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J Biol Chem, Vol. 274, Issue 27, 19389-19396, July 2, 1999
From the Transcription of the mb1 and
B29 genes is initiated when lymphoid progenitors enter the
B cell differentiation pathway, and their transmembrane Ig Membrane-bound immunoglobulin (Ig) molecules are non-covalently
bound to the transmembrane Ig Expression of Ig The physiological role of Ig Antibodies--
Mouse monoclonal antibodies (mAbs) included
SA-DA 4;4 ( Cells--
Human cell lines included the RS4;11 (25) and Nalm16
(26) pro-B cells, the 697 (27) and Nalm6 (26) pre-B cells, and Ramos
(28) B cells. These were maintained in stationary culture in RPMI 1640 medium supplemented with L-glutamine (2 mM),
penicillin (100 units/ml), streptomycin (100 mg/ml), and 10% fetal
calf serum at 37 °C in 5% CO2.
Cell Surface Biotinylation and Biosynthetic Protein
Labeling--
Viable cells (5 × 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 × 108) were also metabolically labeled with
[35S]Met and [35S]Cys (400 µCi each) for
6 h, then washed and lysed. For pulse labeling and chase analysis,
cells (1-2 × 108) were preincubated in Met- and
Cys-free RPMI 1640 for 2 h to deplete internal pools, and then
labeled with 300-500 µCi of both [35S]Met and
[35S]Cys for 15 min. Labeling was terminated by addition
of 100-fold excess of cold Met and Cys. The pulsed cells were incubated
for various intervals before harvesting.
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 × 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 Western Blots--
Anti-Ig 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 Northern Blots, Reverse Transcription-Polymerase Chain Reactions
(RT-PCR), and DNA Sequencing--
Total RNA was prepared using the
guanidinium isothiocyanate method, electrophoresed on 7% formaldehyde,
1% agarose gels, and blotted onto nitrocellulose filters (Schleicher & Schuell). Northern blots were hybridized with random-primed
32P-labeled DNA fragments. Ig Densitometric Analysis--
Radiolabeled protein intensities
were determined relative to cell surface µHC levels using a
densitometer (UltraScan XL) and a software program (GelScan XL;
Amersham Pharmacia Biotech, Uppsala, Sweden). Background readings of
the autoradiographic films were designated zero.
Characterization of Ig
Pulse-chase analysis of the RS4;11 and Nalm16 pro-B cells indicated
that newly synthesized Ig
These results indicate that the Ig Ig Ig
Ig Association of Ig
An in vitro kinase assay was conducted using unstimulated
and pervanadate-treated Nalm16 pro-B cells. The anti-Ig
The above data suggest that Lyn preferentially associates with the
minor population of Ig Comparison of Ig
EndoF digestion of the anti-Ig In these studies, we observed a remarkable diversity of the Ig Two Ig The molecular interactions and functional potential of the truncated
Ig A functional potential for the Ig The Ig Ig The Ig We thank Drs. Peter Burrows and Jean-Claude
Brouet for review of the manuscript and helpful suggestions, and Ann
Brookshire and Muriel Bargis-Touchard for help in preparing the manuscript.
*
This work was supported in part by Grant 6661 from the
Association pour la Recherche sur le Cancer; Grant 94043 from the
Société Française d'Hématologie, Contrat de
Recherche Clinique AP-HP; and Grant AI39816 from NIAID, National
Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence and reprint requests should be addressed:
Laboratoire d'Immunopathologie, Institut d'Hématologie, Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75475 Paris Cédex 10, France. Tel.: 33-1-53-72-21-64; Fax: 33-1-53-72-21-58; E-mail: kaiss.lassoued{at}sa.u-picardie.fr.
The abbreviations used are:
BCR, B cell
receptor;
LC, light chain;
HC, heavy chain;
mAb, monoclonal antibody;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel
electrophoresis;
endo, endoglycosidase;
RT, reverse transcription;
PCR, polymerase chain reaction;
bp, base pair(s).
Modifications of Ig
and Ig
Expression as a Function of
B Lineage Differentiation*
,
, and**
Laboratoire d'Immunopathologie, Institut
d'Hématologie, Hôpital Saint-Louis, 75475 Paris
Cédex 10, France, § Institut de Génétique
Moléculaire, 34293 Montpellier cedex 05, France, and
¶ Developmental and Clinical Immunology, Departments of
Medicine, Pediatrics, Pathology and Microbiology, Howard Hughes Medical
Institute, University of Alabama at Birmingham,
Birmingham, Alabama 35294
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Ig
products constitute essential signaling components of pre-B and B cell
antigen receptors. We analyzed Ig/Ig 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 Ig form
and three principal Ig forms, transient 33/40-kDa species and a
mature 44-kDa glycoprotein. Deglycosylation revealed a major Ig core
protein of 25 kDa and a minor 21-kDa Ig protein, apparently the
product of an alternatively spliced mRNA. In pro-B cells, the Ig
and Ig 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 Ig/Ig
molecules in pro-B cells. Greater heterogeneity of the Ig and Ig
molecules in pre-B and B cell lines was attributable to increased
glycosylation complexity. Finally, the Ig/Ig heterodimers
associated with fully assembled IgM molecules as a terminal event in B
cell receptor assembly.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(CD79a) and Ig (CD79b) proteins, respectively the products of the mb1 and B29
genes, to form the B cell antigen receptor
(BCR)1 complex (1).
Ig/Ig heterodimers are also integral components of pre-BCR
complexes composed of surrogate light chain (
LC) and µ heavy
chains (HC) on the surface of pre-B cells (2-8). Ligation of BCR and
pre-BCR initiates cytoplasmic signals via the Ig and Ig molecules
whose cytoplasmic domains contain immunoregulatory tyrosine-based
activation motifs. BCR aggregation thereby promotes interaction with
protein-tyrosine kinases and resultant immunoregulatory tyrosine-based
activation motif phosphorylation, hydrolysis of phosphatidylinositol,
sustained intracellular calcium elevation (9-12), and the activation
of multiple signaling pathways (13-15).
and Ig transcripts begins very early in B
lineage differentiation prior to the onset of
DH-JH rearrangements in the µHC locus (16,
17), and Ig-deficient mice are unable to generate µHC-producing
pre-B cells (18). Surprisingly, B cell development in
Ig
/ mice appears to be compromised as early as the
pro-B stage when VH-DJH rearrangements are
occurring, thereby suggesting an Ig role in B lymphopoiesis even
prior to µHC 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).
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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
) anti-human µH chain (21), CB3-1 (1), and
CB3-2 (1) anti-human Ig (4), HM57 (1) anti-human Ig
(22), SLC1 (1) anti-human LC (8), 4G10 (1)
anti-phosphotyrosine (Upstate Biotechnology Inc., Lake Placid, NY),
4D10 (2a) anti-human Syk (Santa Cruz Biotechnology, Inc. CA), and
Fyn (1) anti-human Fyn (Santa Cruz Biotechnology). The JH3
(1) anti-human Ig idiotype (23) and CT4 (1) anti-chicken
CD4 (24) mAbs were used as controls. In immunoprecipitation
experiments, mouse mAbs were either directly coupled to Sepharose 4B
beads or incubated with rat anti-mouse LC-coupled beads (Interchim,
France). Rabbit antibodies to human Syk (a kind gift of Urlich Blanc,
Institut Pasteur, Paris, France) and Lyn (Upstate Biotechnology Inc.)
were also employed, with rabbit -globulins (Pentex, Miles
Laboratory, Kankakee) serving as a control. Phycoerythrin-conjugated
goat anti-mouse Ig was obtained from Southern Biotechnology Associates
(Birmingham, AL).
or anti- Ig mAbs, and bound
materials resuspended in 500 µl 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.
, anti-Ig, and anti-µ
immunoprecipitates were resolved on one-dimensional SDS-PAGE and
two-dimensional diagonal gels, blotted onto nitrocellulose membrane
(Schleicher & Schuell), before incubation with anti-µ, 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.
, 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 MnCl2, 5 mM MgCl2, 1% digitonin) before resuspension in
50 µl of kinase buffer containing 1 mCi of [-32P]ATP
(>4000 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 µl of SDS sample buffer and
boiled for 5 min or submitted to reprecipitation as follows: elution in
50 µl of 0.5% SDS lysis buffer instead of digitonin, incubation for
10 min at 60 °C, 10-fold dilution in 500 µl of 1% Triton X-100,
then immunoprecipitation. In one series of experiments, Nalm16 cells
were incubated with the CB3-1 anti-Ig antibody (30 µg/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 anti-phosphotyrosine 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.
and Ig transcripts were
further analyzed by RT-PCR. The first strand cDNA synthesis
employed Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Inc.) for 50 min at 42 °C in the presence of 5 µg of
total RNA and 100 ng of primers complementary to the coding sequence of Ig mRNA (Ig-R, 5'- CTGGACATCTCCTATGTTGA-3') or Ig mRNA
(Ig-R1, 5'-CTCCTGGCCTGGGTGCTCAC-3'). The cDNAs were amplified in
the presence of both the 5' and 3' primers with Taq
polymerase (Cetus, Emeryville, CA) through 28 cycles involving
denaturation at 92 °C for 1 min, annealing at 55 °C for 1 min,
and elongation at 72 °C for 1 min. Ig forward and reverse primers
were Ig-F (5'-ATGCCTGGGGGTCCAGGAGTC-3') and
Ig-R; Ig primers were Ig-F
(5'-ATGGCCAGGCTGGCGTTGTC-3') and Ig-R2 (5'-
AATGTCCAGGCCCTCGTAAGG-3'). The RT-PCR protocol was essentially that of
Hashimoto et al. (30). Amplified PCR products were analyzed
on 2.5% agarose gels. DNA fragments corresponding to the different PCR
products were eluded from the agarose gel, cloned in the PCR 2.1 vector
(Invitrogen, Carlsbad, CA), sequenced using dye terminator chemistry,
and analyzed with an automated ABI DNA sequencer (Perkin Elmer, Foster
City, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 two-dimensional 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.
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Fig. 1.
Analysis of Ig and
Ig expression in pro-B cell lines.
A, Anti-Ig (lanes 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 anti-mouse 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.
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).
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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 [35S]Met and [35S]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.
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.
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.
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.
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Fig. 3.
Analysis of mb1 (Ig )
and B29 (Ig) transcripts in cell lines
representative of different stages in human B lineage
differentiation. Ig (A) and Ig (B)
RT-PCR products separated on a 2.5% agarose gel. The sequences
determined for the indicated 660- and 550-bp Ig cDNAs and for
the 600- and 290-bp Ig cDNAs matched the mRNA regions
indicated by closed bars in the schematics and
described under "Experimental Procedures." Hybridization
experiments and sequence analysis indicated that the 500-bp band
observed in some Ig reactions did not correspond with an Ig gene
product.
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 3' 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 (41-45).
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 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).
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Fig. 4.
Ig /Ig
association with Lyn in pro-B cells. A, digitonin
lysates of metabolically labeled RS4;11 pro-B cells were incubated with
anti-Lyn antibody and the eluate submitted to a second precipitation
with anti-Ig (lane 1). Conversely, the
anti-Ig eluate was reprecipitated with anti-Lyn (lane
2). B, kinase activity of pro-B cells
phosphoproteins associated with Ig. Pervanadate-treated
(P+) and unstimulated (P
) Nalm16 cells were
examined for kinase activity; anti-Ig (lanes 3 and 4) and anti-Lyn (lanes 1 and
2) precipitates of digitonin cell lysates were subjected to
an in vitro kinase reaction in the presence of
[-32P]ATP. C, analysis of the tyrosine
phosphorylation status of Ig-associated Lyn in pro-B cells. Whole
cell lysates (L, lanes 1 and
2) and anti-Ig precipitates (lanes
3 and 4) of pervanadate (P)-stimulated
(+) and unstimulated () Nalm16 cells were separated by SDS-PAGE
before immunoblotting with an anti-phosphotyrosine antibody.
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.
/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.
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 two-dimensional 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, µHC were coprecipitated
with the Ig and Ig molecules. Comparative analysis of the
anti-µHC precipitate (Fig. 5E) with the anti-Ig and
anti-Ig precipitates (Fig. 5, C and D)
indicates that the (µHC)2/(
LC)2 complexes
could be coprecipitated with antibodies against the Ig and Ig
molecules, while the µHC/LC subunits could not.
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Fig. 5.
Analysis of the Ig
and Ig molecules produced by pre-B and B
cell lines. Anti-Ig (A and C), anti-Ig
(B and D), and anti-µ (E)
immunoprecipitates of metabolically labeled 697 pre-B cells
(A and B) and Ramos B cells
(C-E) were assessed by two-dimensional gel
electrophoresis. Note that the µHC and LC components representing the
unassembled µHC/LC subunits (small arrows) are
seen in the anti-µHC immunoprecipitate (E), whereas only
components of the assembled (µHC)2/(LC)2
units (large arrows) are evident in the
anti-Ig and anti-Ig immunoprecipitates of pre-B and B cells. The
surrogate light chain components (16/18-kDa Vpre-B, and 22-kDa
5/14.1) in the pre-B cells are not shown in A and
B.
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Fig. 6.
Analysis of
Ig /Ig association and
heterogeneity in human pre-B and B cells. Anti-Ig (A
and C) and anti-Ig (B and D)
immunoprecipitates of lysed, unlabeled 697 pre-B (A and
B) and Ramos B cells (C and D) were
resolved by two-dimensional SDS-PAGE and blotted with antibodies
against Ig and Ig as described in Fig. 1. Note the greater
heterogeneity of Ig (C) and Ig (D)
molecules in the B cells, and the subfractions of smaller Ig and
Ig species present in both the pre-B and B cells.
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-µ 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).
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Fig. 7.
Pulse-chase analysis of Ig
and Ig biosynthesis and association in
pre-B and B cell lines and comparative analysis of
Ig/Ig heterogeneity
in the pre-BCR and BCR. A, Pre-B and B cell lines, 697 and Ramos, were pulse-labeled with [35S]Met and
[35S]Cys and chased with cold Met and Cys for varying
time intervals before cell lysis and analysis of anti-Ig or
anti-Ig immunoprecipitates by SDS-PAGE under reducing conditions.
The different molecular forms of Ig/Ig seen in pro-B cells (Figs.
1 and 2) are also present in the pre-B and B cells, in which Ig and
Ig association is already evident at time 0. B,
two-dimensional gel analysis of anti-Ig immunoprecipitates at
various intervals following pulse labeling of the 697 pre-B cells. Note
the relative homogeneity of Ig and Ig molecules as late as 2 h after biosynthesis. C, comparable heterogeneity of
Ig/Ig molecules on the surface of 697 pre-B cells and Ramos B
cells. Cells were surface-biotinylated, lysed in digitonin, then
incubated with an anti-µ antibody. Immunoprecipitates were resolved
by two-dimensional SDS-PAGE.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
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 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 transcripts, 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).
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 µHC 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).
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 µHC 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.
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 µHC 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 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 VDJH 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-DJH
rearrangements (18). In contrast, V-DJH rearrangement
proceeds normally in mice that have a cµ mutation that
prevents membrane-bound µHC 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 µHCs 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.
/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
(µHC)2/(LC)2 units, but not with the
precursor µHC/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 µHC in
the BCR complex (1), it may be important to determine whether when the
Ig/Ig-µHC association occurs immediately before or after
assembly of the membrane-bound IgM subunits.
ACKNOWLEDGEMENTS
FOOTNOTES
Investigator of the Howard Hughes Medical Institute.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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