Vol. 173, No. 2,1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 554-560

December 14, 1990

FRACTIONATION OF THE RIBOSOME INACTIVATING PROTEIN PREPARATIONS WITH TRIAZINE DYES Saturnino M. Mufioz 1, Maristeila Caldera 2, Silvana Canevari 2, Emanuela Tosi 2, Tiziana Cogliati 2, Maria I. Colnaghi 2 and Francisco P. Conde 1

1Secci6n de Inmunoqufmica, Dpto. Investigaci6n, Hospital "Ram6n y Cajal", Ctra. de Colmenar, Km 9.1, 28034 Madrid, Spain 2Oncologia Sperimentale E, Istituto Nazionale dei Tumori, Milan, Italy Received

October

5,

1990

SUMMARY. Aspergillins are ribosome-inactivating proteins (RIPs), isolated from several strains of Aspergillus. T h e interaction between Cibacron Blue F3GA and two members of this family, alpha sarcin and mitogillin, and other RIPs of type I, was studied. Alpha sarcin retention depended on pH and ionic strength. By chromatography on Affi-Gel Blue in mild experimental conditions, mitogillin and PAP-I did not interact with the dye, whereas 40% of alpha sarcin and 70-90% of briodin, RTA and gelonin were recovered in the bound fraction. In all cases, the major fraction showed a higher toxicity level in protein synthesis inhibition assays. The unbound alpha sarcin, conjugated with the anti-ovarian carcinoma monoclonal antibody MOvl7, showed on OVCA 432 a cytotoxicity which was 900 times higher than that exerted by the alpha sarcin alone. ®199oAcaderai....... I n c .

The ribosome inactivating proteins (RIPs) are potent cytotoxic agents which inhibit protein synthesis by enzymatic activity on ribosomes(l). Beside plants, also filamentous fungi Aspergillus have been reported to produce RIPs, one named alpha sarcin (2) was isolated from a strain of A. giganteus, and other two, restrictocin and mitogillin (3,4), from A.restrictus. Due to the high level of immunological cross-reactivity and of sequence homology (5,6) they could be considered to belong to the same family of RIPs hereafter named Aspergillins. Two different mechanisms of RIP action have been reported; Aspergillins are able to cleave a single phosphodiester bound between G-4325 and A-4326 of the 28S rRNA (5-7), whereas ricin and other toxic lectins, such abrin and modeccin, act by modifying the A-4324 N-glycosidic bound of the same rRNA (8). Triazine dyes have been reported to interact with several types of enzymes such as kinases, dehydrogenases and nucleotide-dependent enzymes, and are now widely used in their isolation (9). More recently it has been reported that RIPs, including ricin and alpha sarcin, were retained by one of the triazine dyes: Cibacron Blue F3GA (10-12) and it has been hypothesized that the interaction takes place through the RIP enzyme region. A large number of plant RIPs have been used for the generation of immunoconjugates with monoclonal antibodies (MAbs) directed against human carcinoma cell lines (13,14). More recently we reported that restrictocin could successfully be used as a toxic agent for immunoconjugate generation with MAbs against ovarian and mammary carcinoma cell lines (15,16). Since it 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

554

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

has previously been reported that immunoconjugates could be purified by exploiting RIP retention on Cibacron Blue F3GA (11), in this report we studied the interaction between aspergillin toxins and triazine dyes and compared the results with the retention of other RIPs on Cibacron Blue F3GA.

MATERIAL AND METHODS Toxins: Alpha sarcin was purified from the culture medium of the Aspergillus giganteus strain MDH 18894, as described by Olson et al (2). Mitogilllin and briodin were kindly provided in purified form respectively by Dr. D. Vazquez (Centro de Biologia Molecular, Spain) and by Dr. F. Dosio (Chimica Farmaceutica, University of Turin, Italy). The ricin-A chain (RTA) and PAP-I were purchased from Calbiochem (La Jolla, CA, USA) and gelonin was obtained from Boehringer (Mannheim, GmbH,W.Germany). Target cells: The human tumor cell lines MCF-7 (mammary carcinoma), MeWo (melanoma) and OVCA 432 (ovarian carcinoma) were respectively provided by the ATCC (Rockville, MD, USA) by the late Dr. J. Fogh (Memorial Sloane-Kettering Cancer Center, N.Y.) and by Dr. R. Knapp (Dana Farber Institute, Boston, Mass, USA). The cell lines were regularly checked for mycoplasma contamination. Triazine dye immobilization: Cibacron Blue F3GA (Sigma, USA) or Procion Red (Sigma, USA) were covalently coupled with Sepharose CL-4B (Pharmacia, Uppsala, Sweden) according to the method described by Dean and Watson (9), and the obtained resins were respectively named Blue Sepharose and Red Sepharose. Already immobilized Cibacron Blue F3GA dye (Affi-Gel Blue, 50-100 mesh) was purchased from BIORAD (Richmond, CA, USA). Chromatography on immobilised Cibacron Blue F3GA dye: Affinity chromatography was performed at room temperature on either Blue Sepharose, Red Sepharose or Affi-Gel Blue according to the experimental conditions respectively reported in Tables 1 and 2. The RIPs were applied and eluted first with the loading buffer (unbound fraction) and then with the same buffer plus 0.5-1 M NaC1 (bound fraction). The obtained fractions were dialyzed against Tris-HC1 0.01 M, NaC1 0.15 M pH 7.4 buffer and analyzed by SDS-PAGE as described (5). In each case both fractions migrate similarly to the unfractionated material. Conjugate preparation: Conjugation between the monoclonal antibody MOvl7 (16) and the unbound fraction of alpha sarcin, obtained after affinity chromatography on Affi-Gel Blue, was performed essentially as described by Conde et al (16). To avoid the alteration of the MAb binding activity due to SPDP (17) a 10x molar ratio SPDP/MAb was employed. Protein synthesis inhibition assays: The cytotoxic activity of the RIPs, of their fractions after affinity chromatography, and of the immunoconjugate was evaluated on in vitro cultured cells by protein synthesis inhibition, as previously described (16). The activities were expressed as the concentration required to inhibit 50% protein synthesis (IC~t~). Inhibition of the cell-free translation of the Tobacco Mosaic virus mRNA (Amersliffm International, Buckinghamshire, England) was measured in the Translation Kit Reticulocytes Type II (Boehringer Mannheim, GmbH, W. Germany). In all cases ICs0 was derived from dose-response curves.

RESULTS. The interaction of alpha sarcin with Blue Sepharose was studied under various experimental conditions of pH and ionic strength, and the relevant results are shown in Table 1. In experimental conditions A, i.e. those previously adopted by Sperti et al. (12), the toxin was entirely retained. In the experimental conditions B, i.e. low ionic strength and change of pH, the retention capability of Cibacron Blue for alpha sarcin was modulated. In fact, at pH values below 5, the alpha sarcin retention was total, whereas at values greater than 5 an unbound fraction, which represents 13-17% of the total recovered material, was obtained. The more consistent unbound fraction was recovered at pH 10, a value which 555

Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1. Alpha sarcin fractionation on Blue Sepharose under various experimental conditions. Samples of 2 ml of alpha sarcin (3 mg/ml) were loaded on Blue Sepharose

columns (15 x 46 mm) in the above-mentioned buffer and the columns were eluted at a flow rate of 25 ml/h, with 0.5-1 M NaC1 added to the same buffer, as described in Material and Methods. Total recovery ranged from 70 to 95%. Buffer characteristics pH

Relative fraction yield

material and molarity (mM)

ionic strength (mS/cm)

Unbound (~1

Bound (~)

Experimental conditions A: 7.4 Tris HC1 10 NaC120

2.68

2.2

97.8

Experimental conditions B: 2-3 Citrate 50 4 Citrate 25 5 Acetate 25 6 Acetate 25 7 NaPhosphate 25 8 NaPhosphate 10 9 Tris HC1 20 10 Tris HC1 200

1.75 1.93 1.61 2.09 2.20 1.85 0.24 0.37

0 0 0 13 16 17 13 39.2

100 100 100 87 84 83 87 60.8

Experimental conditions C: 7.4 NaPhosphate 100

10.30

48

52

approached the alpha sarcin isoelectric point. When Red Sepharose, equilibrated in Tris HC1 0.02 M p H 9 was used similar results were obtained, since an unbound fraction, which represents 20% of the total recovered material, was obtained. On the other hand, in experimental conditions C, i.e. higher ionic strength and p H 7.4, alpha sarcin was recovered equally distributed in bound and unbound fractions. When compared to conditions B, an increase of molarity on the Na-phosphate buffer (from 25 to 100 mM), corresponded to an

TABLE 2. Fractionation of various RIPs on Affi-Gel Blue. A sample of 1 ml of each RIP

(0.5 mg/ml) was loaded on Affi-Gel Blue columns (10 x 25 ram), equilibrated in NaPhosphate 0.1M, pH 7.4 buffer. The columns were eluted at 30 ml/h and the retained protein was eluted with Na-Phosphate 0.1 M, pH 7.4 buffer, 0.5 M NaC1, as described in Material and Methods. Relative fraction yield Toxin

RTA Briodin Gelonin PAP-I Alpha sarcin Mitogillin

Total recovery (%)

86 66 65 88 91 80

556

Unbound (%)

Bound (%)

19 29 4 100 59 90

81 71 96 0 41 10

Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 3. Protein synthesis inhibition activity of various RIPs after Affi-Gel Blue fractionation. Fractions obtained by affinity chromatography on Affi-Gel Blue, as

described in table 2, were assayed for their cytotoxic activity on in vitro cultured cells and inhibition of cell-free translation. Toxin

RTA Bound 2 Unbound Briodin

Bound 2 Unbound

Cytotoxicity (ICs0xl0 -6 M)

cell-free

MCF-7

MeWo

ICs0 (x 10-11 M)

0.11 0.085

0.2

0.9 2.5

0.08 0.60

_1

1.5

0.095 N.D.

0.34 N.D.

2.9

0.65

0.36

0.44

0.24

0.51 0.11

30 0.3

1.3

N.D. N.D.

8.7 1.2

Gelonin

Bound 2 Unbound PAP I Unbound Alpha sarcin

Bound Unbound 2 Mitogillin

Bound Unbound 2

1 A dash (-) indicates protein synthesis inhibition < 20%, even at the maximal dose tested, 10-~M and 10-gM on cytotoxicity and cell-free translation respectively. 2 Prevalent fraction ( see Table 2).

increment of the unbound fraction from 17 to 48%. Experimental conditions C were used in the following chromatographies. Several RIPs were tested for their capability to interact with Affi-Gel Blue and the results are reported in Table 2. The toxins PAP-I and mitogillin were not retained by the column, whereas RTA, briodin and gelonin were mainly recovered in the bound fraction. The retention values of gelonin and R T A were comparable to the previously reported ones (11,12). Alpha sarcin showed a behaviour similar to that exhibited on Blue Sepharose (see Table 1). Noteworthy in the case of AffiGel Blue was a further increase in the unbound fraction (from 48% to 59% respectively on Blue Sepharose and Affi-Gel Blue), probably due to the different Cibacron Blue crosslinker matrix. As reported in Table 3, the Affi-Gel Blue bound and unbound fractions were tested for in vitro toxicity on the human cancer cell lines MCF7 and MeWo. In every case, the fraction which exhibited the highest level of toxicity was the prevalent one. For all RIPs, except alpha sarcin, the activity of the major fraction was comparable to that of the unfractionated toxin (data not shown). The IC50 of the unfractionated alpha sarcin (3.5 x 10-7M) on MeWo cells was comparable to the mean of the values obtained with the bound and unbound fractions (see Table 3). The toxin activity in cell-free translation was also determined (Table 3). All fractions tested showed inhibitory activity and again the prevalent fraction exhibited the highest level of protein synthesis inhibition. For each RIP the ratio between the IC50 obtained with the minor versus the prevalent fraction ranged 557

V o l . 173, N o . 2, 1990

o

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

100-

o

"6

A

50¢n

o 13.

OVCA432 n=4