµHC in chicken B cells may reflect the absence of a {lambda}5 counterpart.

VpreB3 is glycosylated in DT40 cells. Glycosylation likely takes place at residue 49, a unique consensus N-linked glycosylation site (Asn-Leu-Ser) that is absent in mouse and human VpreB3 (Fig. 1A). Although the majority of VpreB3 synthesized in DT40 cells is endoH-sensitive, two endoH-resistant isoforms of molecular mass 16.5 and 18 kDa were observed (Figs. 2 and 6), the latter being secreted. Nevertheless, VpreB3 was never found at the cell surface and could not substitute for the IgLC to restore maturation and surface expression of IgµHC in VJL–/– cells (data not shown and Fig. 6B).

In VpreB3-overexpressing cells the major role of VpreB3 is to limit maturation and secretion of free IgLC through their retention in the ER (Figs. 7 and 8). IgM surface expression was not affected by VpreB3 overexpression (Fig. 2), underlying the role of VpreB3 in the control of free IgLC rather than fully assembled IgM. The mechanism by which most VpreB3 proteins and VpreB3-IgLC dimers are retained in the ER remains to be elucidated, but it may involve some interaction with ER-resident chaperones and/or a thiol-mediated retention mechanism (31). It will be interesting to determine whether the covalent association of VpreB3 with IgLC is required in this process. Clearly, Cys-99 (see Fig. 1) is not involved in this covalent association or in the ER retention of IgLC (data not shown).

The secretion of free IgLC under physiological conditions is well documented and has often been reported in pathological situations such as myelomas. In the latter case, secretion of free monoclonal IgLC results in specific complications related to precipitation or deposition in different tissues, including heart, lungs, liver, and kidneys (32, 33). In addition, increased free IgLC levels in different body fluids has been reported in several immunopathological conditions (34). A recent study has provided evidence for a role of antigen-specific free IgLC in mast cell-dependent hypersensitivity-like responses (35). Furthermore, because mast cells play a key role in coordinating the early phases of autoimmune diseases (36) and increased levels of free IgLC are found in these pathologies (37, 38), the hypothesis that IgLC-induced mast cell activation might be involved in the progression of some autoimmune diseases has been proposed (34). Whether the presence or absence of VpreB3 proteins plays a role in this context is an interesting matter for future investigation. In this view, the lack of expression of VpreB3 (20) in myeloma cell lines might be related to the frequent occurrence of excess IgLC secretion in myelomas.

Finally, the retention of most VpreB3 proteins in the ER could suggest a function in the context of the ER-initiated unfolded protein response (UPR) (39, 40). It was shown that membrane µ chain is able to elicit a UPR in transfected COS cells (41). Some secretion-incompetent IgLC are known to bind the chaperone Bip in the absence or in the presence of limiting quantities of heavy chains (42–44). This is likely to cause a UPR that could be detrimental to the cell through translation attenuation or induction of apoptosis (45, 46). In this context, VpreB3 may compete with Bip to bind free IgLC and inhibit a UPR that is known to be induced by Bip mobilization (47).

In conclusion, our study provides the first description of a VpreB3 function in B cells. Further experiments are now required to better characterize this now less enigmatic evolutionarily conserved B cell-specific molecule.


   FOOTNOTES
 
* This work was supported by INSERM, CNRS, and a grant from the Association pour la Recherche contre le Cancer (ARC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a fellowship from ARC and previously from Ministère de l'Enseignement Superieur et de la Recherche. Back

Present address: Dept. of Virology, University of Freiburg, D-79104 Freiburg, Germany. Back

{ddagger} To whom correspondence may be addressed: Centre d'Immunologie de Marseille-Luminy, Campus de Luminy, Case 906, 13288 Marseille Cedex 09, France. Tel.: 33-4-9126-9448; Fax: 33-4-9126-9430; E-mail: rosnet{at}ciml.univ-mrs.fr or schiff{at}ciml.univ-mrs.fr.

1 The abbreviations used are: BCR, B cell receptor; pre-B, precursor B; endoH, endoglycosidase H; LC, light chain; HC, heavy chain; ER, endoplasmic reticulum; PBS, phosphate-buffered saline; Ab, antibody; mAb, monoclonal antibody; Ig, immunoglobulin. Back


   ACKNOWLEDGMENTS
 
We thank M. Milili, B. Rossi, and L. Gauthier for discussions and help, Agnès Mistral for excellent technical assistance, H. Arakawa and J. M. Buerstedde for the DT40 VJL–/– cells, and D. Birnbaum, M. Fougereau, and L. Leserman for critical reading of the manuscript.



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