Identification of Three Oligosaccharide Binding Sites in Ricin

Abstract

The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been clearly established in the ricin B chain by X-ray crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A method was developed using thiol-specific labeling of the ligand combined with subsequent immunoaffinity chromatography which allowed the isolation of ricin B chain peptides covalently linked to the ligand from proteolytic digests of purified blocked ricin. The sites of covalent attachment of the two ligands in blocked ricin were inferred from sequence analysis to be Lys 62 in domain 1 of the B chain and Tyr 148 in domain 2. A minor species of blocked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferred that Tyr 67 in domain 1 is the specific site on the ricin B chain where a third reactive ligand becomes covalently linked to the protein. These results are interpreted as providing support for the notion that the ricin B chain has three oligosaccharide binding sites.

Crystal Structure of Himalayan Mistletoe Ribosome-inactivating Protein Reveals the Presence of a Natural Inhibitor and a New Functionally Active Sugar-binding Site

“…The other aromatic residue, Tyr 64 , which stacks with the pyranose ring of the penultimate sugar (glucose of lactose), is also conserved. The above observations provide evidence for the functional studies carried out on ML-I and ricin (55)(56)(57) and suggest that the 1␤-site may be active in ML-I and ricin. The overall architecture of the 1␤-site of ebulin is also similar to that of HmRIP, ML-I, and ricin (Fig.…”

Genome Sequence and Comparative Analysis of the Solvent-Producing BacteriumClostridium acetobutylicum

“…In particular, a predicted operon located on the C. acetobutylicum megaplasmid (CAP0114 to CAP0120) consists mostly of genes encoding xylan degradation enzymes. Similarly to the cellulosome components, these enzymes possess complex domain architectures, with the oligosaccharidebinding ricin domain (74) typically present at the C terminus; the addition of ricin domain is (so far) a unique feature of this postulated novel system for xylan degradation in Clostridium (Fig. 5B).…”

Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids

“…Ricin lipolytic activity on pNPC 6 , pNPC 8 , pNPC 10 and pNPC 12 Immediately before the experiments, pNPC ' , pNPC ) , pNPC "! or pNPC "# (300 mM in acetonitrile) was added to 1 ml of 50 mM phosphate buffer, pH 7.4, containing 100 µl of 2% (v\v) Triton X-100 in water, to give a final concentration of 30 mM.…”

“…Since its endocytotic properties are very limited, RTA alone shows a very low level of cytotoxicity [7]. RTB is a galactose-specific lectin containing three galactose-binding sites [8][9][10]. Mutation or deletion of any one of these sites reduces the ability of ricin to penetrate into the cell [11].…”

“…More recent studies also showed that RTB [15] and ricin [16] could induce membrane fusion on liposomes. The ability of ricin and RTB to interact with lipids is Abbreviations used : RSA 60 , 60 kDa Ricinus sanguineus ricin ; RCA 60 , 60 kDa Ricinus communis ricin ; RTA, ricin toxin A-chain ; RTB, ricin toxin Bchain ; ATCL6B, acid-treated cross-linked sepharose 6B ; pNPC 2 , p -nitrophenyl acetate ; pNPC 4 , pNP butyrate ; pNPC 6 , pNP hexanoate ; pNPC 8 , pNP octanoate ; pNPC 10 , pNP decanoate ; pNPC 12 , pNP dodecanoate ; DTNB, 5,5h-dithiobis-(2-nitrobenzoic acid) ; BAL-TC 4 , 2,3-dimercapto-1-propanol tributyrate ; DC 10 , dicaprin ; E 600 , diethyl p -nitrophenylphosphate ; τ, lag time. 1 To whom correspondence should be addressed (e-mail pieroni!marseille.inserm.fr).…”