Ricin B-chain

Ricin is a lectin-type globular glycoprotein and consists of A and B chains (Doan, 2004 Li and Pestka, 2008). Ricin is considered a Type 2 ribosome-inactivating protein (RIP) (Millard and LeClaire, 2008). The ricin B chain attaches to the plasma membrane at the galactose receptors, pits are... 

Swimmer, C., Lehar, S.M., McCafferty, 1., ChisweU, D.J., Blattner, W.A., and Guild, B.C. (1992). Phage display of ricin B chain and its single binding domains system for screening galactose-binding mutants. Proc. Natl. Acad. Sci. USA 89, 3756 3760. 

Here we report the results obtained using MIANS-labeling in conjunction with affinity chromatography to map free thiols in reduced, native ricin A-chain. We further describe the results obtained when utilizing this method to isolate ligand-bound ricin B-chain peptides. 

Extinction coefficients used for ricin A-chain and blocked ricin B-chain at 280 nm for 0.1% solutions were 0.765 and 1.48, respectively (Olsnes and Pihl, 1973). The extinction coefficient for MIANS is 20,000 M cm" at 322 nm (Gupte and Lane, 1979). The empirically determined contribution of MIANS to the absorbance of the labeled protein at 280 nm was 0.9 x A320. 

Abbreviations MIANS, 2-(4 -maleimidylanilino)naphthalene-6-sulfonic acid PBS, phosphate buffered saline 20 mM sodium phosphate containing 150 mM sodium chloride, pH 7.2 EDTA, ethylene diamine tetraacetic acid DTT, dithiothreitol GuHCl, guanidine hydrochloride 2-ME, 2-mercaptoethanol RB2L-MIANS, ricin B-chain covalently blocked by two affinity ligands and MIANS labeled TEA, Trifluoroacetic Acid. 

C. Peptide mapping of the Ricin B-chain attachment sites... 

COS cells have been used for the production of recombinant proteins, including HSV-1 glycoprotein B, ricin B chain, placental alkaline phosphatase, thrombomodulin, CD7, von Willebrand factor, human dihydropteridine reductase, human P-glucuronidase, interleukin 5 and human interferon-... 

Sundan, A., Sandvig, K. and Olsnes, S. (1983) Preparation and properties of a hybrid toxin of modeccin A-chain and ricin B-chain. Biochim Biophys Acta, 761, 296-302. 

Ferrini J-B, Martin M, Taupiac M-P et al. (1995). Expression of functional ricin B chain using the bac-ulovirus system. Eur J Biochem, 233,112-111. 

Villafranca JE and Robertas JD (1981). Ricin B chain is a product of gene duplication. J Biol Chem, 256, 554-556. 

With cloned ricin genes available, the most definitive way of altering ricin B chain to make it suitable for inclusion in intact ricin ITs will be by genetic manipulation. In this way the B chain will be structurally modified so that it loses completely its ability to bind galactose but retains its ability to translocate A chain across a membrane. An important indication of the feasibility of this approach was the recent demonstration that these two functions of the B chain reside on separate domains in the molecule. Thus while chloramine-T-treated ricin B chains showed a dramatic reduction in their ability to bind galactose, their ability to potentiate the killing of cells treated with an A-chain IT was virtually unimpaired [112]. 

J. L. Asensio, F. J. Canada, M. Bruix, A. Rodriguez-Romero, and J. Jimenez-Barbero, Studies of the bound conformations of methyl-lactoside and methyl beta-allolactoside to ricin-B chain using transferred NOE experiments in the laboratory and rotating frames, assisted by molecular mechanics and dynamics calculations, Eur. J. Biochem., 230 (1995) 618-630. 

Wales R, Richardson PT, Roberts LM, Woodland HR, Lord JM. Mutational analysis of the galactose binding ability of recombinant ricin B chain. J Biol Chem. 1991 266 (29) 19172 19179. 

Afrin LB, Gulick H, Vesely J, Willingham M, Frankel AE. Expression of oligohistidine-tagged ricin B chain in Spodoptera frugiperda. Bioconjug Chem. 1994 5(6) 539-546. 

Yabe R, Suznki R, Kuno A, Fujimoto Z, Jigami Y, Hirabayashi J (2007) Tailraing a novel sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein by natural evolutirai-mimicry. J Biochem 141 389-399... 

Ricin B-chain (RTB) is a 262-amino acid protein domain that conforms to a barbell-shaped architecture. 

Guglielmo-Viret, V, ThulHer, R, 2007. Comparison of an electrochemilumines-cence assay in plate format over a colorimetric ELISA, for the detection of ricin B chain. J. Immunol. Methods 328,70-78. 

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