(Received for publication, October 12, 1995; and in revised form, November 30, 1995)
From the
Four human IgE isoforms produced by alternative splicing of the
epsilon primary transcript were expressed as chimeric mouse/human anti
5-dimethylamino-1-naphthalenesulfonyl antibodies in the murine myeloma
cell line Sp2/0. The four isoforms include the classic secreted form
and three novel isoforms with altered carboxyl termini. All of these
isoforms lack the transmembrane region encoded by the M1/M1` exon and
are therefore predicted to be secreted proteins. When expressed in
Sp2/0 cells, three of the IgE isoforms are assembled into complete
molecules of two Ig heavy chains and two Ig light chains, whereas the
fourth isoform is predominately assembled into half-molecules of one Ig
heavy chain and one Ig light chain. All four isoforms are secreted with
similar kinetics. In contrast, when the isoform containing the C4
domain joined directly to the M2` exon (IgE
grandé) is expressed in the J558L cell line, it
is degraded intracellularly, suggesting a cell line-dependent
regulation of secretion. These data show that these novel isoforms of
human IgE, predicted to occur from in vivo and in vitro mRNA analysis, can be produced and secreted by mammalian cells.
The different forms of IgE may have physiologically relevant but
distinct roles in human IgE-mediated immune inflammation. The
availability of purified recombinant human IgE isoforms makes it
possible to analyze the functional differences among them.
Alternative RNA splicing determines the production of secreted versus membrane-bound forms of immunoglobulins(1, 2) . This is accomplished in mammals by the alternative usage of either a secreted terminus at the end of the last constant region domain or two downstream exons (M1 and M2) that encode the transmembrane and intracellular amino acids. Splicing to the M exons removes from the transcript the nucleotides that encode the hydrophilic COOH terminus and polyadenylation signal for the smaller, secreted form of the Ig.
The one functional genomic
locus encoding human epsilon heavy chain contains four Ig domain exons
(C1 to C
4) and the two membrane exons (M1 and M2). We (3, 4, 5) and others (6, 7) have previously shown that RNA prepared from
the IgE-producing human cell line AF-10 and from fresh B lymphocytes
stimulated to make IgE contain a variety of epsilon mRNAs produced by
alternative splicing. In contrast to what is observed with other
isotypes, the most common form of mRNA encoding membrane IgE is
produced by splicing to a novel splice acceptor 156 base pairs upstream
of the normal M1 acceptor site(4, 7) . The M1` exon
produced using this splice acceptor encodes 52 novel amino acids that
are largely hydrophilic followed by the amino acids normally encoded by
M1.
Other alternatively spliced epsilon mRNAs are present that
encode a series of potentially secreted proteins. The splicing events
that generate these mRNAs utilize several novel exons including M2`,
M2", and C5 in addition to the classic secreted form (see Fig. 1A). The M2` exon is created by splicing directly
from C
4 to the normal M2 splice acceptor. The omission of M1
results in a frameshift in M2, which creates an open reading frame
encoding 136 hydrophilic amino acids (i.e. M2`). M2" is a
short tail (8 amino acids) created by splicing from C
4 to a splice
acceptor located within the M2` exon. The reading frame of M2" is
different from that of M2`(4, 5) .
Figure 1:
A, structure and alternative splicing
at the 3` end of the human epsilon locus. Locations of classic membrane
exons M1 and M2 and of novel exons M1`, M2`, M2", and C5 are
depicted at the top of the figure. Below are shown
the splicing events that lead to the production of the isoforms
investigated: IgE classic, IgE grandé, IgEtp, and
IgE
CH4. The dotted line at the far 3` end of splicing
diagrams indicates uncertainty regarding downstream splicing events. B, strategy used to generate isoform-specific IgE constructs.
The top portion of B depicts the third and fourth
constant domain exons of the genomic human epsilon gene and a
3`-untranslated region,
3 3`-UT (not drawn to scale),
from the human
3 immunoglobulin heavy chain locus. The mutations
used to generate the XbaI and NheI sites in C
4
are shown above the C
4 exon. The XbaI and NheI
sites in the three RT-PCR clones (at the bottom of the figure)
were introduced with the same mutations. None of the mutations
introduced amino acid substitutions. The three RT-PCR clones were then
fused to the genomic exons at the XbaI and EcoRI
sites or at the NheI and EcoRI sites. C,
diagram of the pSV2 gpt expression vector for IgE
grandé highlighting relevant features. The
immunoglobulin is encoded by an anti-dansyl variable domain exon (VDJ), epsilon constant domain exons (1, 2, 3, and 4), and sequence encoding the novel carboxyl
terminus (NOVEL). Two orphan J segments are also present (J). Expression is driven by an Ig heavy chain promoter/leader
sequence (P) and an Ig heavy chain enhancer element (ENH). The wavy arrow indicates the direction of
transcription. 3`-UT indicates the human
3
3`-untranslated region. Amp
and gpt indicate the
-lactamase and xanthine-guanine
phosphoribosyltransferase genes used for selection in procaryotic and
eukaryotic cells, respectively. The XbaI, NheI, and EcoRI sites introduced by PCR mutagenesis are shown. Also
shown is the BamHI site used to subclone the different
constructs into expressions vectors. The SalI and XhoI sites used to subclone into expression vectors were
destroyed in the ligation and are indicated here by a
.
It is of great interest whether these novel mRNAs encode functional proteins and whether these various forms of IgE play distinct roles in the immune response. Using polyclonal anti-peptide antibodies, we have detected the protein product of one of these novel splice variants in the supernatant and cytoplasm of AF-10 cells and in serum from a patient with IgE myeloma(3) . However, the low level of IgE present in normal serum makes it impossible to isolate sufficient quantities with adequate purity for definitive functional studies. Human IgE is the least abundant Ig with average serum concentrations (125 ng/ml) generally 100,000-fold less than IgG in normal individuals. Purification from serum would be further confounded by the similar molecular size of several of the splice variants. Therefore, we have focused on developing expression systems for the production of each epsilon splice variant.
In a recent study, Batista et al.(8) report the expression in J558L murine myeloma cells of five constructs that encode individual splice variants of IgE(8) . The conclusions of this study were that only one form of IgE is detectable on the surface of the transfected cells and only one form of soluble IgE is secreted by myeloma cells. These forms were found to correspond to the CH4-M1` membrane-bound and the classic secreted (CH4-S) forms of IgE, respectively. Although Western blotting of protein secreted by the cell line U266 revealed heterogeneity of epsilon chains, Batista et al.(8) conclude that this is the result of differential glycosylation. They further conclude that the novel epsilon isoforms produced by alternatively spliced mRNAs are degraded intracellularly and therefore cannot constitute functionally relevant forms of IgE.
In the present study, we demonstrate the
expression and secretion of four soluble isoforms of human IgE by the
murine myeloma cell line Sp2/0. The isoforms examined are classic
secreted (CH4-S), CH4-M2`, CH4-M2", and CH4`-CH5. We designate these
proteins as IgE classic, IgE grandé, IgE
tailpiece (IgEtp), and IgE chimeric CH4 (IgECH4), respectively.
All four isoforms show similar kinetics and efficiency of assembly and
secretion, although one of the isoforms (i.e. IgE
CH4) is
secreted predominately as HL (
)half-molecules. Furthermore,
the production of secreted protein is shown to depend on the murine
myeloma cell line used for expression.
Isoform-specific epsilon chain genes were created
by first using PCR mutagenesis to create novel XbaI or NheI restriction sites within the C4 coding region of
each of these RT-PCR clones and an EcoRI site at the 3` end of
each clone (before the polyadenylation addition signal). PCR
mutagenesis was then used to create the identical mutations within the
C
4 exon of the human genomic epsilon gene. The XbaI site
was created at arginine 520; the NheI site was added at
alanine 543. In all cases introduction of the restriction sites did not
alter the amino acid sequence. The XbaI or NheI sites
were then used to fuse the various downstream sequences with the
epsilon gene. The 3`-untranslated region (UTR) from human
3 heavy
chain gene had been provided with an EcoRI site 5` of the
polyadenylation addition signal (9) and was substituted for the
3`-UTR of the human epsilon gene by ligation at the EcoRI
sites created at the 3` ends of the RT-PCR clones. The genes encoding
the different epsilon isoforms were then cloned into pSV2 gpt
containing the coding sequence for a heavy chain variable domain
specific for the hapten dansyl chloride under control of the Ig heavy
chain promoter and Ig heavy chain enhancer(9) . This was done
by ligation at the BamHI site at the 3` end of the 3`-UTR (see Fig. 1C) and by ligation of the XhoI site
immediately 5` of the C
1 exon with a SalI site 3` of the
Ig heavy chain enhancer in the expression vector. The SalI and XhoI sites were destroyed in the ligation and are indicated by
in Fig. 1C. The anti-dansyl light chain used is a
chimeric kappa chain consisting of a murine variable (V
) domain and human C
domain.(10) .
To
precipitate the Ig protein, 2.5 µl of rabbit anti-human Fab
antiserum (R27) was added to each culture supernatant, and the
supernatant was incubated at 4 °C for 2-18 h. 100 µl of
IgGsorb Staph A (The Enzyme Center, Inc., Malden, MA) was then added,
and the supernatant was incubated at 4 °C for 15 min to 1 h. Immune
complexes were spun through a pad of 30% sucrose + 0.15% SDS
+ 0.5X NDET (1 NDET = 1% Nonidet P-40, 0.4%
deoxycholate, 66 mM EDTA, and 10 mM Tris, pH 7.4),
and the pellet was washed sequentially in 300 µl of NDET +
0.3% SDS and 400 µl of distilled H
O. The pellet was
then resuspended in sample loading buffer (loading buffer = 25
mM Tris, pH 6.7, 2% SDS, 10% glycerol, and
0.1 µg/ml
bromphenol blue) and boiled for 2 min. The samples were analyzed on
polyacrylamide gels(12) . For two-dimensional gel analysis,
samples were first electrophoresed on 5% polyacrylamide
gels(12) , and the lane containing the sample of interest was
excised and incubated in sample loading buffer containing 5%
dithiothreitol (Boehringer Mannheim) for 20 min at room temperature.
The lane was then embedded in 12.5% polyacrylamide. Electrophoresis in
the second dimension was then done as described (12) .
Cells were separated from the
supernatant by centrifugation for 5 min at 225 g at 4
°C. Cell lysates were prepared by resuspending the cell pellet in
0.5 ml of NDET, centrifuging at 4 °C for 15 min at 15,000
g, and discarding the pellet. IgE was precipitated from
supernatants and cell lysates with a mixture of rabbit anti-human Fab
(R27 antiserum) and rabbit anti-human epsilon (ICN) as described above.
For densitometry of IgE assembly intermediates, nonreducing gels from pulse-chase experiments were visualized by autoradiography and scanned on a Hewlett/Packard ScanJet IIcx scanner. The images were analyzed at 600 dots/inch using the NIH Image software package.
Figure 2:
Immunoprecipitation of
[S]methionine-labeled IgE isoforms. A,
cell lines producing IgE classic, IgE grandé,
IgEtp, or IgE
CH4 were labeled 6-18 h with
[
S]methionine, and IgE was precipitated from the
secretions using rabbit anti-human Fab followed by Staph A. Samples
were loaded on a 5% gel under denaturing, nonreducing conditions. IgE
classic, IgE grandé, and IgEtp are secreted as
species of 190 kDa. IgE
CH4 is secreted primarily as HL, which
migrates at 75-90 kDa, and free light chain of 25 kDa. B, samples prepared in a similar fashion were loaded onto a
12.5% gel under denaturing, reducing conditions. The 190-kDa species of
IgE classic, IgE grandé, and IgEtp dissociate
into two species of approximately 25 and 75 kDa upon treatment with
2-mercaptoethanol. The 75-90-kDa species of secreted IgE
CH4
migrates as light chain of approximately 25 kDa and heavy chain of
55-75 kDa after treatment with reducing
agent.
Figure 3:
Intracellular assembly and secretion of
IgE isoforms. 2-8 10
cells/time point were
incubated in methionine-deficient medium for 1 h to deplete
intracellular methionine. Cells were pulsed with
[
S]methionine (15 µCi/10
cells)
for 5 min at 37 °C and then chased with a
100-fold excess of
unlabeled methionine. Samples were taken at various time points
following addition of the chase. Epsilon and kappa chains were then
immunoprecipitated from the cytoplasms and secretions at each time
point and run on 5% gels under nonreducing conditions. A, IgE
classic cytoplasms. B, IgE classic secretions. C, IgE
grandé cytoplasms. D, IgE
grandé secretions. E, IgEtp cytoplasms. F, IgEtp secretions. G, IgE
CH4 cytoplasms. H, IgE
CH4 secretions. The open and closed
arrows in C denote non-IgE proteins co-precipitating with
IgE grandé; the asterisk in F denotes a secreted species that we propose but have not proven to
be the H
form of IgEtp.
Because the protein secreted by the
cell line producing IgE grandé does not migrate
as slowly as one would predict based on amino acid translation, we
undertook experiments to verify that the protein was indeed complete
and intact. Initially, the expression construct for IgE
grandé was subjected to extensive restriction
analysis, which showed that the entire coding region had been retained
in the construct (data not shown). Due to concern that the novel
136-amino acid tail of the IgE grandé could be
post-translationally cleaved, the protein was analyzed by ELISA and
Western blotting using antibodies specific for the C2/C
3
boundary (CIA-7.12) and for the COOH-terminal ten amino acids of IgE
grandé (
-2331). As expected, both IgE
classic and IgE grandé were recognized by
CIA-7.12, whereas
-2331 recognized IgE grandé but failed to recognize classic secreted IgE ( Table 1and
data not shown). Neither CIA-7.12 nor
-2331 recognized an IgG
control. Recognition of IgE grandé by
-2331
indicates that the large secreted terminus of IgE
grandé is not removed by proteolytic processing.
In addition, recognition of IgE grandé by
monoclonal antibody CIA-7.12 indicates that the C
2-C
3
interface (the epitope that CIA-7.12 recognizes) is intact; the ability
of IgE grandé to bind antigen confirms that V
is present.
Fully assembled IgE classic, IgE
grandé, and IgEtp are detectable in the
secretions starting at 60 min post-chase (Fig. 3, B, D, and F). Quantitative analysis of the gels shown in Fig. 3indicates that in the case of IgE classic and IgE
grandé, approximately 25% of the total Ig
produced during the 5-min pulse is secreted as HL
during the course of the experiment (Fig. 4A).
Following reduction, the secreted proteins migrate as heavy and light
chains of approximately 75 and 25 kDa (Fig. 2B and Fig. 5and data not shown). IgEtp is the most efficiently
secreted of the isoforms examined, with approximately 40% of the Ig
secreted as H
L
during the course of the
experiment (Fig. 4A). HL is secreted in varying amounts
for each of the isoforms examined (Fig. 3, B, D, F, and H). Secretion of small amounts of
HL was also reported for IgE classic in the recent study by Batista et al.(8) . In the case of IgE
CH4 (Fig. 3H), the HL form constitutes the majority of IgE
secreted by the transfectoma, with a smaller amount secreted as
H
L. The efficiency of secretion of these two forms is shown
in Fig. 4B and indicates that by 3 h after the pulse,
approximately 20% of the labeled Ig is secreted in HL form, whereas
only
10% is secreted as H
L. All four transfectants
synthesize excess light chain that is secreted as free L and as L
dimers (Fig. 3, B, D, F, and H).
Figure 4:
Kinetics of secretion of IgE isoforms.
Data are from densitometric analysis of the pulse-chase experiments
shown in Fig. 3. A, for IgE classic, IgE
grandé, and IgEtp, the amount of
HL
secreted is expressed as a percentage of the
total heavy and light chain produced during the pulse. B, for
IgE
CH4, the amount of H
L and HL secreted is expressed
as a percentage of the total heavy and light chain produced during the
pulse.
Figure 5:
Migration of 50- and 200-kDa
co-precipitating species from the cytoplasm is unaffected by treatment
with reducing agent. Cytoplasmic and secretion samples from the IgE
grandé pulse-chase experiment were reduced with
2-mercaptoethanol. The IgE grandé in both
cytoplasm and secretion reduces to a heavy chain of 75 kDa and a
light chain of
25 kDa. The co-precipitating species from the
cytoplasm, however, still migrate as 50 and 200 kDa following
reduction. The 50-kDa light chain dimer in the secretions (see Fig. 3, B, D, and F) reduces to light
chain.
Two species of 50 and 200 kDa (closed and open arrows in Fig. 3C) co-precipitate with
intracellular IgE grandé and (to a lesser extent)
IgEtp (Fig. 3E). The mobility of these two
coprecipitating proteins is not affected by treatment with
2-merceptoethanol (Fig. 5, cytoplasm). It is also
noteworthy that the 200-kDa species is present at zero time. From these
data, we conclude that the 200- and 50-kDa proteins are not assembly
intermediates of IgE but instead represent non-IgE proteins that are
coprecipitated. The 200-kDa protein is not secreted, although a band at
50 kDa is seen in the secretions (Fig. 3, B, D, and F). However, the latter is no longer
detectable after treatment with 2-merceptoethanol (Fig. 5,
secretion) and most probably represents light chain dimers
(L
). Thus, the co-precipitating 50-kDa cytoplasmic protein,
like the 200-kDa protein, does not appear to be secreted.
These
findings are in marked contrast to those of Batista et
al.(8) , who concluded that the novel isoforms IgE
grandé (CH4-M2`) and IgECH4 (also
designated CH4`-CH5 and CH4`-I) are not secreted by plasma cells. One
difference between the present and earlier studies is the murine
myeloma cell line used for expression. To determine if the cell lines
could account for the different results, we transfected our expression
vector for IgE grandé into J558L, the murine
myeloma cell line used by Batista et al. No positive clones
were identified when cell culture supernatants of several hundred
selection-resistant transfectomas were screened by ELISA (data not
shown). However, several clones demonstrating high levels of
intracellular epsilon chain were identified by ELISA using anti-IgE to
capture epsilon chain from cell lysates and alkaline
phosphatase-conjugated anti-IgE to detect bound epsilon chain.
Pulse-chase analysis indicates that large amounts of epsilon chain are
produced by this cell line but are degraded intracellularly. Fig. 6A shows that the IgE grandé is assembled in J558L into an HL form that is seen by 5 min
post-chase. However, assembly appears to stop at this intermediate
form, and very little H
L
is formed. A band that
migrates slightly faster than the H and HL forms is seen and may
represent a degradation product. When these samples were
electrophoresed under reducing conditions and analyzed by densitometry,
the total intracellular epsilon chain was found to decrease to less
than 5% of the original level by 180 min post-chase (data not shown).
The reduced samples also demonstrate the existence of additional,
labile species that likely represent degradation products. No
H
L
is detectable in the supernatant of the
J558L transfectoma during the same interval, although small quantities
of HL may appear in the supernatant (Fig. 6B and data
not shown). When selection-resistant transfectomas of J558L were
screened by ELISA of cell lysates, a large percentage (
50%) of the
clones were positive for intracellular epsilon chain, but none secreted
a detectable amount of IgE. Therefore, the intracellular degradation of
IgE grandé appears to occur whenever it is
expressed in J558L. These results demonstrate that different cell lines
can vary in their ability to assemble and secrete the IgE isoforms.
Figure 6: The J558L myeloma cell line does not assemble or secrete IgE grandé. Pulse-chase analysis was done as described in the legend to Fig. 3. A, cytoplasms. B, secretions.
Secreted IgE functions via its ability to bind to specific
IgE receptors. These receptors make it possible for IgE to act as a
very sensitive trigger for initiating both afferent and efferent immune
reactivity in the presence of low doses of antigen. Three such
``receptors'' have been identified; the high affinity IgE
receptor (FcRI), the low affinity IgE receptor (Fc
RII or
CD23), and galectin 3 (formerly known as epsilon-binding protein) (13) .
IgE mediates immediate type hypersensitivity
primarily through its association with the high affinity IgE receptor
present on the surface of mast cells and basophils. These cells release
a variety of soluble mediators upon cross-linking of their
receptor-bound IgE by a cognate antigen. There is also evidence that
IgE bound to the high affinity receptor on mast cells can be
cross-linked by member(s) of a broad class of IgE-dependent histamine
releasing factors(14) . Studies involving histamine releasing
factors have led to the suggestion of a functional heterogeneity of
IgE. The basis for this heterogeneity is not understood but has been
speculated to be the result of differential glycosylation(14) .
IgE has also been suggested to participate in a variety of other immune
processes such as antigen recognition, antibody-dependent cellular
cytotoxicity, and B cell growth via binding to the high affinity IgE
receptor (FcR I), the low affinity IgE receptor (Fc
R II), or
galectin 3. The existence of splice variants of IgE provide an
additional possible explanation for the functional heterogeneity of IgE
and suggests that IgE may differ in its primary protein structure as
well as glycosylation. Splice variants have also been observed in the
mRNA of human IgA (15) and avian IgY(16) .
The low
serum levels of IgE and the similar molecular size predicted for many
putative splice variants make purification of the individual protein
isoforms of IgE from serum problematic. Our approach using recombinant
DNA transfection has the advantage that vectors can be constructed that
encode a single isoform of IgE, guaranteeing the homogeneity of the
isoforms produced. In the current study, we have constructed vectors to
express four isoforms of IgE in an Sp2/0-derived murine cell line. The
kinetics and efficiency of the assembly and secretion are similar for
each of these isoforms. The results indicate that of the four secreted
isoforms examined, IgE classic, IgE grandé, and
IgEtp are fully assembled by the Sp2/0 cells, whereas IgECH4 is
secreted predominately as HL half-molecules. The incomplete assembly of
IgE
CH4 is not entirely unexpected given that the constant regions
of Igs are stabilized by noncovalent interactions between the
COOH-terminal domains of both heavy chains. In the IgE
CH4
isoform, the 3` portion of the C
4 exon is removed by splicing from
a cryptic splice donor within that exon and replaced by sequence from
the C
5 cryptic exon. It is noteworthy that among the residues
removed from IgE
CH4 is a cysteine that is universally conserved
in immunoglobulin domains(5) . The domain structure of
IgE
CH4 is therefore likely to be disrupted. Speculation as to the
physiological relevance of this structural variation must await a
detailed functional comparison of these IgE isoforms.
In previous
studies, we have reported that IgE grandé is
detectable in the supernatant of the IgE-producing cell line AF-10, an
IgE-stable, mycoplasma-free subclone of U266 (4) in the serum
of a patient with an IgE myeloma and in the serum of highly atopic
persons with very high serum levels of IgE(3, 4) .
Because IgE grandé is the only known isoform with
an M sufficiently different from IgE classic to
resolve by SDS-polyacrylamide gel electrophoresis, it is impossible to
ascertain from the previous data whether the other described isoforms
were also present in the U266/AF-10 supernatant or the serum IgE from
myeloma or highly atopic patients, although heterogeneity of bands in
the appropriate size range was evident(3) . In a recent study
by Batista et al.(8) , Western analysis and
immunoprecipitation of the supernatant of U266 showed two species
similar in size to the epsilon chain. However, treatment with
glycosidase PNGase F caused the two bands to be reduced in size and
comigrate as a single species, and it was concluded that only one
isoform of IgE (i.e. the ``classic secreted''
isoform) is secreted by B cells(8) . The same study had found
that when the murine myeloma cell line J558L was transfected with
expression vectors encoding individual splice variants of epsilon heavy
chain, the recombinant epsilon chains could be detected in the cytosol
of the transfectants but not in the secretions.
Secretion of the
four isoforms of IgE reported in the present study is clear. In light
of the aforementioned report, this raises questions concerning cell
line-dependent factors affecting protein expression. Batista et al.(8) expressed their isoform-determined IgE genes in the
J558L myeloma cell line using the pRc/CMV expression vector, whereas we
expressed the proteins in the Sp2/0 myeloma cell line using the pSV2
gpt expression vector. Because adequate levels of epsilon chain were
evident in the cytoplasm of the transfectants in both studies, it is
unlikely that the different expression vectors account for the
difference in secretion. Indeed, when we expressed IgE
grandé in J558L using the same pSV2 gpt-based
expression vector, the amount of epsilon heavy chain produced by the
J558L transfectant was much greater than that produced by Sp2/0, but
the J558L transfectant fails to efficiently assemble and secrete it (Fig. 3, C and D, and Fig. 6, A and B). Quantitation of the epsilon heavy chains on a
12.5% gel under reducing conditions (not shown) indicates that nearly
all of the epsilon chain produced by these cells during the 5-min pulse
is degraded within 180 min. Although the Sp2/0 IgE
grandé transfectoma produces far less epsilon
chain than its J558 counterpart, readily detectable levels of IgE
(25% of the epsilon and kappa chains labeled during the 5-min
pulse) are secreted in the H
L
form by 180 min
post-chase, and there is no evidence for heavy chain degradation ( Fig. 4and data not shown). Additionally, the kinetics of
assembly and secretion of the IgE grandé is very
similar to that of IgE classic and IgEtp ( Fig. 3and Fig. 4A).
Several factors could cause cell
line-dependent variation in protein secretion. One such factor is
glycosylation. We have some evidence ()that IgE
grandé is degraded when the producing cell line
is labeled in the presence of tunicamycin. Glycosylation-dependent
differences in post-translational proteolytic processing have been
described for the soluble form of CD23 (17) . Also, cell
line-dependent variation has been described in the utilization of N-linked carbohydrate addition sites. (
)It is
possible that the altered exon usage of some IgE isoforms alters the
accessibility of certain addition sites to some
glycosylases-glycosidases and that some cell lines are better able to
process these carbohydrate addition sites in their altered molecular
context. Another possible explanation lies in chaperone proteins. It
has been shown that during Ig assembly, Igs interact in a sequential
fashion with at least two chaperones, BiP and GRP94(18) .
Although we do not observe co-precipitation of either of these two
chaperones with IgE under the conditions used, it is interesting to
note that both IgE grandé and IgEtp are seen to
co-precipitate with two species of
50 and
200 kDa ( Fig. 3and 5). We have as yet taken no steps to identify these
proteins; however, they have been observed to co-precipitate with other
antibody isotypes including IgA. (
)
We have shown that the protein products of three novel messages for human IgE are translated by the murine myeloma Sp2/0 and are efficiently assembled and secreted. Alternatively spliced mRNAs have been described for one more potentially secreted epsilon protein (5, 6, 7) as well as two forms of membrane epsilon chains. It is quite likely that all the described forms of epsilon mRNA are expressed at the protein level.
On the basis of the
present and previous studies, it appears that human IgE is comprised of
a family of proteins generated by alternative RNA splicing. Individual
secreted members of this family may have some unique properties as
circulating, cytophilic Igs and may differ in their ability to carry
out IgE-mediated functions through binding to FcRI, Fc
RII, or
galectin 3. Although the contact residues for the former two receptors
appear to be intact in the secreted epsilon isoforms, the isoforms may
nevertheless function differently because the CH4 domain may be
critical in constraining the three-dimensional shape of the IgE
constant region. IgE
CH4 merits special attention because this
isoform is secreted primarily as HL half-molecules (Fig. 2A and Fig. 3H), although we have not ruled out
noncovalent interactions between half-molecules. If IgE
CH4 binds
to Fc
R I as a half-molecule, it would be less able to cross-link
the receptors. It may therefore be less able to arm mast cells and
basophils for antigen-triggered release and may in fact inhibit the
release. Our findings gain added significance in that the relative
levels of mRNAs encoding various isoforms are altered in allergic and
parasitic diseases(3) . Additionally, we have demonstrated a
cell line-dependent variability in the efficiency of assembly and
secretion of IgE grandé, suggesting that
additional, as yet uncharacterized regulatory mechanisms of
intracellular IgE assembly and/or trafficking may exist. The
availability of highly purified IgE protein isoforms will now allow us
to determine if the different isoforms have unique functional
properties.
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