Ebulin 1, a nontoxic novel type 2 ribosome-inactivating protein from Sambucus ebulus L. leaves.

T Girbés,L Citores,R Iglesias,J.M Ferreras,R Muñoz,M.A Rojo,F.J Arias,J.R García,E Méndez,M Calonge

doi:10.1016/s0021-9258(17)46829-3

Abstract

A novel type 2 ribosome-inactivating protein (RIP) that we named ebulin 1 has been isolated from leaves of Sambucus ebulus L. (Caprifoliaceae). In vitro ebulin 1 strongly inhibited protein synthesis by rabbit reticulocyte lysates, rat brain, and rat liver cell-free systems but did not affect in vitro plant nor bacterial protein synthesis. Ebulin 1 is composed of two subunits, a catalytic A subunit (M(r) 26,000) and a D-galactose-binding lectin B subunit (M(r) 30,000). Amino-terminal amino acid sequence homology revealed the novelty that the ebulin 1 A-chain shares a high degree of homology not with the A-chain of other type 2 RIPs but rather with the Cucurbitaceae type 1 RIP briodin S and the anti-human immunodeficiency virus type I proteins trichosanthin and TAP 29. Upon treatment with acid aniline the rRNA from ebulin 1-treated rabbit reticulocyte ribosomes released the RNA fragment which is diagnostic of RIP catalytic action. Ebulin 1 was nontoxic to mice up to 2 mg/kg of body weight and did not inhibit protein synthesis in cultured NHC human epithelial cells which are highly sensitive to ricin.

Highlights

Mice up to 2 mg/kg of body weight and did not inhibit 29 [16] are agentsthat aredirectly active against anti-human protein synthesis in cultured NHC human epithelial immunodeficiency virus type I
In the present work we report that the leaves of S. ebulus L. contain a new type 2 ribosome-inactivating protein (RIP), named ebulin 1, which strongly inhibited protein synthesis, displayed the rRNA N-glycosidase activity on mammalian ribosomes, which is characteristic of RIPs, and is not toxic to mice nor to cultured mammalian cells
During preliminary screening studies on protein synthesis inhibition by plant extracts in our search for RIPs we found that S. ebulus L. crude proteinextracts strongly inhibited protein synthesis antdhat itagglutinated red blood cells(data not shown)

Summary

To whom correspondence should be addressed

Dept. de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Valladolid, E-47005 Valladolid, Spain. Sepharose 6B was from Pharmacia Iberica (Madrid, Spain), and the treatmentwith acid was performed at 50 “C for 3 h with 0.1 N HCl. Ricin and the pokeweed antiviral protein from roots (PAP-R)were generous gifts from Prof. Preparation of the mammalian cell-free systems and the assays of polypeptide synthesis by rabbit reticulocyte lysates, rat brain, and rat liver, measured as incorporation of ~ - [ ~ ~ S ] methionine into nascent peptides, were performed as described elsewhere [21, 22]. Preparation of the E. coli ribosomes and high speed supernatant andpoly(Phe) poly(U)-directed synthesis was carried out as described elsewhere [25].Incorporation of radioactivity into proteins was assessed by the addition of 0.5 ml of 0.1 N. The membranes were washed in methanol:water:acetic acid (4847:5),and the protein bands were cut out of the poly(viny1idenefluoride) and applied to a Knauer sequenator with on-line phenylhydantoin-derivative analyzer according to manufacturer’s instructions. 11 2 3 4 5 6 7 1 nine for 2 h at 37 “C. The radioactivity incorporated into proteins was determined as described elsewhere [28]

RESULTS

DISCUSSION

Findings

40. Stirpe