Biological activity of recombinant Ricinus communis agglutinin A chain produced in Escherichia coli

Abstract

DNA encoding Ricinus comtunis agglutinin A chain was ligated into the E. co/i expression VCCIO~ pDS 513. Induced E. coii 71.18 cells which had been transformed with this plasmid express Ricitrus cottrtmmis agglutinin A chain in M soluble and biologically active form. Recombinant Riclnur cmtmnis agglutinin A chain had ribosomal RNA N.glycosidase activity and was approximately IO-fold less active than ricin A chain in a cell-free protein synthesis inhibition assay.Rihtrs cotnmcnis agglutinin A chain; Ricin A chain; Expression; E. co/i

Ribosome-inactivating proteins: progress and problems

“…The first non-toxic type 2 RIP was identified in castor beans which contain, besides ricin, a much less toxic tetrameric Ricinus agglutinin (RCA) whose A and B chains are similar, but not identical, to the corresponding A and B chains of ricin (94 and 83% homologies, respectively [91]). Both the native [92,93] and the recombinant [94] A chains of the agglutinin are 15-and 11-fold, respectively, less active in inhibiting cell-free protein synthesis than the A chains of native and recombinant ricin. The reduced toxicity of RCA may be due to its reduced capacity to translocate [95], and that of ebulin, another nontoxic type 2 RIP, from S. ebulus, to its reduced affinity for galactose [96].…”

Isolation and partial characterization of nigrin b, a non-toxic novel type 2 ribosome-inactivating protein from the bark ofSambucus nigra L.

“…However, in this case, there are not yet data indicating that M. charantia agglutinin is an rRNA N-glycosidase [4], though it probably is if we take into account that the recombinant Ricinus communis four-chain agglutinin A chain has rRNA N-glycosidase activity [28 ]. Whether protein synthesis inhibitory agglutinins must be considered as RIPs or not requires a further study.…”

Molecular mechanism of inhibition of mammalian protein synthesis by some four‐chain agglutinins

“…The fact that both toxic RIPS, ricin and abrin, and the RCA A-chain are ~-~y#sidases acting on the eukaryotic rRNA [3,71, raised the question of whether the native four-chain agglutinins might trigger ribosomal inactivation by the same mechanism, i.e. depurination of the largest rRNA.…”

“…The best known is R communis agglutinin which is formed of two enzymic A chains of nearly 32 kDa each and two galactose-binding B chains of nearly 34 kDa each [1,2]. The R. communis agglutinin A chain has also been shown to be inhibitory to both in vitro rabbit reticulocyte lysates and plant protein synthesis at concentrations of 1.5 ngml-' [3] and 580 pgml-' [4], respectively. The A and B chains of R communis four-chain agglutinin share good sequence homology with the corresponding A and B chains of the ribosome-inactivating protein (RIP) ricin, a highly poisonous dimeric toxin isolated from Ricinus communis [5].…”

“…This suggested the consideration of R. communis four-chain agglutinin as a RIP [6]. In fact, the recombinant R communis agglutinin A chain has recently been proved to be a true RIP [3]. With the exception of this report on the recombinant protein, there are no data on the molecular mechanism of protein synthesis inhibition by the native four-chain agglutinins and consequently, in a recent general review of plant RIPS, they were not compiled as such [7].…”