Structural comparison of murine T-cell (B151K12)-derived T-cell-replacing factor (IL-5) with RIL-5: Dimer formation is essential for the expression of biological activity

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Molecular Immunology, Vol. 27. No. 9, pp. 91 I-920, 1990 Printed in Great Britain.

STRUCTURAL COMPARISON OF MURINE T-CELL (B15 1K12)-DERIVED T-CELL-REPLACING FACTOR (IL-5) WITH rIL-5: DIMER FORMATION IS ESSENTIAL FOR THE EXPRESSION OF BIOLOGICAL ACTIVITY* TAKEO TAKAHASHI,?~

NAOTO YAMAGUCHI,~ SEIJI MITA,? YUJI YAMAGUCHI,$ TOSHIO SUDA,$ AKIRA ToMINAGA,t YUJI KIKUCHI,t YASUSADAMIURA~ and KIYOSHI TAKATsUt 11

tDepartment Honjo,

of Biology, Institute for Medical Immunology, Kumamoto University Medical School, 2-2-l Kumamoto 860. Japan and SDivision of Hematology, Department of Medicine, Jichi Medical School, Tochigi-Ken. Japan (First rewired 22 December

1989; accepted in reaised,form 2 March

1990)

Abstract--T-cell-replacing factor (TRF)/IL-5 is a T-cell-derived glycoprotein which has pleiotropic activity on lymphoid and myeloid cells. IL-5 polypeptide translated into Xenopus oocytes are heterogeneous in molecular size (40,000 to 60,000 under nonreducing conditions) and yields a monomeric form (M, of 25,000 to 30,000) under reducing conditions (J. Immun., 140, 1175-1181. 1988). We purified T-cell-derived TRF and rIL-5 using anti-TRF/IL-5 antibody-coupled affinity column from supernatants of a T-cell hybridoma B151K12 and supernatants of HeLa cells, respectively, which had been transfected with murine IL-5 cDNA, and determined their partial N-terminal amino acid sequence (27 residues for BISI-TRF and 13 residues for rIL-5). A single amino acid sequence of each sample was obtained beginning from methionine that was identical to that predicted from IL-5 cDNA. This finding supports the notion that secreted B151-TRF polypeptide consists of 113 amino acids. Purified BISl-TRF supported eosinophilopoiesis of human bone marrow cells as effective as mouse rIL-5 and human rIL-5. BISl-TRF competitively inhibited %-labeled rIL-5 binding to target cells to the same extent as rIL-5. Treatment of purified rIL-5 and BISI-TRF with reducing reagents such as 2-ME, sodium borohydride or dithiothreitol produced a monomeric form of IL-5 which did not exert a biological activity. Reduction and alkylation of rIL-5 caused the loss of binding to its target cells. These results strongly suggest that BISl-TRF exists as a homodimer and its primary structure and secondary structures are identical to those of rIL-5. Moreover. the formation of inter-molecular disulfide bond(s) linked by two pairs of cystein residues is essential for the expression of the biological activity of mouse IL-5.

INTRODUCTION

1972). We detected TRF activity in the Sup of PPD-stimulated Mycobacterium tuberculosis-primed T cells and in the Sup from a T-cell hybridoma (B 15 1K 12), established by means of fusion between Mycobacterium tuberculosis-primed T cells and BW5147 (Takatsu et al., 1980a, b). TRF activity was initially assessed by the ability to stimulate DNPprimed B cells to induce anti-DNP IgG antibody production. Later on, we also noticed that the same molecules seem to mediate the BCGFII activity (Harada et al., 1985; Swain et al., 1985). This was further proved by using affinity purified rTRF that is derived from the translation product of cDNA encoding TRF (Kinashi et al., 1986). The cDNA cloning of TRF has also clarified characteristics of this lymphokine to be active in the process of growth and differentiation of eosinophils (Yamaguchi et al., 1988a, b; Sanderson et al., 1988; Yokota et al., 1987) and of cytotoxic T cells or LAK cells (Aoki et al., 1989; Ramos. 1989; Takatsu et al., 1987). Based on these diverse activities on various target cells, TRF is now accepted as IL-5. Molecular analysis of rIL-5 revealed that it is a glycoprotein with an apparent molecular weight (M,)

IL-5 was formerly called T-cell-replacing factor (TRF). B cell growth factor II (BCGFII), eosinophildifferentiation factor (EDF), or IgA-enhancing factor (Takatsu, 1988; Takatsu et al., 1988; Coffman et al., 1988; Sanderson et al., 1988; Swain et al., 1988). TRF activity was defined as a T-cell-derived late-acting B-cell differentiation factor that is initially found in supernatants (Sup) of alloantigen-stimulated T cells or Con A-stimulated T cells (Schimpl and Wecker,

*This work was supported in part by a Grant-in-Aid for Cancer Research and for Scientific Research from the Japanese Ministry of Education. Science and Culture, and by a Grant-in-Aid from the Osaka Foundation of Clinical Immunology. $Present address: Olympus Co. Ltd, Hachi-oji, Tokyo 192, Japan. IiTo whom correspondence should be addressed. Abbreviations: TRF. T-cell-replacing factor: BCGFII, B-cell growth factor type II; EDF, eosinophil differentiation: PFC, plaque-forming cells; B151, a T-cell hybridoma Bi 5 I K I2 which constitutively produces IL-5; Sup, conditioned media or supernatants; M,, molecular weights. 911

912

T.4mo

TAKAHASHI

of 40,000 to 60,000 (Tominaga et al., 1988, 1990). Since core IL-5 polypeptides predicted by IL-5 cDNA sequence have two cystein residues that may be involved in the dimer formation (Kinashi ef al., 1986), we can envisage the role of the dimer formation in the IL-5 activity. Although we reported that the reduction and alkylation of partially purified BISI--TRF caused the decrease in TRF activity (Takatsu er ul., 1985) it is still not clear whether or not dimer formation of IL-5 is obligatory for the expression of its biological activity. In order to evaluate the role of dimer formation of TRF and rIL-5 in the expression of IL-5 activity, we purified T-cell hybridoma (Bl5 1K 12)-derived TRF and rIL-5 separately using anti-TRF/IL-5 antibodycoupled immunoaffinity columns, and evaluated IL-5 activity of the monomeric form of each preparation. Here we will provide evidence to show that rIL-5 and T-cell-derived B 15 1-TRF consist of homodimers, and a monomeric form of Bl51-TRF and rIL-5 does not fully express biological activity, probably because of loss of binding activity to target cells. MATERIALS

AND

METHODS

Mice

Female BALB/cCrSlc mice (pathogen-free) were obtained from the Japan SLC, Inc. (Hamamatsu), and were used when 2-3 months of age. Culture

medium

RPMI-1640 (Sigma Chemical Co., St. Louis, MO) medium was supplemented with 50pM 2-ME, penicillin (100 U/ml), streptomycin (100 pgg/ml) and 10% FCS (Flow Laboratories, McLean, VA). For TRF assay. we used serum-free medium (ASF-101, Ajinomoto. Tokyo) supplemented with 50 PM 2-ME, penicillin streptomycin and BSA (0.5%). Cell lines TRF-producing murine T cell hybridoma Bl5 I K 12 was established and was maintained as described (Takatsu et al., 19800). The HeLa cells transfected with murine IL-5 cDNA were established and were maintained in r:itro as previously described (Noma et t/l.. 1987). Their Sup were collected and were stored at -80 C until use. BCL, cells (in ciro line) were maintained by in ciw passage in BALB/c mice as previously described (Takatsu et ul.. 1985).

m7G(5’)ppp(S’)G was obtained from New England Biolabs (Beverly, MA). RNasin was purchased from Promega Biotec (Madison, WI). Cloned SP6 RNA polymerase and restriction endonuclease Sal1 were the products of Takara-Shuzo (Kyoto) and were used according to the manufacturer’s recommendation. “S-Methionine (1000 Ci/mmol) was obtained from New England Nuclear (Boston. MA). A rat-mouse B cell hybridoma (NC17, IgG, class) that produces monoclonal anti-TRF/IL-5 antibody was established

et

al.

as described (Harada et al., 19876). The purified NC17 antibody was coupled to formyl-cellulofine beads (Seikagaku Kogyo, Tokyo) and was used for the immunoaffinity matrix. PuriJication of IL-5

TRF-containing BlSl-Sup (30 liters) were applied to six columns of NCI 7-coupled formyl-cellulofine beads and were eluted with IO ml per column of 1 M acetic acid after extensive washing by each of the following solutions: 1 M NaCI, 0.5% nonidet P-40, PBS pH 7.2 and distilled water. TRF-containing fractions were concentrated up to 200 ~1 with the use of Speed Vat Concentrator (SAVANT Instruments Inc., Hicksville. NY). The concentrate thus obtained was used for bioassay and a portion of it was applied to two successive Superose 12 columns for a gelpermeation column chromatography which had been directly connected in a FPLC system (Pharmacia Fine Chemicals, Uppsala, Sweden) and had been equilibrated with PBS (pH 7.2). Elution from the column was conducted with PBS at the rate of 0.3 ml/min. Fractions showing TRF activity after a gel permeation step (M, 40,000 to 60,000) were re-applied to a column of NC 17-coupled beads and eluted with 2 ml of 1 M acetic acid. rIL-5 was also purified from HeLa Sup (5 liters) according to the same procedures as those for BlSl-TRF, excepting the second NC17-column step. To prepare samples for amino acid analysis, the material from the gelpermeation column chromatography step was applied to a reverse phase HPLC (RP-HPLC) column (Protein C,; Vydac, Hesperia, CA), which had been equilibrated in 0. I % trifluoroacetic acid (TFA). After sample application, the column was washed with 0.1% TFA, and a gradient of t&80% acetonitrile in 0.1% TFA developed over a 60 min period. The conditions for chromatography were: flow rate of 0.5 ml/min. fraction size of 0.5 ml, and column effluent monitored for absorbance at 214nm. IL-5 thus purified was subjected to analysis with respect to the amino acid sequence and amino acid composition. Preparution

qf‘ ‘SS-lubeled

rlL-5

IL-5 mRNA (1 mg/ml) was injected into Xenopus oocytes in a volume of 50ml and incubated in modified Barth’s buffer as batches of IO oocytes in 100 ~1 for 48 hr at 2O.C (Tominaga et ul., 1988). To obtain internally labelled IL-5, 100 PCi of “Smethionine in 10 ~1 was added to each batch of 10 oocytes in 100 ~1 of modified Barth’s buffer at the initiation of the incubation. After the incubation, 35S-labeled IL-5 was purified from Sup, using the NC17-coupled immunoaffinity column as described (Mita et al., 1988). Treatment

qf‘ IL-5

with reducing reugents

A sample from the last immunoaffinity step was treated with 5% 2-ME for IO min at 37 C, with 0.1 M

Dimer formation is essential for IL-5 activity sodium borohydride (NaBH,) for 10min at room temperature, with various concentrations of dithiothreitol (DTT) for 30 min at room temperature, with 8 M urea for 18 hr at room temperature, or with 3 M KSCN for 30min at room temperature. Then the sample was treated with 10% excess of iodoacetamide as previously described (Takatsu et al., 1975). As a control, another set of samples was treated with iodoacetamide in the absence of reducing agents. After the treatment all samples were dialyzed against PBS, and subjected to bioassay for SDS-PAGE analysis Assessment

of IL-5 a&cities

TRF assay. Purified BCL, cells (in aiuo passaged line) were cultured in aitro in a serum-free medium (ASF-I) with a sample to be tested for 2 days in a 96-well microplate (No. 29836, Corning Glass Ware, Corning, NY). Cultures were assayed for total IgM-secreting plaque-forming ceils (PFC) by the reverse PFC assay (Takatsu et al., 1985). TRF activities were expressed as units/ml. One unit of IL-5 was determined by the amounts of IL-5 required for half-maximal responses in the IgM PFC assay. BCGFII assay. BCGFII activity was determined using BCL, cells or B cell line. T-88 (Tominaga et al., 1989). Either BCL, cells (1.5 x 105/0.2 ml) or T-88 cells (5 x 104/0.2 ml) were cultured with samples to be tested for 48 hr and pulsed with 0.2 PCi ‘H-thymidine for 12 hr. After the culture, cells were harvested and incorporated ‘H-thymidine was counted using a liquid scintillation counter (Aloka Type III). Clonal cell culture Cultures of 5 x IO4 human non-phagocytic bone marrow cells/ml were prepared in 35-mm non-tissue culture dishes (Falcon Labware, Oxnard, CA) using methylcellulose medium. Cells were suspended in 1 ml of 1.2% methylcellulose (Fisher Scientific Co., Pittsburgh, PA) in a medium containing 30% FCS and IOmg BSA (Sigma), and were cultured with appropriate amounts of BlSI-TRF, with mouse rIL-5, or with human rIL-5 as previously described (Yamaguchi et al., 1988a, 6). The cultures were incubated at 37 C in a humidified atmosphere of 5% CO? in air. The numbers of colonies were lifted with a 3 ~1 centrifuge (Cytospin; Shandon Southern Instruments Inc., Sewickley. PA) and stained with May-Grunwald-Giemsa. Sodium dodecyl sulfate phoreses (SDS-PAGE)

polyacrylamide

gel electro-

The system used was the discontinuous SDS method of Laemmli (1970). In some experiments, samples were treated with reducing reagents such as 2-ME, DTT, or NaBH, before the analysis. Proteins were made visual by the silver staining method, using a staining kit obtained from Kanto Chemicals Co. Inc.. Tokyo. Marker proteins used were lysozyme (M, 14,400). soybean trypsin inhibitor (21,500) car-

913

bonic anhydrase (31,000), ovalbumin (42,700) BSA (66,200) and phosphorylase b (97,400). After electrophoresis, the gel was stained with Coomassie brilliant blue R-250, dried on Whatman 3 mm paper, and was exposed to AIF new RX 50 film (Fuji Photo Film, Tokyo) at -80°C. Binding assay jor radiolabeled IL -5 IL-5-binding assay was carried out under highaffinity conditions according to the procedures previously described (Mita et al., 1988, 1989), with the use of IL-5-dependent T88-M cells that had been established and had been maintained according to the procedures as previously described (Tominaga et al.. 1989). T88-M cells were washed twice and incubated for at least 24 hr at 37-C in RPM1 1640 medium containing 25 mM Hepes (pH 7.2) and then were washed again three times with the same medium to remove any endogenous IL-5. After washing, cells were immediately used for a binding assay. To determine the level of binding, serial dilutions of “Slabeled rIL-5 (a dimeric or a monomeric form) (IO-80 PM) were incubated at 37 C for 10 min with II10 x IO6 T88-M cells in a total volume of 200~1 binding medium, using 1.5 ml micro-test tubes (Assist Trading Co., Tokyo). At the end of incubation, the reaction mixture was layered onto a 200-,ul mixture of 84% silicone oil (SHSSO, Nakarai Chemical Co., Tokyo) and 16% paraffin oil (No. 7162, Merk & Dohm Co., Inc., Rahway, NJ). The mixture was then centrifuged at 85OOg for 90 s to remove the small amounts of unbound IL-5 in the cell pellets. The tips of the tubes were cut off, placed in vials. and their radioactivities counted as previously described (Mita et al., 1988). The specific binding was defined as the difference between total binding and nonspecific binding, which was obtained in the presence of lOO-fold molar excess of unlabeled IL-5. The amount of “S-labeled IL-5 was determined by the ELISA, using NC-17 (monoclonal anti-IL-5 antibodies) and polyclonal rabbit anti-IL-5 antibodies. (This data will be published in the future.) Specific activity of “S-labeled rIL-5 was 11,328$- 758 cpm/fmole. Estimation

of protein concentrations

The amount of protein in the sample concentrated after the RP-HPLC column step was determined by fluorescamine methods (Yamaguchi et al., 1989). Integration of the absorbance profile was used to estimate the protein eluted from the RP-HPLC column assuming e” “‘O= I .O. Protein in fractions from the gel filtration column step analyzed by SDS-PAGE was quantitated by comparison with serially diluted M, standards (Pharmacia Fine Chemicals, Tokyo, Japan). Amino acid analysis Protein samples (10 pg) were hydrolyzed in evacuated tubes for 24 hr at 1 IO’C in 0.15 ml of 6 N HCl.

914

TAKEO

TAKAHASHI

The amino acid analysis was carried out with Hitachi amino acid analyzer, Model 835-50.

a

Peptide sequence analysis Fractions from RP-HPLC column step were pooled and partially evaporated (5 pg). These results were applied to an Applied Biosystems (Foster City, CA) 477A “gas-phase” sequencer for amino acid sequencing (Tsujimoto et al., 1989). The phenylhydantoin analysis was identified by RP-HPLC with an Applied Biosystem 120A on-line system (Hewick et al., 1981). RESULTS

PuriJication and characterization

cf IL-5

T-cell-derived BlSI-TRF was purified from the Sup of B151Kl2 cells according to the procedures described in Materials and Methods, above. First from approximately 6 x IO9 of all, BlSl-Sup cells in 30 liters were applied to the NCl7-coupled column, and the absorbed Bl5l-TRF was eluted with 1.0 M acetic acid. This step was crucial to recover most of TRF-active molecules and to separate BlSI-TRF from most of the components of the culture medium. The recovery of TRF activities monitored by TRF assay after this step was more than twice the initial activity. Since the samples after this step still contained higher m.w. contaminants, they were concentrated up to 200~1 and were applied to two successive Superose 12 columns for a gel permeation chromatography. TRF active fractions with the molecular mass of 40.000 to 60,000 were recovered. By this procedure, more than 90% of TRF activities were recovered. accompanied by the massive elimination of contaminants. The materials thus obtained were reapplied to a column of NC17-coupled beads. IL-5 active fractions were again eluted with I M acetic acid from the column and were neutralized with I M Tris. This step permitted two conclusions. First, the sample was free from contaminating proteins. Second, protein with TRF

Table

I, Purllication

of naturally

produced

TRF

A

activity was concentrated and appeared diffused in an area equal to an apparent M, of 40,000 to 60,000. Samples thus obtained were used as the purified BlSI-TRF. According to similar procedures described above, rIL-5 was purified from the Sup in HeLa cells, which had been transfected with IL-5 cDNA, except that the second NC17-affinity column step was not included because of the satisfactory purity of IL-5 after the first immunoaffinity chromatography step. Table 1 summarizes the recoveries and the specific activities after each purification step. The specific activities of BISl-TRF and rIL-5 after the last step of purification were approximately 9.4 x IO6 units/mg protein and 8.0 x 106/mg protein, respectively. Recoveries of BISl-TRF and rIL-5 were 227% and 40%, respectively, and both had approximately 6000-fold purification (Table I). In summary, the purification procedures described here are practical, reproducible and not timeconsuming. The sample after the gel permeation column chromatography step was more than 90% pure. The sample after the gel permeation column chromatography step was subjected to SDS-PAGE analysis. Figure I shows that under reducing conditions, both BlSl-TRF and rIL-5 showed heterogeneities with a major protein at about M, 25,000 and a minor band at about 28,000. These two bands were broad, which was indicative of further microheterogeneity. When the analysis was performed under nonreducing conditions, two bands at about M, 40,000 to 60,000 were detected. rIL-5 migrated relatively slower than Bl51-TRF. When each of the IL-5 preparations was treated with Nglycanase and analyzed under reducing conditions, only one major band with M, 20,000 was observed (data not shown). The estimate of protein amount in the M, 25,000-28,000 region by silver staining was 5Opg in total. A portion of it was subjected to RP-HPLC to get ultrapure IL-5. We obtained two symmetrical protein peaks, and each peak was used for the further analysis.

from a Bl5

cell culture

et al.

IK

I2 cell culture

medium

and rIL-5

from a CHO

medium

RISI-TRF

VOlUIlX Step

Superose

TOtal activity

(rng x IO

(ml)

Culture sup NCl7-column

Total protein

30.000 20

‘1

(U

220 31 5

x IO

specific PldiCdOll

XtiVity ‘1

(U

30 80

x IO

%ng)

(fold


Recovery

increase) (1) 17

(%I (100) 267

I?

6

021

71

338

2253

237

NC I7-column

3

0.07

68

944

6287

227

Total

Specilic

B. rIL-5 Total Volume

XtiVily

protein

strp

(InI)

(mg x IO

Culturesup

5000

55

‘1

(U

x IO 2.5

XtiVity J)

(U

x IO

Purification %llg)

(fold


increase) I

Recovery (%

1

100

NCl7-column

7

0.14

2.8

20

142

I58

Superose-

I

0.00125

I .o

800

5714

40

I?

One U ml IS defined

as the concentration

requred

for half maximal

IgM

production

in the assay system used.

915

Dimer formation is essential for IL-5 activity

rIL-5

BlSl-TRF

Predicted: cDNh

1 10 Met-Arg-Arg-Met-Leu-Leu-llls-Leu-Ser-Val11 Leu-Thr-Leu-Ser-Cys-Val-Trp-Ala-Thr-Ala-

20

cDNA

21 Met-Glu-Ile-Pro-Met-Ser-Thr-Val-Val-Lys-

30

cDNA

31 Glu-Thr-Leu-Thr-Gln-Leu-Ser-Ala-llls-Arg-

40

cDNA cDNA

41 Ala-Leu-Leu-Thr-Ser-Asn-Glu-Thr-Met-Arg-

50

Observed:

2-ME

-

+

-

qf N-terminal

1 Met-Glu-Ile-Pro-Met-Ser-Thr-Val-Val-Lys-

rIL-5

Met-Glu-Ile-Pro-Met-Ser-Thr-Val-Vel-Lys-

B151-TRF

11 Glu-Thr-Leu-Thr-Cln-Leu-Ser-Ala-)lls-Arg-

rIL-5

Glu-Thr-Leu-

BlSl-TRF

21 Ala-Leu-Leu-Thr-Ser-

10

+

Fig. 1. SDS-PAGE analysis of purified BlSI-TRF and rIL-5. Purified BlSl-TRF and rlL-5 were subjected to SDS-PAGE analysis in the presence or absence of 5% 2-ME, followed by silver staining. Molecular weight markers are expressed as kDa.

Determination puriJied IL -5

BlSl-TRF

amino acid sequence of

BISl-TRF (1Opg) was analyzed for amino acid composition. As can be seen in Table 2, the amino acid composition of B15 1-TRF obtained was consistent with that of rIL-5 predicted from the cDNA, although one tryptophan residue and two cystein residues were not detected. Next, we determined a partial amino acid sequence of both BlSl-TRF (5 pg, about 250pmol as a monomer) and rIL-5 (2 pg), using an automated gas-phase sequenator. The detected amino acid after each determination is displayed in Fig. 2. Only one sequence of 27 amino acid residues of BlSl-TRF was obtained, except for Asn-26 (see Discussion). We also Table 2. Annno acid analysis of purified

20

27 / -Glu-

Fig. 2. NH,-terminal amino acid sequence of BlSl-TRF and rIL-5. Comparison of the NH,-terminal amino acid sequence was obtained from the cDNA clone (pSP6K-mTRF23) and from the RP-HPLC-purified Bl51TRF and rIL-5. The cDNA sequence shown by underlining corresponds to the amino acid sequence of the amino acids of BlSl-TRF and rIL-5.

obtained a single amino acid sequence of 13 residues of rIL-5. In each case, the amino acid sequence determined exactly corresponded to a sequence deduced from IL-5 cDNA (Fig. 2). This analysis showed that the primary structure of T-cell-derived Bl51-TRF, as well as rIL-5 polypeptide, are highly homogeneous, and when secreted, the first 20 amino acid residues specified in the open reading frame of the IL-5 cDNA are removed from the precursors of IL-5.

BISI-TRF Amino AspjAsn Thr Ser GlU/Ghl Pro GlY Ala Cy5 Val Met Ile LW” Tyr Pht: Lys His Arg TOP

acids

Observed Predicted (residues/molecule) 6.54 7.53 4.53 19.99 3.34 5.75 3.1 I N.D.h 4.50 4.61 4.39 14 2.24 3.21

7 IO 5 19 3 I 3 2 6 6 6 14 2 4

5.71

7

2.30 6.35 N.D.h

3 8 I

“The values were calculated by assuming the number of leucine residues to be 14. %D.: not determined.

Eosinophil rlL-5

d@erentiation

activity

of B 151-TRF

and

Purified Bl51-TRF and rIL-5 exert TRF and BCGFII activities on murine chronic B cell leukemia BCL, cells (Kinashi et al., 1986; Tominaga et al., 1988). They also induce enhanced antigen-specific and polyclonal IgA production of B cells (Matsumoto et al., 1989), and also induce an increase in the expression of IL-2 receptor on B cells (Harada et al., 1987a; Loughnan et al., 1987; Nakanishi et al., 1988). We evaluated the effect of purified BlSI-TRF on non-phagocytic human bone marrow cells for eosinophil-differentiation inducing activity (EDF). Table 3 shows the compositions of the various types of colonies formed in the presence of 8 U/ml of BlSI-TRF, mouse rIL-5, and human rIL-5. We obtained 9, 18 and 10 colonies in cultures of 5 x lo4 human non-phagocytic bone marrow cells to which

916

TAKEO TAKAHASHI et

Table 3. Effect of BISI-TRF

on non-obaeocvtic

Bl51-TRF Colony No.

e

I 2 3 4 5 6 I 8 9

100 100 91 91 95 93 88 86 84

n

5

human

m

Bl

C010lly No. 2 3 4 5

I 2 2 6 12 14 9

I 2

6 I x 9 IO II 12 13 14 IS 16 17 18

e 100 100 100 99 98 96 95 95 95 95 95 94 89 87 85 69 64 48

n

m

Bl

Others

Colony NO.

I 2 4 3 2

3

I 2

2 3 5 5 2 4 6

I 2 6 I6

8 21 7 30

we added BlSl-TRF, mouse rIL-5, and human rIL-5, respectively. In mixed colonies containing eosinophils, the proportion of eosinophils was different in each colony, ranging from 84 to 100% in the culture with BlSl-TRF. This clearly demonstrates that Bl51TRF supports the colony formation of eosinophils from human bone marrow cells, at the same rate of efficiency as mouse and human rIL-5 do. B151-TRF also supported the growth and differentiation of mouse eosinophilic progenitor cells in bone marrow (data not shown). bonds on the biological activity

To evaluate the dimer formation in the IL-5 activity, BlSl-TRF and rIL-5 were treated with various reagents and were subjected to SDS-PAGE to analyze the formation of a monomeric IL-5. Treatment of BlSl-TRF with 5% 2-ME, with 50 mM DTT and with 0.1 M NaBH,, followed by alkylation

ceils

Human

e 96 87 86 86 16 75 66 63 50 40

rIL-5

n

m

81

4 3 14 16 16 20 6 21 4

5 19 7 40

8 25 9 12 22 16

I 3 12 3 4 3 22

All colonies in each dish were lifted and examined for morphology. 200 cells stained with May-Grunwald-Giemsa. e. eosinophil; n. neutrophil; m. macrophages; and BI, blast ceils.

Significance qfdisul$de qf IL-5

bone marrow

Mouse rIL-5

I

2 I 3

al.

2

Differential

counts were performed

on

with 20% excess of iodoacetamide, gave a monomeric form of IL-5, while treatment with I % SDS, with 8 M urea, with 3 M potassium isothiocyanate (KSCN), with 5 mM Na,CO, (pH 10.8) or with 0.8 M CH,COOH (pH 2.3) (all of which are nonreducing agents) failed to do so (Fig. 3), indicating that BlSI-TRF exists as a dimer uia inter-molecular disulfide bonds. Figure 3 also displays SDS-PAGE analysis of NaBH,-treated and KSCN-treated Bl51TRF. BlSl-TRF, reduced with NaBH, and followed by alkylation, show protein bands corresponding to a M, of about 25,000, even under nonreducing conditions. Moreover, alkylated B15 I-TRF was eluted from the Superose 12 column at a point corresponding to an apparent M, of 22,000 (data not shown), indicating that after the treatment of T-cell-derived IL-5 with a reducing reagent such as NaBH, and followed by alkylation, IL-5 results in a monomeric form. It was also clear that molecular size of a monomeric form of B15 I-TRF is very similar to that

Treatment

BlSl-TRF

SDS pH 10 pH 2.3 Urea KSCN 2ME DTT NaBH4

40-60 kDa 40-60 40-60 40-60 40-60 25-30 25-30 25-30

Fig. 3. SDS--PAGE analysis of NaBH,-treated BlSl-TRF and KSCN-treated BlSl-REF. Purified BlSI-TRF was treated with 0.1 M NaBH, 3M KSCN, 2.3% SDS, 5mM NaZCO, (pH 10.8), 0.8M CH,COOH, 8 M urea, 5% 2-ME or 50 mM DTT, according to procedures described in the Materials and Methods above. After the treatment, samples were subjected to SDS-PAGE analysis. The left hand side of the panel summarized M, and the right hand side of the panel displayed SDS--PAGE analysis of representative samples.

Dimer

formation

is essential

Table 4. Effects of reduction and alkylation TRFIIL-5 Treatment Assay

DTT

Iodoacetamide

for IL-5 activity

917

of biological activity of Bl51-

Concentration

of BISI-TRF (%)

0.6

2.5

627(1.16) 170(1.45) 733(1.07) 143(1.22) 2859(1.13) 1419(1.19) 2842 (I .06) 1425(1.15)

1656(1.11) 203 (I .07) 1543(1.10) 125(1.06) 6012(1.06) 1631 (1.05) 3789(1.12) 1404 cI .03)

mM

TRF

BCGFII

0 50 0 50 0 50 0 50

0 0 I20 120 0 0 120 120

BI 51.TRF (IO U/ml) was treated with DTT, lodoacetamide or both. After the treatment TRF and BCGFII activities were determined separately, using various dilutions of the sample, according to the methods described in the Mareriols and Methods above. In the TRF assay, results are expressed as mean IgM PFC of triplicate cultures and SE. In the absence of IL-5, I I4 IgM PFC per culture were observed. In the BCGFII assay, results are expressed as mean cpm and SE. Background cpm in the absence of IL-5 were 2358 cpm per culture.

of the rIL-5 monomer. In both cases, we could see two bands, probably due to differential glycosylation. N-glycanase treatment of each IL-5 specimen gave only one band (data not shown). To examine the significance of disulfide bonds on the biological activity of IL-S, the monomeric form of BISI-TRF was used to titrate IL-5 activity in standard TRF and BCGFII assays, using BCL, cells. As shown in Table 4, reduced and alkylated Bl Sl-TRF lost more than 95% of its TRF and BCGFII activities. Treatment with iodoacetamide alone had little effect, strongly suggesting that the dimer formation via inter-molecular disulfide bond(s) is essential for the biological activity of BISI-TRF. Failure of binding of a monomeric

IL-5 to target cells

Binding assay for ‘S-labeled rIL-5 to T88-M cells was then carried out. As we reported (Mita et al., 1989) T88-M cells proliferate in an IL-5-dependent manner and express two classes (high-affinity and low-affinity) of IL-5-binding sites. Since BlSl-TRF and rIL-5 can cause proliferation of T88-M cells at concentration levels accessible only to high-affinity receptors, biological activity of IL-5 seems to be mediated through such high-affinity receptors. Binding assays were therefore conducted under the highaffinity conditions of ?S-labeled rIL-5. First, the competitive inhibitory effect of BISI-TRF on 35S-

Table 5. Competitive inhibition for the binding of “‘l-labeled rIL-5 by unlabeled rIL-5 and BISI-TRF Expt

Inhibitor

Specific binding of “‘I-labeled rIL-5

I

None rIL-5 BISI-TRF None BISI-TRF

2879 256 35x 3372 416

2

Binding assay to T88-M cells was conducted at 37 C for IO min incubation under high-affinity conditlons (50 pM “‘I-labeled IL-5). Fifty-fold excess amounts of unlabeled rIL-5 or BISI-TRF were added as inhibitors.

labeled rIL-5 binding to T-88 cells was tested. As a control, unlabeled rIL-5 was also used. As can be seen in Table 5, a 50-fold molar excess of unlabeled B 15 1-TRF competitively inhibited the binding of 35S-labeled rIL-5 to T88-M cells to the same extent as that of unlabeled rIL-5. When 3SS-labeled rIL-5 was reduced with various concentrations of DTT and then was followed by alkylation with iodoacetamide, its specific binding to T88-M cells was decreased along with the concentrations of DTT employed for the reduction (Table 6). More than 70% of the binding ability of j5S-labeled rIL-5 was lost by treatment with 50 mM DTT, and most of the binding activity was lost by treatment with 200mM DTT. Most of ‘5S-labeled rIL-5 was shown to be a monomer after treatment with 200mM DTT, as determined by SDS-PAGE and autoradiography, while treatment of 35S-labeled rIL-5 with 50 mM DTT left intact rIL-5 molecules, to a certain extent as a dimer form (up to 15%) (data not shown). DISCUSSION

Our earlier observations on the diverse biological activities of B151-TRF prompted us to define this lymphokine at the molecular level. We previously reported the partial purification of B151TRF from BISI-Sup, using DEAErzellulose column Table 6. Comparison labeled dimeric IL-5

of specific binding of ‘%with “S-labeled monomeric rIL-5

DTT used for reduction (mW 0 50 100 200

Specific binding of “S-labeled rIL-5 3366 I396 1024 599

“S-labeled rIL-5 were reduced with various concentrations of DTT for 30 min at 4 C followed by alkylatlon with 20% excess of iodoacetamide. After extensive dialysis against a binding buffer. bmding assay at 5OpM was carried out according to procedures described in Table 5.

918

TAKEOTAKAHASHI

chromatography, Blue-Sepharose column chromatography, hydroxylapatite chromatography, gel permeation column and disc chromatography, polyacrylamide gel electrophoresis (Takatsu et al., 1985; Harada ef al., 1985). Over all, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity. However, this was not enough to allow for analysis of amino acid sequencing, partly because of poor purity. In this study, BISI-TRF and rIL-5 have been purified with more than satisfactory yields by using anti-TRFjIL-5 antibody-coupled beads. The use of anti-IL-5 antibody-coupled immunoaffinity column enabled us to purify IL-5 in a practical, easy, reproducible, and efficient manner. The reason we could recover more than expected purified BI51-TRF/IL-5 may be due to the elimination of unknown proteins that may inhibit IL-5 activity from BlSl-Sup. Purified B15 I-TRF/IL-5 was shown to be heterogeneous in its molecular size. Two forms with apparent M, of 28,000 and 25,000 were detected by SDS-PAGE in the presence of 2-ME. Both forms of Bl51-TRF were determined to have average M, values of around 43,000 in the absence of reducing reagents. These results are fully compatible with our previous data (Takatsu et af., 1985) and the data obtained using rIL-5 (Tominaga et al., 1988, 1990; Tavernier ef al., 1989; Rolink et al., 1989), indicating that BISI-TRF and rIL-5 exist in a dimeric form uiu inter-molecular disulfide bond(s). Determination of the location of the disuhide bond(s) in mouse IL-5 is important in order to understand whether cystein residues form parallel or antiparallel dimers. As we will describe later (Tominaga et al., 1990), secreted rIL-5 molecules are heterogeneous in p1 and in size, and degiycosylated rIL-5, prepared either by the treatment of rIL-5 with N-glycanase or by the translation of IL-5 mRNA in oocytes in the presence of tunicamy~in, shows limited heterogeneity in p1 and in molecular sizes. Although we do not give data here, treatment of BISI-TRF with N-glycanase under reducing conditions yielded dramatically reduced heterogeneity. Furthermore, the 26th asparagine from the N-terminal end (Asn-26) seems to be N-glycosylated, because we could not detect amino acid in its sequencing analysis. These results strongly suggest that heterogeneities of BlSl-TRF and rIL-5 are caused by differential glycosylation. In contrast to this M, heterogeneity, BISI-TRF shares a common-terminal sequence that matches precisely to a partial sequence delivered from the cDNA clone of IL-5 implying that the first 20 residues in the open reading frame of that cDNA constitute the processed signal peptide, leaving a maximum mature polypeptide length of 113 residues and a M, of about 12,300. Coffman ef al. (1988) reported the purification and characterization of an IgA-enhancing factor from the Sup of cloned helper T cells (D10.36.69). Its partial N-terminal amino acid

et al.

sequences are identical to those deduced from IL-5 cDNA. McKenzie et al. (1987) reported the N-terminal amino acid sequencing of murine BCGFII produced by cloned T cells. Their sequences are also precisely identical to the one described in this study, except for one amino acid change (Leu-16 to Asp-16). Taking all of the results together, the structure of TRF/BCGFII/IgA-enhancing factor produced by T cells is identical to that of rIL-5. Using purified BlSl-TRF and rIL-5, a significance of dimer formation of IL-5 in a biological function was observed. Previously, we reported that B151TRF treated with 20mM DTT and followed by alkylation with iodoa~etamide exerted BCGF II activity on BCL, cells, while it lost TRF activity (Harada et al., 198.5). However, we could observe little, if any, BCGF II and TRF activity in a monomeric form of purified BlSl-TRF in this study, as shown in Table 4. Several possibilities account for the discrepancies between this study and our previous one. First of all, purities of the BISI-TRF used in the previous study could have been lower than that we had predicted. Therefore, the cleavage of S-S bonds by DTT might have been insufficient, allowing a dimeric form of BISI-TRF to be left. Second, the concentrations of DTT (10 mM) mainly used for the previous study were lower than those used in this study. DTT at 50mM did not give a complete monomeric form of B151-TRF, and 200 mM DTT was required to get a satisfactory recovery of monomeric BISl-TRF (data not shown). In the previous study, we had tested the effects of DTT up to 10 mM on TRF activity and had not known what proportions of dimeric B151-TRF became a monomeric form. Therefore, reduced and alkylated BlSI-TRF preparations in the previous studies may have contained enough amounts of dimeric IL-5 It is of interest to note that the treatment of BISI-TRF with 50mM DTT itself, in the absence of iodoacetamide, caused the striking decrease in IL-5 activity (Table 41, suggesting that cleavage of inter-molecular disulfide bonds may create irreversible structures. Taking all of the observations collectively, the inhibition we observed in the previous paper may have been caused by insufficient cleavage of disulfide bonds. The observation that a monomeric form of B151TRF shows decreased binding to IL-5-responsive T88-M cells under high-affinity conditions (Table 6) is very interesting. The data shows that initial translation product from IL-5 mRNA must be processed in order to bind to the IL-5 receptor and hence express biological activity. This is similar to IL-I/I and its receptor system and is different from IL-la and its receptor system (Mosfey er al., 1987). As we reported (Mita et al., 1988, 1989) there are two classes of IL-5 binding sites, and numbers of highaffinity binding sites correlate with IL-5-responsiveness. IL-5 signals can be transduced through high-affinity receptors. It is therefore important to clarify in future the reason why the inter-molecular

Dimer

formation

is essential

for IL-S activity

919

dimer formation is essential for IL-5 to bind to high-affinity IL-5 receptor. In conclusion, a monomer form of BlSl-TRF neither exerts IL-5 activities nor does it exert binding to high-a~nity receptors, suggesting that intermolecular dimer formation of IL-5 is essential for both the binding to high-affinity receptors and the expression of its activities..

not class switching of surface InA-uositive B cells into IgA-secreting cells: Immunology $32-38. McKenzie D. T.. Filutowicz H. I.. Swain S. I. and Dutton R. W. (1987) Purification and partial sequence analysis of murine B cell growth factor II (interleukin 5). J. Irnni~~o~. 139, 2661-2668. Mita S., Harada N., Naomi S., Hitoshi Y., Sakamoto K., Akagi M., Tominaga A. and Takatsu K. (1988) Receptors for T cell-replacing factoriinterleukin 5: snecificitv. quantitation, a&l its jmplicaiion. J. exp. Med. 168,

are grateful to Drs Kaoru Onoue and Hideo Hayashi for their continuous encouragement throughout this work. We are also grateful to Drs Tasuko Honio and Tatsuro Nishihara for orovidine HeLa cells transfected with IL-S cDNA and amino acid sequencing, respectively. Mr Donald Ellis is acknowledged for his critical reading of this manuscript.

Mita S., Tominaga A., Hitoshi Y., Sakamoto K., Honjo T.. Akagi M., Yamaguchi N. and Takatsu K. (1989) Characterization of high-affinity receptors for interleukin 5 (IL5) on IL-5 dependent cell lines. Proc. nafl. Acad. Sci. USA

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