Expression of the murine interleukin-5 receptor on Xenopus laevis oocytes

Vol. 172, No. 2, 1990

October 30, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 570-575

EXPRESSION OF THE MURINE INTERLEUKIN-5 RECEPTOR ON XENOPUS LAEVIS OOCYTES

RENE DEVOS, JAN TAVERNIER, GEERT PLAETINCK, JOSE VAN DER HEYDEN, ANTONIUS ROLINK*-x and WALTER FIERS*

ROCHE RESEARCH GENT, J. Plateaustraat 22, B-9000 GHENT, BELGIUM

-2% BASEL INSTITUTE FOR IMMUNOLOGY, CH-4005 BASEL, SWITZERLAND

4:. STATE UNIVERSITY GHENT, Laboratory for Molecular Biology, K.L. Ledeganckstraat 35, B-9000 GHENT, BELGIUM

Received August 24, 1990

In this study we describe the use of Xenopus laevis oocytes for the detection of mRNA coding for a murine interleukin-5 (mIl$) receptor. When injected with sucrose gradient fractionated polyA RNA derived from the murine Il&dependent pre B cell line B13, these oocytes could specifically bind S-methionine labeled mI15. A sise of approximately 4000 nucleotides (255) was estimated for the mRNA corresponding to the mIL5-binding activity. This binding was not blocked by a monoclonal antibody R52 specific for the mI15-receptor, suggesting that the oocytes express a different form of this receptor. 0 1990 Rcadrmlc FWSS. 1°C

Interleukin-5 is a 45.000 dalton homodimer glycoprotein produced by antigen

or mitogen activated T lymphocytes, for review see (1). In the murine

system it is synthesized by TH2 cell clones (2). cDNA encoding mI15 has

been isolated on the basis of its B lymphocyte proliferation and

differentiation activity (3), and its IgA enhancing activity (4). I15 also

induces cytotoxic T lymphocytes (5) and enhances 112-mediated

lymphokine-activated killer activity (6). Beside its biological activities

on B and T lymphocytes, mI15 also stimulates the differentiation of

eosinophil precursor cells in bone marrow cultures from parasite infected

mice (7). This property is of primary importance since it can be

reproduced in the human system using either bone marrow or cord blood as a

source of eosinophil precursor cells (8), while hI15 had little effect on

human B cell responses (9) . Moreover it was recently shown that a

monoclonal antibody recognizing m115 blocks the hypereosinophilia in

parasite infected mice (10).

115 exerts its biological function via cell-surface receptor proteins.

Cross-linking of radiolabeled mI15 to cell surface proteins revealed that two polypeptides with molecular masses 45.000 dalton (a -chain) and 130.000

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved. 570

Vol. 172. No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dalton (p-chain) comprise the receptor (11). These results suggested that

the two classes of binding sites with kd of approximately 10 nM (low

affinity) and 60 pM (high affinity) identified on mI15-dependent cell

lines, correspond to a receptor comprised of the (a-chain) 45.000 dalton (mI15-binding) polypeptide and the (fl -chain) 130.000 dalton (signal

transducing) or a complex of both polypeptides respectively. A monoclonal antibody R52, to the mI15-receptor, which inhibit the binding of mI15 to

the high affinity receptor also inhibits the mI15-dependent proliferation

(12). This antibody specifically immunoprecipitates the 130.000 dalton and

to a lesser extent the 45.000 dalton polypeptides from 35 S-methionine labeled or surface labeled mI15-dependent cells.

Oocytes have been used for expression of neurotransmittors (13, 14, 15,

16), ion channels (17, 18), Fc-receptors (19), the low density lipoprotein

receptor (20), the C3b/C4b receptor (21) and recently the mIFNY receptor

(22). In this study, we show that a mI15-binding activity can be expressed

on the surface of Xenopus laevis oocytes injected with mRNA derived from mI15-dependent cells.

MATERIALS AND METHODS

Production and radiolabeling of recombinant mI15 mI15 was produced by injection i.n Xenopus laevis oocytes of SP6-RNA polymerase-transcripts made using a pSP64T-mI15 plasmid DNA construction (23). After 3 days incubation at 18~, the supernatants derived from batches of 5 oocytes in 50 pl were pooled, centrifug

"51- and stored at -20°C

until further '5"".

Such preparations yielded to 2x10 units/ml of mI15 as measured by radiolabeling, 5 ~1 of

H-thymid~3~-m~~~~~~~~~t~~~ c$, ~~~~ha~e~~lsOl:p?&e adz::

at the beginning of the incubation period after which the supernatants were collected (0.5ml) lyophilized to half its original volume and passed over a 2ml Sephadex-G25 column, equilibrated in binding medium (Iscove modified Dulbecco's medium, 5% S~~~t;;~~i,s.rum, 25 pg/ml gentamycine sulfate) to remove unincorporated . The void fractions were pooled and aliquots analyzed by SDS-polyacrylamide gele on a 15% gel. Fig. 1 shows an autoradiogram of different

&ctrophoresis S-labeled mI15 preparations

electrophoresed in the 3P

ence and presence of 2-mercaptoethanol. The specific activity of the pCi/pg -

S-m115 was calculated to be between 100 and 300 Unlabeled recombinant mI15 which was used for growing the Bl3

cells and for competition experiments was produced in a Baculovirus expression system (23).

Isolation and fractionation of RNA from B13 cells B13 cells were grown in roller bottles (IMDM, 5% heat inactivated foet 1 calf serum, 25 pg/ml gentamycine sulfate, 0.03% L-glutamine, 5-10 -3 M 2-mercaptoethanol) in the presence of 100 U/ml recombinant mI15. Total RNA was isolated u+sing guanidinium thiocyanate according to Chirgwin et al. (24) and polyA RNA selected by two chromatographic steps on oligo dT cellulose (Collaborative Research, type 3). Sucrose gradients were prepared in 10 mM Tris HCl (pH7.5)-1mM EDTA by sequentially adding 2.75ml sucrose solutions (40%, 30%, 20%, Beckman Instruments)

and 10%) in Ultra-Clear tubes (14x89 mm, and freezing in liquid nitrogen. Gradients were

571

Vol. 172, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

thawed at 4OC, loaded with O.Zml polyA' RNA and spun, at 40.000 rpm in a SW41 rotor at 4W for 16 hours. Fractions (0.4ml) were collected with an ISCO gradient fractionator and RNA precipitated with ethanol and one tenth volume 3M NaAc pH5.5. Xenopus laevis oocytes.

RNA was taken up in 25 ~1 water for injections into

Microinjection of RNA into oocytes and binding of 35S-m115 Collagenase treated lh room temp., oocytes were injected with days at 180~.

For ,1,,, 55"l p01yA~"~g (clor$$na::d zub::dd%:; S-mI15, a group of ten healt8y ooc,tes were

selected and incubated in O.lml binding medium with 2.10 cpm S -mI15 for 90 min at room temperature. Every 15 minutes the oocytes were suspended very gently by rocking the tubes, after which the oocytes were washed four times with 1,5 ml binding medium. Each oocyte was then transferred individually to a 0.65d tube, excess medium removed and 30~1 1% SDS was added. After disruption of the oocyte in a sonicator bath, 300 ~1 Ready-solv. HP/b (Beckman Instruments) was added and the radioactivity measured in a Beckman ~~2800 Scintillation counter.

RESULTS AND DISCUSSION

m115 radiolabeled to a high specific radioactivity ( > 100 ,@i,$ug) with

retention of its dimeric structure is a prerequisite for testing whether

the receptor for mI15 can be detected on the surface of Xenopus laevis

oocytes, injected with mRNA derived from a mI15-dependent cell line. Since

m115 contains ten methionine residues, five in each chain of the disulfide

linked homodimer, we reasoned that biosynthetic labeled mI15 could be used

for this purpose. X. laevis oocytes were therefore used for both the

production of the ligand 35S-mI15 (Fig. 1) and expression of the

mI15-binding activity. Polyadenylated RNA was isolated from a

mI15-dependent murine pre B cell line B13 (25), fractionated on sucrose

-1.6

-JO

Fig. 1. SDS-polyacrylamide (15%) gel electrophoresis of 35S-m115 produced b X. laevis oocytes. SP6-RNA polymerase transcripts from plasmid pzP64T-mIl5 DNA (23) were injected in oocytes derived from

After incubation for 3 days in the presence of

non-reducing (a) the oocyte supernatants were elec;ro;;i$sed under

and reducing (b) conditions. protein molecular weight markers (Amersham, rainbdw,

C-labeled low molecular

weight range).

572


a b c d e

fraction no

Fig. 2. Expression of mI15-binding activity on oocytes injected with mRNA from B13 cells fractionated by sucrose gradient centrifugation. ggd o;"Yf es were incubated for two days after which the

S-m115 was measured as described in materials and methods. The histogram is the average of 10 oocytes.

Fig. 3. Competition for binding 35 S-m115 on injected oocytes by an excess of unlabeled ml15 (c) and lack of inhibition of mI15-binding activity by monoclonal antibody R52 (d and c). Uninjected oocytes ‘(a) and oocytes injected with fraction 19 + 20 (b, c, d and e) from sucrose gradient fractionated B13 mRNA (similar as in incubated for 4 days after which the binding of 3f;-;;j5 ";,'; measured alone (a and b) or in the presence of 1.5mg/ml unlabeled m115 (c) or in the presence of 50 pg/ml R52 (d) or 160 pg/ml R52 (e). The histogram is the average of 10 oocytes with standard deviation.

gradients and microinjected into oocytes. After two days at 180~ the

binding activity was measured by incubating groups of 10 oocytes for 90

min. at room temperature with 1 nM 35S-m115 (2~10~ cpm). These conditions

of binding were necessary to detect both the high and low affinity mI15

receptors known to be present on mIl$dependent murine B cell lines (11).

Due to occasional leaking of some oocytes and therefore non-specific

retention of label ( ) 1000 cpm), each oocyte had to be counted

individually. The experiment presented in Fig. 2 shows that oocytes

injected with mRNA corresponding to a size of approximately 4000

nucleotides (25s) bound two times more label than control oocytes.

Furthermore, this binding is specific since it was blocked by addition of

cold m115 (Fig.3). Longer incubation of the oocytes increased the specific

binding of mI15 which could still be detected 9 days after injection of

mRNA (Table I). From the specific radioactivity of the 35S-mI15, we

calculated the number of m115 molecules bound per oocyte tc be 1.2 x 107.

This number is of the same order of magnitude as the number of other receptors expressed on oocytes after injection of mRNA (19, 22).

Since cross-linking of m115 bound on mI15-dependent cells demonstrated that

the mI15 molecule interacts with 45.000- and 130.000 dalton surface proteins (11, and our unpublished results), the question arises which of

573


Table I. Kinetics of expression of mIl$binding activity on X. laevis oocytes

35S-m115 bound

days incubation after injection

2

5

9

cpm oocyte Lcpm/oocyte

Mock-injected Injected with mRNA

93.8 _c 21.7 196.6 + 44.0 102.8 + 65.7

145.5 _+ 14.8 332.1 z 67.4 186.6 k 82.2

55.5 + 13.4 350.3 - + 100.4 294.8 + 113.8

The oocytes used were either mock-injected or injected with fraction 19 from sucrose gradient fractionated B13 RNA (Fig. 2). The values shown in the table are the average of 10 oocytes with standard deviation.

these molecules is expressed on the surface of X.laevis oocytes injected

with mRNA derived from B13 cells, and which is responsible for binding

mI15. Efforts in cross-linking 35 S-m115 onto injected oocytes however

failed to demonstrate a band after gel electrophoresis. This is not

surprising seen the very low efficiency of cross-linking mI15 on the

mI15-receptor. Fig. 3 shows that the binding of 35S-m115 on injected

oocytes is not blocked by a monoclonal antibody R52. This antibody

recognizes the high affinity mI15-receptor and precipitates a 130.000

dalton protein (b-chain) from surface labeled B13 cells (12, and our

unpublished results). This result suggests that the oocytes most probably express the 45.000 dalton protein (a-chain), and that perhaps this molecule

correspond to the most abundant, low affinity mI15-binding receptor. The

availability of monoclonal antibodies directed towards the 45.000 dalton

chain or cDNA recombinants corresponding to the mI15-binding activity will

allow further characterization of the mI15-receptor expressed on oocytes.

Acknowledgments

We wish to thank R. Bauden, T. Tuypens, S. Dewaele and I. Fache for their

skillful technical assistance, Dr. R. Palacios for the generous gift of the

B13 cell line and C. Vermeire for her secretarial assistance.

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Expression of the murine interleukin-5 receptor on Xenopus laevis oocytes