The EMBO Journal vol.6 no.1 pp. 109 -114, 1987
Cloning and expression of the cDNA coding for a human
lymphocyte IgE receptor
Christoph Ludin, Hans Hofstetter, Marie Sarfatil,
Claude A.Levy, Ueli Suter, Danielle Alaimo,
Erich Kilchherr2, Heiner Frost2 and Guy Delespessel
Departments of Biotechnology and 2Biology, Ciba-Geigy Ltd., 4002 Basel,
Switzerland, and 1MRC Group for Allergy Research, Department of
Immunology, University of Manitoba, Winnipeg R3E OW3, Canada
Communicated by M.Birnstiel
Low-affinity receptors(FcER)and secreted factors (IgE-BF)
which bind to immunoglobulins ofthe IgE isotype play a key
role in the regulation ofhuman IgE synthesis. We report here
the cloning of a cDNA coding for theFc,Rof the human B-
lymphoblast cell line RPMI 8866. The nucleotide sequence
ofthis cDNA predicts a polypeptide with 321 amino acids and
a mol. wt of 36 281 daltons. A functional FcR capable of
binding IgE was expressed in Chinese hamster ovary cells
after stable transformation with the cDNA which had been
cloned into a mammalian expression vector. Amino acid se-
quence analysis of IgE-BF purified from RPMI 8866 cells
revealed an amino-terminal sequence of 19 residues which
coincides with the predicted amino acid sequence of theFcER,
starting at residues 148 and 150. A computer search with the
translated amino acid sequence of theFcERrevealed a do-
main of 120 amino acids having striking homology to the
human asialoglycoprotein receptors.
Key words: lymphocyte IgE receptor/IgE-binding factor/cDNA
mol. wts of 45 and 65-96 kd. Both components bind to IgE-
Sepharose independently and react with all monoclonal antibodies
directed against FcER, suggesting that they have at least some
amino acid sequences in common (Nakajima and Delespesse,
1986). The present experiments were undertaken to isolate cDNA
clones which encode a human lymphocyte FcER. We have ident-
ified one type of cDNA coding for a protein which reacts with
FcER-specificmonoclonal antibodies and binds to human IgE.
The human B-lymphoblastoid cell line RPMI 8866 which ex-
presses FcER (Sarfati et al., 1984a; Peterson and Conrad, 1985)
and secretes IgE-BF (Sarfati et al., 1984a) was used as the source
of poly(A) mRNA. This was fractionated on sucrose density
gradients and then injected into Xenopus laevis oocytes. After
overnight incubation, the synthesis of Fc>R was monitored by
allowing oocyte homogenate to react with Fc R-specific mono-
clonal antibodies in a radioimmunoassay. The 17S mRNA frac-
tion produced a strong signal (Figure 1). It was used to prepare
a cDNA library in a pUC9-based plasmid vector. Since no protein
sequence was available initially, the cDNA library was screened
by hybrid-selection and translation in X. laevis oocytes. One clone
(pCL1) was identified which hybridized to mRNA coding for
FcER. DNA sequencing revealed that the cDNA insert ofpCL1
contained only 420 bp. It was used to prepare a radioactive DNA
probe in order to select by hybridization several additional clones
with longer cDNA inserts containing up to
Immunoglobulins of the IgE isotype are responsible for the im-
mediate hypersensitivity reactions that occur in diseases such as
hay fever, allergic asthma and anaphylaxis (Ishizaka and Ishizaka,
1967). As shown in animal models, the production of IgE anti-
bodies is regulated not only by antigen-specific mechanisms but
also by isotype-specific and non-antigen-specific mechanisms
(Ishizaka, 1985). The latter are mediated by lymphocytes capable
of expressing low-affinity receptors for the Fc region of IgE
(Fc,R)and secreting IgE-binding factors (IgE-BFs), which may
selectively potentiate or suppress IgE synthesis. Similar mech-
anisms have been identified in human IgE regulation. Particular
B-cell lines bearingFc,Rwere shown to produce IgE-BFs
capable ofmodulating the in vitro synthesis ofhuman IgE (Sarfati
et al., 1984a,b). In particular, FcER and IgE-BF expressed by
the human B-lymphoblast cell line RPMI 8866 are structurally
related proteins which share several antigenic determinants and
which have identical peptide fragments after proteolytic digestion
(Delespesse et al., 1986b; Suemura and Kishimoto, 1986).These
results strongly suggest that human IgE-BFsareprocessed prod-
ucts of FcER.
Several different monoclonal antibodies have been prepared
which react with FcERon the B-cell line RPMI 8866 (Rectoret
al., 1985). They have been used to identify, in the Nonidet-P40
lysates of RPMI 8866 cells, two main protein components with
IRL Press Limited, Oxford, England
1 5 20 25 30
Fig. 1. Sedimentation analysis of human FcERmRNA. Poly(A)mRNA
isolated from RPMI 8866 cells was centrifuged througha 5-23% sucrose
gradient. RNA from individual fractions was injectedinto X. laevisoocytes
and the synthesis ofFcER protein was measured in aradioimmunoassay.
rRNA from RPMI 8866 cells was centrifuged in a paralleltube as a size
C.Ludin et al.
40 h incubation at 20°C, the oocytes were homogenized and the cleared
homogenate assayed for Fc,R receptor in a radioimmunoassay, essentially as
described previously (Sarfati et al., 1986). Briefly, a 96-well microtiter plate was
coated with a first monoclonal antibody raised against theFc,Rreceptor protein
onto which the oocyte homogenate was adsorbed, followed finally by a second
1251-radiolabelled monoclonal antibody directed againstFc.R.Bound125I was
determined in a -y-counter.
Molecular cloning and DNA sequencing
2.8jigof single-stranded cDNA was synthesized from 614gmRNA of fraction
15 in Figure 1, elongated by dCMP residues and finally converted into double-
stranded cDNA by using oligo(dG)12-18 as primer for DNA polymerase
(Klenow fragment). The double-stranded cDNA was elongated with dCMP
residues and molecules longer than 1300 bp were selected by electrophoresis
through a1 % agarose gel. The size-selected cDNA was inserted into the PstI
site of the plasmid pUC-KO by the standard G/C-tailing method. pUC-KO is
a derivative of pUC9 wherein the lac promoter and operator have been deleted
between the HaeH
dIlI restriction site within the polylinker. After transformation of Escherichia coli
HB101 by the hybrid plasmids, -4500 colonies were screened by hybrid selec-
tion and frog oocyte injection (Nagata et al., 1980). Individual colonies were
grown to saturation in 2ml LB broth. Groups of 96 cultures were pooled and
the plasmid DNA was isolated. From each pool, 100 ,tg of alkali-denatured plasmid
DNA was bound covalently to APT cellulose and hybridized overnight with total
mRNA isolated from RPMI 8866 cells (Seed, 1982; Odink et al., 1984). The
hybridizedmRNA was eluted, twice precipitated with ethanol and injected into
X. laevis oocytes. The oocyte homogenates were tested forFc,Rin a radioim-
munoassay as described above. After screening of -50 pools, one pool gave
a positive signal. Its 96 colonies were divided into 12 new pools of eight col-
onies and rescreened. Finally, a single colony containing a Fc R cDNA was iden-
tified (clone pCLI). It was used to screen the remaining cDNA library by
hybridization. Several additional clones with longer cDNA inserts were found.
Their nucleotide sequence was detenmined by the dideoxy chain termination method
(Sanger et al., 1977) after subcloning of DNA restriction fragments into M13
bacteriophage derivatives mp8 and mp9.
Expression ofFc,RcDNA in CHO cells
A 1257-bp HinclIRsaI DNA fragment obtained from theFc,RcDNA (nucleotide
positions 82-1338) was inserted into the HincIl site within the polylinker of
plasmid pSP64 (Melton et al., 1984). This construction added a HindIll and a
BamHI site to the 5' and 3' end of theFc,RcDNA, respectively. Partial cleavage
with HindIll and complete cleavage with BamHI restriction endonucleases pro-
duced a 1284-bp DNA fragment which was inserted between the HindIII and
BgIII sites of plasmid pSVd, thereby yielding the expression plasmid pSVd-ER.
The plasmid pSVd was derived from plasmid pSVneo291
1986) by cutting with SmaI and Bglll restriction endonucleases and replacing the
released DNA fragment with a BglII-oligonucleotide linker. 10 jigof pSVd-ER
and1 jig ofpSV2neo plasmid DNA (Southern and Berg, 1982) were co-precipi-
tated with calcium phosphate and used to transfect sub-confluent CHO cells
(dhfr--mutant DXB-11; Urlaub and Chasin, 1980). The transformed cells were
maintained in a-MEM medium supplemented with 5% fetal calf serum, gen-
tamycin and 0.5 mg/ml G418. After 2 weeks, G418-resistant colonies grew out.
Several colonies were picked and enlarged individually. FcER was visualized by
successive incubation of glutaraldehyde-fixed cells with Fc R-specific monoclonal
antibodies (10 jig/ml)at 37°C for 1 h, and rabbit anti-mouse antibodies coupled
to peroxidase (Dakopatts P260; 1:50) at 37°C for 1 h. One positive colony was
selected and established as a permanent Fc R-bearing cell line. IgE-specific
rosettes were prepared essentially as described for Fc,R-positive lymphocytes
(Sarfati et al., 1984a; Yodoi and Ishizaka, 1980). Briefly, - 104 CHO cells were
seeded per well of a 96-well microtiter plate. After 2 days, the cells were wash-
ed and incubated for 2 hon ice with phosphate-buffered saline(PBS) supplemented
with 3% bovine serum albumin alone, or in addition supplemented with human
IgE (1 mg/ml) or Fc R-specific monoclonal antibodies (20jig/ml). Bovine
erythrocytes coated with human IgE were added and sedimented onto the CHO
cells by centrifugation at 200 g. After incubation on ice for 2 h, unbound
erythrocytes were removed by gentle washing.
Purification and N-terminal sequence analysis ofIgE-BF
Ten litres of culture supernatant from RPMI 8866 cells were filtered through
a 20 ml column of Affi-Gel 10 (Bio-Rad) coupled with Fc R-specific monoclonal
antibodies. After extensive washing with PBS, the bound material was eluted
with 50 ml 0.1 M glycine-HCl (pH 2.6). Fractions were collected in tubes con-
taining an equal volume of 1 M Tris-HCl (pH 8.0) and 0.5% Tween 20. IgE-BF-
containing fractions (as assayed by radioimmunoassay) were pooled, dialyzed
against 10 mM Tris-HCl (pH 7.4) and loaded on a SynChropak AX 300 anion-
exchange column (SynChrom Inc.). After washing the column with 10 mM
Tris-HCI (pH 7.4), the protein was eluted with a gradient of 0-1 M NaCI.
and assayed forFc,RmRNA by injection into X laevis oocytes. After
restriction site just upstream of the lac promoter and the Hin-
(Asselbergs et al.,
Fractionscontaining IgE-BFwere collected into 0.1% octylpyranoglucoside
(Sigma) and concentrated by lyophilization. Reversed-phase chromatography was
performed on a SynChropak RP-4 (SynChrom Inc.) column in 0.1 % trifluoroacetic
acid (TFA)/5% acetonitrile. IgE-BF was eluted by applying a gradient of 5-54%
acetonitrile in 0.1 % TFA during 30 min
were collected into 0.05% SDS and assayed for IgE-BF with the radioim-
munoassay. The purity of the material was analysed by SDS-polyacrylamide
gel electrophoresis (Laemmli, 1970). The N-terminal amino acid sequence was
analysed with a positive-phase protein sequencer model 470 (Applied Biosystems)
according to the method of Hunkapillar and Hood
at a flow-rate of 0.5mil/min. Fractions
We thank ourcolleagues Joseph Briggen, Karel Odink, Fred Asselbergs, Norman
Hardman and Jui YoaChangfor their support and advice; Hans Rink for the
initial finding of the protein sequence homology; Rene Knecht for the analysis
of the amino terminus of the IgE-BF; and Doris Ruegg, Elisabeth Edelmann,
Colette Kristofic and Silvie Antz for excellent technical assistance. M.S. is the
holder of an MRC postdoctoral fellowship.
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Received on 20 October 1986