Purification and characterization of biologically active human recombinant 37 kDa soluble CD23 (sFcεRII) expressed in insect cells

Journal ofhnmum+,l,'.,gical ~h'thod.~. 149 (lt)t~2)215-221~

215

:?) Iq92 ElsevierScience PublishersB.V. All rights rcscrvett1)1122-17511/q2/$1151)11

JIM ()b273

Purification and characterization of biologically active human recombinant 37 kDa soluble CD23 (sFceRII) expressed in insect cells Pierre G r a b e r , Kathrin J a n s e n , Sibylle P o c h o n , J o h n Shields, Nicole A u b o n n c y , G e r a r d o Turcatti and J e a n - Y v e s B o n n c f o y Glaro bz.~titutc for Moh,ctdar Ihoh~gq" S.A.. 1227¢Phm h,~-O,utc~ / (;cnct a. Swttzcrhmd

(Received t) October 1991, revised received25 Nt)vcm~cr 19~Jl.acccplcd 2 December 1991) Human recombinant soluble 37 kDa CD23 has been expressed in insect cells and secreted into the culture medium using the IL-2 leader sequence. The 37 kDa CD23 was purified 60()-fold to homogeneity by monoclonal antibody affinity chromatography and gel fihration. The pure protein is monomeric. glycosylated, dep'. -ted of one N terminal amino acid and contains four disulphide bonds. It degrades into smaller fragments of 33, 29 and 25 kDa if purified in the absence of protease inhibitors. The same pattern of proteolytic fragments is observed when the pure preparation is incubated at room temperature for 3 weeks. Physical characterization of the 37 kDa CD23 by ctrcular dichroism indicates that the protein contains mainly ~ sheet and 20% of a helical structures. Specific binding of IgE to natural CD23 (low affinity lgE receptor) was inhibited by purified recombinant 37 kDa CD23. Moreover, purified recombinant 37kDa CD23 and intcrleukin-I promoted the survival of germinal centre B cells. Key word.~: CD23 (Fc~RII); Baeulovirus; Apoptosis;IgE

Introduction The CD23 antigen (the low affinity receptor for lgE) (Bonnefoy et al., 1987; Yukawa et al.,

Correspondence to: J+-Y. Bonnefoy, Glaxo Institute fl~r Molecular Biology S.A, 14. (_'hemin des Aulx, Case Postale 674, 1228 Plan-les-Ouutcs/Geneva.Switzerland(Tel.: 22-711b 96 66; Fax: 22 794 69 65). Abbrectatiom: BSA, bovine serum albumin; CD, circular dichroism; DTNB, 5,5'-dithiobis(2-nitrobcnzoicacid): D'VI'. 1,4-dithiotiJreitol: GuHCI, guanidinium chloride: FCS, foetal calf serum; mAb, monoclomd antibody; NTSB. disodium 2nitro-5-thiosulphobcnzoute;PBS, phosphate buftk:rcd salinez PMSF, phenylmethylsulphonylfluoride; PT|l-aa. phcnylthiuhydantoin amino acid; TLCK. Na-losyl-t-lysyl-chloromethanhydrochloride: IL, interleukin; ClIO cells, Chinese hamster ovary cells.

1987), is found on a variety of hemopoietic cells including T and B lymphocytes (Gonzalez-Molina et al., 1976; Spiegelberg, 1981), monocytes (Melewicz el al., 1982), eosinophils (Capron et al., 1981) and platelets (Joseph eta[., 1983). The eDNA coding for CD23 has recen6y i+cen cloned from human B cells by several laboratories and has been expressed in a variety of mammalian cell lines (Kikutani ct al., 1986; lkuta et al., 1987; Liidin c t a l . , 1987). The polypeptide encoded by the eDNA is an integral membrane glycoprotein with an apparent molecular weight of 45 kDa. CD23 shows an inverted membrane orientation where the amino terminus is intracytoplasmically located and the carboxyterminus is cxtracellular (for recent reviews, see Delcspesse c t a l . , 1989; Gordon ct al., 1989). The IgE-binding region is

216 confincd to the carboxy-terminal domain (Bottler et al., 1989) which shows substantial homology with several Ca2~-dependcnt animal lectins (Kikutani et al., 1986; lkuta ctal., 1987; Liidin et al., 1987) although no lectin aetivily has yet been shown for CD23. Membrane CD23 undergoes further processing to yield a variety of soluble fragments with molecular weights ranging from 37 to 12 kDa (Delcspcssc ctal., 1989). Studies with natural or recombinant material suggest that CD23 a n d / o r its soluble fragments have several roles, including modulation of IgE synthesis (P~:ne et al., 1988; Sarfati et al., 1988), mediation of lgE-dependent cytotoxicity against parasites (Capron et al., 1986), IgE-dependent antigen focusing (Kehry et al., 1989), B lymphocyte proliferation (Gordon et al., 1984; Swendeman ct al., 19d7; Jansen et al., 1991a) and germinal centre B cell survival (Liu et al., 1991). Since baculovirus infected Spodoptera fn,giperda (Sf 9) cells are able to perform many eukaryotic post-translational modifications (including glycosylation), we expected that they would produce recombinant 37 kDa CD23 antigenically and functionally similar to its authentic counterpar:. For the same reasons, this system was previously chosen successfully for the expression of the full length membrane CD23 (Jansen et al., 1991a). In order to understand better the mode of action of CD23/soluble CD23 and the generation of proteolytic fragments, workable quantities were obtained using the baculovirus expression system with thc aim of undertaking functional studies with biochemically characterized 37 kDa CD23. We report the production of recombinant 37 kDa soluble CD23 in insect cells, its purification to homogeneity, its physical characteristics, its ability to bind lgE and to promote with interleukin-I the survival of germinal centre B cells.

Materials and methods

Reagents Protein standards for gel filtration and electrofocusing, Superdex 75 HiLoad 16/60, SDS gels

8-25% and tEF gels 3-9 for the Phast system were from Pharmacia (Uppsala, Sweden). Ultrogel AcA44 was from 1BF Biotechnics (VilleneuveLa-Garenne, France). Affi-Gel 10-activated affinity support was from Bio-Rad Laboratories (Glattbrugg, Switzerland). The glycan detection kit was obtained from Boehringer (Mannheim, Germany).

Antibodies, cell lines The anti-CD23 monoclonal antibody EBVCS #1 (lgGl) was a generous gift of Dr. B. Sudgen (Kintner et al., 1981). The anti-CD23 monoclonal antibody Mab25 (Bonnefoy et al., 1987) (IOB8, lgG1) was purchased from lmmunotech (Luminy, France) and coupled to Affi-Gel-10 (Bio-Rad) at 3 m g / m l resin using the manufacturer's instructions. Human IgE myeloma protein was obtained from The Binding Site (Birmingham, UK) and was purified as described by Bonnefoy et al. (1986). Polyclonal FITC-labelled goat anti-human IgE and peroxidase-coupled goat anti-mouse lgG were purchased from Sigma (St. Louis, USA). The polyclonal antibody Rb33 was obtained by immunisation of a rabbit with the CD23 decapeptide (position 248-258 of the CD23 sequence) cross-linked to bovine serum albumin (BSA) by glutaraldehyde. The Epsteia-Barr virus (EBV)transformed human lymphoblastoid cell line RPMi 8866 was obtained from Dr. K. ishizaka (Baltimore, USA) and was cultured in complete medium consisting of RPM1 1640 (Seromed, Berlin, Germany) supplemented with 10% heatinactivated FCS and 2 mM L-glutamine.

Protein purification The 37 kDa CD23 was obtained from Sf9 cell cultures infected with recombinant baculovirus clone F4 as described by Jansen et al. (1991b). All the chromatography steps were carried out at 4°C and monitored by SDS-PAGE and immunoblotting. Ammonium sulphate (453 g/I) was added slowly to 4 iitres of infected insect cell supernatants corresponding to 71.9% saturation at 0°C and stirred for 1 h. The precipitate was removed by centrifugation at 10,000 x g for 30 min using a GSA rotor (Sorvall) and dissolved in 50 ml PBS (140 mM NaCI, 3 mM KCI. 8 mM Na2HPO 4, 1.5

217 mM KH2PO 4) supplemented with 0.1 M NaCI, 2 mM PMSF, 0.4 mM TLCK, 40 mM iodoacetamide. 10 mM benzamidine-HCI, 2 mM CaCI2, 2 mM MgCl z, oH 7.1; sonicated for 10 s and centrifuged at 130,000 × g for I10 rain using a Beckman Ti 50.2 rotor. The supernatant (100 ml) was then filtered through GS 0.22/zm filter (Millipore, Bedford, USA) to remove the non-pelleted particulate material and applied in batches of 25 ml to an UItrogei AcA44 column (95 cm × 2.5 cm diameter) equilibrated in PBS, 0.1 M NaCI, pH 7.1, The monomcric 37 kDa sCD23 was identified by immunoblotting as the fraction eluting after the albumin peak. Positive fractions were pooled (250 ml) and 0.5 mM TLCK, 10 mM iodoacetamide, ! mM PMSF and benzamidine-HCI were added. The AcA44 pool was applied at 50 m l / h with constant recycling for 15 h to the 20 ml Mab25-Affi-Gel 10 column pre-ehtted with 0.2 M glycine-HCI, pH 3.0 and equilibrated in PBS, 0.1 M NaCl, pH 7.1. The immunoaffinity resin was washed with 40 ml PBS, 0.1 M NaCI followed by a 600 ml wash of PBS, 0.5 M NaCI, pH 7.1 to remove material which had bound non specifically. 37 kDa CD23 was eluted with 50 ml of PBS, 0.1 M NaCI, 3.0 M NH4SCN, pH 6.5. The eluate was concentrated to 3 ml on a Centriprep 10 concentrator unit (Amicon) and gel filtration was performed at 60 m i / h using a Superdex 75 column (16/60) equilibrated in PBS, pH 7.1. Prior to protein sequencing, small aliquots (5(1 tzg) were further purified by RP-HPLC on an Aquapore RP-300 C4 (30 cm × 2.1 mm diameter) column ABI (Applied Biosystems, Foster City,

USA) equilibrated in 0.1% ( w / v ) trifluoroacetic acid (TFA) in water (solvent A). Samples in PBS were diluted with 2 vols. of solvent A before injection. The protein was eluted with a gradient of 0 - 7 2 % (v/v) acetonitrile in (I.1% (w/v) T F A / H 20.

Analytical separation methods SDS-PAGE (8-25% gradient gels) and isoelectric focusing (pH 3-9) was performed using the PHAST System (Pharmacia) anti the gels were silver stained according to the manufacturer's instructions. Densitomctric scanning of silver-stained gels was performed on a Chromoscan 3 densitometer (Joyce-Loebl).

lmmunoblotting analysis SDS-PAGE (12.5%) was carried out using the Bio-Rad Mini-Protean li Dual slab cell system. The proteins were electrophoretically transferred onto a nitrocellulose membrane. The non-specific binding sites on the membrane were blocked in PBS, 0.1% ( w / v ) Triton XI00, 1% ( w / v ) casein, I% ( w / v ) gelatin then labelled according to the method of Towbin et al. (19791 with a 1/200 dilution of the Rb33 antiserum. The binding of the first antibody was revealed with peroxidasclabelled goat anti-rabbit lgG at 2 p,g/ml (KPi., Gaithersburg, 'riD, USA) and developed with 0.5% ( w / v ) 3-amino-9-ethylcarbazole (Sigma) in acetate buffer, 0.014% ( v / v ) hydrogen peroxide.

Analysis of amino acid comlrositiot~ The recombinant 37 kDa CD23 (0.3 rag) in 0.1 M phosphate buffer, pH 7.5 was denatured using 6 M GuHCI, reduced and carboxymethylated us-

TABLE I PURIFICATION OF THE RECOMBINANT 37 kDa CD23 Fraction Culture medium (NFI4)2SO,, precipitate Ultrogel Aca44(l~}oledfractions) Mab25-affinity(pooled fractions) Superdex 75 (pooled fractions)

Volume (ml) 3,4011 50 25(1

30 9

Totalprof, (mg)" 5,78(1.1 1,2611.5 1,162.5 1.3 11.7

Total activity

Specificactiviq.

( U I t,

(U//J.g)

1.16x 9.40 X 7.711X l.(lO X 8.40 X

1117 I Or' 1116 1116 IO s

Protein concentrations ',~erc determined using the BCA protein assay (Pierce). h Total activitywas determ!ned by EL1SA for sCD23.

2.(11 7.114 6.62 769.{10 12011.01)

PuriScatkm Yield 1%) IAIO 3.51) 3.30 382.61) 597.00

I(XI~ 141 66c,~,

9~ 7c/r

2h"; T A B L E II A M I N { ) A ( ' I I ) ANAI.YSIS OF R [ - ( ' O M B I N A N T ('I)23

37 kDa

The composilion was estimated from 24 h acid hydrolysates and is the ;P,¢r;IblCo| three eMimzlliOlls.The Trp content wits estimated by tlllraviolcl absorption spectroscopy. The cyslcine content was eslimilled as earhoxymcthyl-cystcinc(C'm(.'ys)derived from protein alkylatcd with iodoacctamidc. The theoretical •cables arc those predicted from the DNA sequence. Amino a c i d ('m('ys A,,x Thr Ser (.ilx Pro Gly Ala Val Me! IIc Lcu Tyr Phe

('omp~sition (residues/tool) 37 kDa ('I)23 Thcorclical 7.8 I 25.15 25 1(I.14 10 21.40 26 34.59 35 12.28 12 18.28 17 14.15 13 0.90 9 4.02 5 4.70 5 23.18 23 3.80 4 &h5 7

tlis I -t,'~,

f~.Oi I 1.3b;

I

A~g Trp

15.53 8.114

16 8

c'~

ing iodoacetamide (Schrimsher et al., 19871. The sample was then desaltcd on Sephadcx G-25 equilibrated in 511 mM NH4HCO.~. Aliquots of the carboxymethylated material were then hydrolysed in 6 M HCI (Piercc) at I 1 I°C Coven temperature) for 22 h and the hydrolysates analysed using a Beckman 6300 amino acid analyser (ninhydrin detection). The values reported are averages of three separate determinations (Table !1). Tryptophan content was d e t e r m i n e d by absorbance at 28(1 nm in the prescnce of 6 M GuHCI as described by Edelhoch (19671.

N terminal amitm acid sequence determination by Edman degradation Samples were loaded onto an ABI 477A pulsed-liquid sequencer equipped with an on-line PTH analyser model 110A (ABI). PTH-aa derivatives were separated on a PTH-C18 column, 5 mm, 220 × 2.1ram (ABI). A T F A activated glass

fibrc filter was coated with polybrenc and precycled in the sequencer. Proteins were sequenced using a standard program.

Thiol and disulphide analysis Sulphydryl groups were measured in 1% ( w / v ) sodium dodecyl sulphate using D T N B (EIIman, 19501. Disulphide bonds were quantitatively determined using NTSB in 6 M guanidine-HCI (Pierce) ( T h a n n h a u s e r et al., 19841.

Glycosylation Denatured, reduced and carboxymethylated 37 kDa CD23 was desalted as described a0ove in 0.1 M sodium acetate, 5 mM CaCI 2, I m M PMSF, 1).2 mM TLCK, pH 6.5. 69 tzg aliquots were deglycosylated for 17 h at 37°C using either Oglycanase ( e n d o - a - N - a c e t y l g a l a c t o s a m i n i d a s e ) from Diplococcu,~ pneumoniae at 0.5 U / m g or N-glycosidase-F (peptide-N-glycosidase F) from Flewobacterium meningosepticum at 50 U / m g (Boehringer). Some aliquots were previously desialylated by t r e a t m e n t with 1 U / m g neuraminidase from Streptococcus (Genzyme, U S A ) for 7 h at 37°C to allow O-glycanase action. Digestion was s t o p p e d by boiling the samples in SDS sample buffer. 2.5/.tg of 37 kDa CD23 were loaded o n t o each lane o f two 12.5% acrylamide gels. O n e gel was stained with Coomassie blue and the o t h e r was transferred c,nto a nitrocellulose m e m b r a n e and stained for glycans using the glycan detection kit (Boehringer).

Protein deterraination Protein concentrations in crude extracts were d e t e r m i n e d using the BCA protein assay (Pierce) using BSA as a standard. T h e concentration of pure 37 kDa CD23 was d e t e r m i n e d from the ultraviolet absorbance spectrum using the extinction coefficient at 280 nm: A~;cm = 18.8. This coefficient was calculated from the translated sequence starting at K82, containing four disulphide b o n d s (Gill et al., 19891.

Circular dichroism Circular dichroism spectra were measured using a Jasco J-600 spectropolarimeter. T h e spectra p r e s e n t e d are averages of four scans with the base line subtracted. All solutions were filtered

21(1 through 0.22 ~ m Millex-GV filter (Milliporc) before use. Spectra werc recorded at 20°C. The mean residue molecular weight was calculated as 113.2 Da.

ELISA for the determination of sCD23 All incubation steps were carried out at 4°C followed by extensive washing of the wells with PBS containing 0.(15% ( w / v ) Tween 20. t,)6 well ELISA plates were coated overnight with 50 ~ l / w e l l of mAb EBVCS#1 at a concentration of 5 ,u.g/ml of PBS. After saturation with 20 m g / m l BSA for 4 h, the plates were incubated with serial dilutions of samples for 16 h. Alkaline phosphatase conjugated Mab 25 ( l(I ,u.g/ml) was added and incubation c~>~tinued for 4 h. Soluble CD23 was detected by enzymatic hydrolysis of pnitrophenylphosphate monitored by the increase in absorbance at 405 nm. The lower detection limit of this assay was 0.2 n g / m l .

Inhibition of lgE bh:ding to membrane CD23 measured by ]7ow cw ")metric analysis Labelling of RPMI 8866 cells (constitutively expressing CD23) way carried out essentially as described by Bonnefoy et al. (1990) with minor modifications. Briefly, samples of 37 kDa CD23 were tested for IgE binding activity by incubation with lgE (7 p.g/ml) for 30 min at 4°C before addition of this mixture to 100 p.I of RPM1 8866 cells (1(I 7 cells/ml) for 20 min. Cells were washed three times and bound lgE was visualized with 50 p.I F1TC-goat anti-human IgE (I/3(10) (Sigma, St. Louis, USA). Fluorescence analysis was performed with a flow cytometer (FACSCAN, Becton Dickinson, Mountain View, CA).

Apoptosis assay Germinal centre B cells wcrc prepared from tonsil mononuclear cells by a protocol similar to that of Liu et al. (1991). Briefly, T cell depleted tonsil B cells were separated into light and heavy cells on a Percoll density gradient. The light B cells were enriched for gcrmir-d centre B cclls by depleting the lgD a n d / o r CD39 positive cells using monoclonal antibodies and immunomagnetic beads (Dynal, Oslo). Cells (2 × 105) were incubated in 200 p.l of RPMI 1640 supplemcnted with 2 mM l.-Glu, gentamicin (50 /.tg/ml) and

10% FCS for 16 h at 37°C with and without the rescuc factors anti-CD40 (5 # g / r o l l (Serotcc, UK) or 37 kDa CD23 ( 100 n g / m l ) plus IL-I ( 1 ng/ml). As control the .same PBS buffer used for the sCD23 preparation was tested with IL-1. Apoptosix was asse:iscd by examination of Giemsa-staincd cytocentrifuge preparations and at least 3(XI cells were counted for each condition.

Results

Purification of tt.e recombinant 37 kDa CD23 Sf9 insect cclls infected with the recombinant baculovirus clor.e F4 secrete the 37 kDa CD23 into the cell culture medium. The major advantages of this secretion system arc the limited contact of the expressed protein with intracellular proteases and the lack of a cell extraction procedure. The disadvantage is the presence of a high concentration of albumin, due to the 10% FCS added to the culture medium since these conditions are required for a good overall growth of the Sf9 cells and good expression of soluble proteins. After concentration with ammonium sulphate, the resulting precipitate was re-dissolved in PBS containing a cocktail of protease mhibitors. Omission of protease inhibitors from this and subsequent purification steps resulted in rapid degradation of the 37 kDa CD23 into smaller fragmcms. Gel filtration using AcA44 permits isolation of monomeric and desalted material which was found (immunoblotting) in the trailing edge of the albumin peak. Further purification of "~7 kDa CD23 monomer on an immunoaffinity column removed most of the contaminating proteins (Figs. IA and IB), resulting in a preparation of 37 kDa CD23 which was 62% purc as assessed by densitometric scanning of a silver stained SDS-PAGE gel, giving a I I0-fold purification factor (Table 1). lmmunoaffinity direct purification as step one resulted in BSA contaminated 37 kDa CD23. The final Superdex 75 gel filtration step removed the last traces of albumin and separated the 37 kDa from other protcolytic fragmcnts yielding 96% pure material a:~ cstimatcd by gcl dcnsitometry measurements of silvcr stained SDS-PAGE gels and RP-HPLC. The

221) -3

Mr x 10

110

-

84

-

47

-

33

-

24

-

16

-

Std

1

2

3

4

5

6

1

2

3

4

5

6

hg. 1. SI)S-PAGE and immunoblotting analysis of 37 kDa. CD23 aliquots from different steps of the purification were subjected to electro'~horesis on a 12.5c; (w/v) polyacrylamide gel and stained with Coomas~ie blue (A) or transferred onto nitrocellulose membrane and immunoslai~,:d with Rb33 polyclonal antibody (materials and methods section) (B). Std, prestained molecular v.eight standards (Bio-Rad); lane I, recombinant Sf9 cell culture medium; lane 2, (NHa),SO 4 insoluble concentrate (discarded); lane 3. (NHaI,SOa soluble concentrate; lane 4, Ultrogel AcA44 fractions; lane 5, mAb25 immunoaffinity fractions after elution with 3 M NHaSCN; lane 6. Superdex 75 final pool. four step purification p r o c e d u r e resulted in a 600-fold purification with a final yield of 7%. T h i s poor yield can be justified by o u r c o n c e r n for the level of purity required for biological assays.

Molecular mass attd charge S D S - P A G E of the 37 k D a C D 2 3 p r e t r e a t e d with DTI" s h o w e d a diffuse b a n d of a p p a r e n t molecular m a s s e s t i m a t e d at 34 + 2 k D a comp a r e d with the value of 29.1 k D a predicted from ',he c D N A s e q u e n c e (Kikutani et al., 1986; l k u t a et al., 1987; Lfidin et al., 1987). U n d e r n o a - r e d u c -

ing conditions t h e protein gave a diffuse b a n d on S D S - P A G E at 29 + 2 k D a indicating the presence of reducible disulphide bridges. By gel filtration on S u p e r d e x 75, t h e m o l e c u l a r m a s s was calculated as 39 kDa. O n native P A G E (data not shown) t h e r e c o m b i n a n t 37 k D a C D 2 3 c a m e o u t as a diffuse b a n d b e t w e e n 33 k D a a n d 40 kDa. T h e calculated p l from t h e a m i n o acid seq u e n c e was f o u n d to be 4.92 u s i n g t h e P C / G E N E C H A R G P R O p r o g r a m (IntelliGenetics, Genofit, Geneva). A p u r e p r e p a r a t i o n r u n as multiple b a n d s on isoelectric focusing having p l values r a n g i n g from 4.7 to p / 5 . 3 ( d a t a not shown). All

Fig.. 2 Glycosylation. 2.5 pg of each 37 kDa CD23 de.~lycosylated sample were run on a 12.5 % (w/v) polyacrylamide gel, stained for proteins by Coomassie blue ( A ) or Iransierre.d onto nitrocellulose membrane and stained for glycans (B) (see material and methods section). Lane a, undigested 3"/ kDa CD23 in deglycosylation buffer incubated for I day at 37°C; lane b. digestion with neuraminidase: lane c. digestion with neurarninidase and O-glycanase; lane d, digestion with neuraminidase and N-glycosidase F; lane ,:. digestion with N-glycosidase F. Std, prestained molecular weight standard (Bio-Rad). The arrows I and 2 indicate the high and low molecular weight bands respectively; band 1 is still glycosylated, band 2 is completely deglycosylated. The arrow 3 indicates the presence of deglycosylated non reducible dimers.

x o

o

DL.

O"

|

o

x

o

222 these results are consistent with a monomerJc protein structure containing intra-chain disulphide bonds and various degrees of glycosylation.

Glycosvlation The recombinant 37 kDa CD23 was shown to bc glycosylated by the method of digoxigcnin labelled glyconconjugates detected after transfer to it nitrocellulose membrane by an enzymatic immunoassay. Partial deglycosylation was obtained when the 37 kDa sample was first treated with neuraminidase followed by O-glycanase (Fig. 2). Under these conditions, the 37 kDa component, which usually appears as a diffuse band on SDS-PAGE, gave two distinct bands. The higher molecular weight moiety showed residual glycosylation but the lower molecular weight band was absent on the transfer stained for glycans, suggesting complete dcglycosylation. Interestingly, we noted the formation of 37 kDa dimers of the deglycosylatcd form of 37 kDa CD23 on SDSPAGE. Neither neuraminidase, N-glycosidase F nor a combination of both affected the appearance of the 37 kDa CD23 band on SDS-PAGE confirming O-linked glycosylation which is theoretically the only possible type in the absence of an A s n - X - S e r / T h r motif in the amino acid sequence of the 37 kDa CD23.

Sequence attalysis N terminal sequence analysis (20 cycles) of 20(1 pmol of the 37 kDa CD23 showed an initial yield of 43% and gave the single sequence K S Q S T Q 1 S Q E L E E L R A E Q O R. This sequence corresponds to K ~ 2 - R . . of the full length CD23 sequence (Kikutani et al., 1986; Ikuta et al.0 1987; Liidin et al., 1987). No ether N terminal sequence was detected, indicating that the IL-2 leader sequence was completely processed. The cleavage point was between Glna~ and Lys~2, that is between the first and second amino acid encoded by the eDNA construct. Clearly, insect cell proteases process the recombinant 37 kDa CD23 to yield a product which is truncated by one amino acid at the N terminus. This is an unusual cleavage site for an endopeptidase, although cleavage on the amino terminal side of lysine has been found ~.oor . . . . . . . . ~

-40O[

. ..,

......

180 0

Sulphydryl and disulphide bond ana6,sis Direct sulphydryl titration of the protein in the presence of 1% ( w / v ) SDS or in 5 M GuHCI with DTNB (materials and methods section) gave a value of 0.2 reel sulphydryl/mol protein. This result was confirmed by amino acid analysis made on denatured, alkylated material under non reducing conditions. Control experiments using reduced samples detected eight sulphydryl groups per mole of protein as predicted by the DNA sequence. The same value was obtained by amino acid ana'.ysis on denatured, reduced and carboxymethylah, J o~otein. Reduction of the disulphide bonds wring NTSB ir 15f: (w/~) SDS further confirmed the presence of four disulphide bridges per molecule. These disulphide bonds were not accessible to NTSB in the absence of denaturant.

A

Wavelencj t h ( n m )

,oo[/-\,

2z,O o

B1 I

00i ...................................... • .00

Wevele~gth

(rim)

J

3&00

Fig. 3. Circular dichroism spectra of .37 kDa CD23. The circular dichroism spectra are the average of four scans with baseline subtracted. 37 kDa CD23 (0.650 m g / m l ) in PBS was analysed nalive (solid line)or denatured in 4 MGuI-ICI (dotted line) at rm)m lemperature. The far ultraviolet CD was measured using a 0.01 crn pathlenglh cell ( A ) and the near ultraviolel CD ( B ) in a lcm pathlength cen both with ] nrn bandwidth. The units for the ordinate are expressed in molecular cllipticity (O) in dog" cm2/dmol.

223 using the myxobacter AL-I p r o t e a s e II ( W i n g a r d et al., 1972).

TABI,E IV EFFECT OF 37 kl)a ('D23 AND IL-I ON GERMINAL ('ENTRE B ('ELL APOPTOSIS

Circular dichroism spectroscopy

Analysis of the far-ultraviolet circular dichroic s p e c t r u m o f the 37 k D a C D 2 3 (Fig. 3 A ) using the C O N T I N p r o g r a m ( P r o v c n c h e r et al., 1981) suggests 25% a-helix content, 74% /3-shec'. a n d 1% r a n d o m coil. T h o u g h the analysis of the far U V s p e c t r u m indicates that the moleculc c o n t a i n s mostly ,8-sheet s e c o n d a r y s t r u c t u r e s , it h a s been s h o w n by Bobba et al. (19911) that the C O N T I N p r o g r a m clearly o v e r e s t i m a t e s the ,O-sheet content o f a p r o t e i n ' s structure, which is not well d i s t i n g u i s h e d from r a n d o m coils. T r e a t m e n t of the 37 k D a C D 2 3 with 4 M G u H C I gave far a n d n e a r ultraviolet s p e c t r a characteristic of a rand o m coil s t r u c t u r e (Figs. 3A a n d 3B). D i s u l p h i d e b o n d s are predicted to give o n e or two C D broad b a n d s (positive in the case of n e u r o p h y s i n - I I ) located above 240 n m a n d a long o n e below 290 nm. T h e latter will t e n d to be negative a n d h)calized in t h e C D b a n d s of the aromatic a m i n o acid side c h a i n s (Strickland, 1974). T h e 37 k D a C D 2 3 n e a r ultraviolet C D s p e c t r u m (Fig. 3 B ) revealed analog characteristics. T h e broad positive ellipticity from 245 to 275 n m a n d the intense negative signal at 280, 288 a n d 297 tin, respectively reflect t h e contribution of the four disulphidc b o n d s a n d t h e eight t r y p t o p h a r s to the n e a r ultraviolet C D spectrum. Biologk'al actirtties

T h e r e c o m b i n a n t 37 k D a C D 2 3 was s h o w n to inhibit l g E binding to the C D 2 3 positive cell line R P M I 8866. By fluorescence activated cell sorting

~;limtJlus Anli-Cl)4{I 37 kDil/ll- I I'BS/I[.-I

'; rc,,cue fr()m ;ipoptusis " Expt. I 39.9 29.5 11

',; rcxcuc from apoplosis " ExpI. 2

21.4 It). I 0

" Ap(~ptosisin the absence of rescue factor was 6gr; (cxpt. I ) and 38'~ (cxpt. 2).

analysis, dose d e p e n d e n t i n h i b i t i o n occurred w h e n purified r e c o m b i n a n t 37 k D a CD23 was incubated with purified lgE (Table liD. This suggests that r e c o m b i n a n t 37 k D a C D 2 3 has the

iO0

•

• 25wgoI • 29WDo.

90

BO

7o

so ,.c 3o.

TABLE lit INHIBITION OF lgE BINDING ON RPM1 8866 BY RF.COMBINANT 37 kDa CD23 IgE

0~. Rct:ombinant 37 kDa CD23 /zg/ml

Mean fluorescence intensiLV 4_+1

+ +

+ +

10

5

3(1+2 21+2

25 311

16+ 1 13+_3

-

Day'. Fig. 4. Stability exp,:riment perfornled with 37 kDa C D 2 3 li~llowed by RP-IIPLC over 21 d:tys..17 kDa C D 2 3 in PBS at II)I),u.g/ml was incul')aI*,.'d at room tcrnl'~craturcfor 21 day~, in th'~ absence of pr(~tc.';l~e Jnhibimr,;. Aliqnots ;',=.-re regularly analy~,ed by RP-HPL(" ~n a bury: column. The percentage of the different CD23 fragments was calculated from the peak

area of the ahsorbancc profile at 214 nm.

224 snme conformation a~ th.- natural moleculc in i~ ability to bind IgE. The purified recombinant 37 kDa CD23 was shown to prevent apoptosis of germinal centre B cells in the presence of IL-1 (Table 11). Anti-CD40 stimulation used as a positive control of germinal centre B cell survival (Liu et al., 1989) was always higher than 37 kDa CD23 with IL-I. Amino acid Glnsl is not involved in the biological activities of the 37 kDa CD23 moiety reported here. The functional epitopes involved are more probably located in the C terminal part of the molecule since the 25 kDa CD23 starting at position 150 binds IgE and together with IL-I prevents B cell apoptosis (Liu et al., 1991).

Stability study Highly purified 37 kDa CD23 in PBS was submitted to a stability study at room temperature for 21 days. Very slow spontaneous degradation occurred (50%) over this period. A kinetic study of the degradation followed by RP-RPLC showed the sequential appearance of the 33, 29 and 25 kDa fragments (Fig. 4). Similarly, when concentrated 37 kDa samples containing no protease inhibitors were run on SDSPAGE and transferred to nitrocellulose the very sensitive immu~ostaining by the Rb33 antibody showed traces of the 29 and 25 kDa degradation products (Fig. 1B).

Discussion

Through the use of recombinant DNA technology and expression in insect cells, monomeric, glycosylated, soluble 37 kDa CD23 has been purified to homogeneity. Our belief that the molecule is in its native conformation is based on four criteria: firstly, it is able to bind lgE, as shown by its ability to inhibit binding of IgE to the membrane CD23-positive cell line RPMI 8866; secondly, it is able to prevent, in association with IL-I. germinal centre B cell apoptosis. Thirdly, it is recogni,,ed in an ELISA assay by two anti-CD23 monoclonal antibodies (EBVCS#1, Mab25), defining two different epitopes (Bonnefoy et al., 1990); finally, the tertiary structure is lost if the molecule is treated with a denaturing agent as

showu by ~ear and far ultraviolet CD spectra. Furthe:more, we have shown that the molecule is O-glycosylated. Though this last criterion strengthens the similarity between the natural and the recombinant form cf the 37 kDa CD23, we cannot expect the insect cell O-glycosylation to be identical to that occurring in mammalian cells. If glycosylation is important for some biological effect, we cannot exclude the possibility that an altered glycan composition would somehow affect activity. Using the present secretion system, much of the product stays in the intracytoplasmic compartment and the poor yield of purified recombinant 37 kDa CD23 reflects our concern with purity rather than quantity. The recombinant 37 kDa CD23 obtained by this expression system has permitted us to examine some of the biochemical characteristics of what is normally a short-lived intermediate species. A full comparison of tl' z physicochemical characteristics of the recombinant and natural molecules has not been possible due to the limited amount of natural material available. However, the recombinant material does exhibit some of the characteristics reported by Delespesse et al. (1989) for the native molecule. Firstly, it degrades to smaller fragments of 33, 29 and 25 kDa, a process which can be inhibited by protease inhibitors, in particular iodoacetamide and TLCK. Whether the degradation of pure preparations is due to an autoproteolytic process as suggested by others or to a contaminating enzyme is still controversial but it was observed in all the cells expressing CD23 (Delespesse e t. al., 1989; Letellier et al., 1990). Interestingly, the same sequence of degradation described for the natural form in vitro was observed with pure recombinant 37 kDa CD23, showing the sequential appearance of the 33, 29 and 25 kDa fragments. The absence of free cysteines suggests that there are four disulphide bridges compared with three predicted in a hypothetical model based on the homology between part of the CD23 sequence and a number of animal lectins. Cysteines at position 160 and 288 have been reported not to be involved in disulphide bridge formation in the published model of Delespesse (1989). However, deletion mutants expressed in C H O cells missing

225 Cyst~ J or Cys28 ~ showed slower mobility on SDSP A G E u n d e r non reducing conditions, thus implicating them in disulphide bridge formation (Bettler et al., 1989). We have shown here that the recombinant 37 kDa as well as the more stable 25 kDa CD23 fragment (Liu et al., 1991), both obtained by the same expression system are active in prevention of apoptosis, therefore sharing the same active domain. The 37 kDa and 25 kDa moieties with IL-I must now be included together with anti-lg and CD40 ligand as signals influencing the survival of germinal centre B cells that are otherwise destined to die by apoptosis. T h e availability of large a m o u n t s of recombinant material should help to determine which fragment of CD23 is mediating which biological activity and will permit us to look for o t h e r activities of this multifunctionai molecule. Finally, purified recombinant CD23 should be a v~Juable tool for the identification of a putative receptor for CD23.

Acknowledgments We thank Ms. Th6r~:se Jomotte, Edith Magnenat and Sybille H e n c h o z for expert technical assistance, Mrs. Nadine H u b e r for typing the manuscript and Dr. T. Wells for helpful discussions.

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