1979 Biochem J. 186, pp. 633-635

by Luigi Barbieri, Enzo Lorenzoni, Florenzo Stirpe
Protein synthesis by a rabbit reticulocyte lysate is inhibited by the heamagglunating lectins from Momordica charantia and Crotalaria juncea seeds…

Inhibition of Protein Synthesis in Vitro by a Lectin from Momordica charantia and by Other Haemagllutinins

Author: Luigi Barbieri, Enzo Lorenzoni, Florenzo Stirpe

Type of Publication: Pre-Clinical

Date of Publication: 1979

Publication: Biochem J. 186, pp. 633-635, 1979

Organization: Istituto di Patologia generale dell’ Universita di Bologna

Protein synthesis by a rabbit reticulocyte lysate is inhibited by the heamagglunating lectins from Momordica charantia and Crotalaria juncea seeds and from the role of Rutilus rutilus, and by a commercial preparation of the mitogenic lectin from Phytolacca Americana. The haemagglutinins from the seeds of Ricinus communis and of Vicia cracca aquired inhibitory activity after theor reduction with 2-mercapoethanol.

Lectins are protein or glycoproteins with binding sites for specific carbohydrates groups (Lis and Sharon, 1973; Liener, 1976; Brown & Hunt, 1978), which have interesting biological properties, in that many of them agglutinate erythrocytes or other cells, and some are mitogenic to lymphocytes. Among lectins there are three potent toxins from plants, namely ricin, brin (review by Olsnes & Pihl, 1977) and modeccin Refsnes et al., 1977; Stirpe et al., 1978), which are potent inhibitors of protein synthesis in cells and in cell-free systems.

Various plant material contain proteins which are non-toxic or scarcely toxic to animals and which inhibit protein stnthesis in cell-free systems but not in whole cells, presumably because they cannot enter into cells (Obrig et al., 1973; Irvin, 1975; Stirpe et al., 1977; Gasperi-Campani et al., 1977; Stewart et al., 1977; A. Gasperi-Campani, L. Barbieri, P. Morelli & F. Stirpe, unpublished work). Some these proteins were purified (Obrig et al., 1973; Irvin, 1975) or semi-prurified (Stirpe et al., 1976; Sperti et al., 1976), and these cases it was ascertained that they inhibit protein synthesis through the same mechanism as do ricin and the other toxins mentioned above, i.e. by inactivating enzymatically the 60S ribosomal subunit, and making it unable to bind the elongation factor 2.

Lin et al.(1978) reported two lectins (mol. wts. 32,000 and 24,000) from the seeds of a Cucurbitaceae, Momordica charantia (bitter pear melon), the latter inhibiting proteins synthesis by Ehrlich ascites cells at concentrations relatively high(from 100 g/ml) as compared with the toxic lectins.

We report that a haemagglutinating lectin from the seeds of Momordica charantia is a potent inhibitor of protein synthesis in acell-free system (a lysate a rabbit reticulocytes). Inhibition of protein snthesis in the same system was obtained also with lectins from other seeds from a fish roe.

Experimental

Materials

Seeds of Momordica charantia, originally from India, were obtained from Mr. F. G. Celo, Zweibrucken, West Germany, and the haemagglutinating lectin was purified by affinity chromatography on Sepaharose 4B as described by Tomita et al. (1972) with minor modifications. Ricinus communis agglutinin was prepared as described by Nicolson & Blaustein (1972), and other lectins were obtained from the sources listed in Table1. Other chemicals were from the same sources as described by Gasperi-Campani et al. (1978).

Methods

Protein synthesis was determined as described by Gasperi-Campani et al. (1978) with a lysate of rabbit reticulocytes prepared as described by Allen & Schweet (1962) or with Yoshida AH-130 ascites cells. Protein was determined by the method of Lowry et al. (1951) or spectrophotometrically (Kalb & Bernlohr, 1977).

Results and Discussion

The lectin purified by affinity chromatography from Momordica charantia seeds a single band on a polyacrylamide-gel electrophoresis in either the presence or the absence of sodium dodecyl sulphate, had mol. wt. 115000, and agglutinated human erythrocytes (group ), the lowest active concentration being 24 ng/ml.

This agglutinin inhibited protein synthesis by a lysate of rabbit reticulocytes: the ID₅₀ (concentration giving 50% inhibition) was 1.74 g/ml, i.e. similar to those of the toxic lectins mentioned above (e.g. modeccin; see Stirpe et al., 1978). The inhibition was unchanged in the presence of 100 mM-galactose, which inhibits haemagllutination by the lectin, as shown by Tomita et al. (1970) and confirmed with our preparation. This indicates that the inhibitory effect on protein synthesis is independent of the haemagglutinating property of the lectin.

The lectin at 100 g/ml inhibited by 30% protein synthsesis by Yoshida ascites cells: 50 g/ml had no effect. The fact that relatively high concentrations, as compared with the toxic lectins, are required to affect protein synthesis by wgole cells suggests that Momordica charantia lectin enters with difficulty into cells, and this in turn could account for its low toxicity to animals: it did not cause any apparent harm to rats when injected intraperitoneally at the dose of 1 mg/mg 100 g body wt.

Momordica charantia lectin is the second example of non-toxic lectin inhibiting protein synthesis in vitro, after the Ricinus communis agglutinin (distinct from ricin), as shown by Saltedt (1976) and by Cawley et al. (1978). This led us to the hypothesis that other lectins could have same property. Therefore 27 lectins were examined, before and after reduction with 2-mercaptoethanol, a treatment which greatly enhances the inhibitory effects of ricn and abrin (Olsnes & Phil, 1972) and of modeccin (Refsnes et al., 1077; Gasperi-Campani et al., 1978), lectins were inhibitory (Table 1), and among the already known agglutinin from Ricinus communis the Momordica charantia lectin prepared by a recent method (Horejsi & Kocourek, 1978) in and laboratory, and the pokeweed mitogen. The inhibitory actitivity of the latter was observed before (Gasperi-Campani et al., 1977), and was attributed to tamination by the powerful pokeweed and peptide (Irvin, 1975). It was comfirmed that mercial preparations of pokeweed mitogen 0.3% of pokeweed antiviral peptide, which is eficeint to account5 for the inhibition(J. D. personal communication). It is noteworthy the lectin from the roe of the fish Rutilus rutilus was inhibitory. Reduction with 2-mercaptoethanol hanced greatly the effects of the lectins from Ricinus communis and from Vicia cracca, and abolished effect of the agglutinin from Ruticulus ruticulus.

It is not known whether all these lectins protein synthesis through the same mechanism. However, our results demonstrate that the capable og inhibiting protein synthesis is common to lectins, presumably to many othersa besides identified by the present experiments, and thus show considered another general property of lectins, the mitogenic activity.

Acknowledgements

We thank Dr. B. Ersson, Dr. A. Falasca, Dr. L. G. Dr. J. Kocourek, Dr. T. Kurokawa and Dr. F. Cessi for generous gifts of lectins. The research supported by a grant from the Consiglio Nazionale Ricerche, Rome, within the Progetto finalizzato trollo della crescitaneoplastica, and by the Pallotti’s for cancer research.

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