Analysis of the functional properties of ricin B chain

Purvis, Anne Rosemary (1987) Analysis of the functional properties of ricin B chain. MSc(R) thesis, University of Glasgow.

Abstract

Ricin is isolated from the plant Ricinus communis, and is an extremely toxic protein. The protein consists of two functionally distinct polypeptides, ricin A chain which will inhibit protein synthesis in eucaryotic cells, and the ricin B chain which acts as a lectin binding to galactose-containing cell surface macromolecules. Ricin has been extensively studied in attempt to determine both the biochemical and physical properties of the protein. Ricin has been attributed with anti-tumorogenic properties, and has recently been used in the synthesis of immunotoxins. Studies on the interactions of immunotoxin molecules have indicated that the ricin B polypeptide may not function simply as a lectin. When cells were treated with a weakly toxic dose of an antibody - ricin A conjugate, the cytotoxicity was less than that of an antibody-ricin conjugate. The cytotoxicity of the antibody-ricin A conjugate can be greatly enhanced by the addition to the culture system of either free ricin B polypeptide or an antibody-ricin B complex. Thus it would appear that the ricin B chain was responsible for promoting access of the ricin A chain into the cytosol. It is this putative transport function of the ricin B chain that this project sought to address. Before the principle experimental investigation could be undertaken a method for the purification of the commercially produced ricin B chain had to be developed, for it was found to contain ricin holotoxin. This was achieved by chromatofocusing for the isoelectric point of ricin is 7.1 and ricin B chain is 4.8, and this difference allowed separation. Affinity chromatography using Sepharose 4B pretreated with 1.0M propionic acid, as the column matrix, was utilised to separate the polybuffer (chromatofocusing buffer) from the purified ricin B chain. This also had the added advantage of confirming that the ricin B polypeptide retained its ability to function as a lectin. The purity of the ricin B chain was ascertained by silver staining of SDS-PAGE gels and cytotoxicity assays. To study the 'hypothetical' transport function of the ricin B polypeptide, a complex was synthesised between bovine pancreatic RNase A and ricin B chain. Bovine pancreatic RNase A was chosen for a number of reasons, the main one being the possibility of determining the internalisation of the RNase A by the ricin B chain due to the enzymatic digestion of cellular RNA which could be measured by a decrease in incorporation of [35S]-methionine or [3H]-thymidine by the cell cultures. The heterobifunctional cross-linking reagent, SPDP, was chosen to link the RNase A molecule to the ricin B polypeptide, so that a disulphide bridge was formed between the two proteins. This duplicates the disulphide linkage normally found between the ricin A and ricin B chains. The formation of the RNase A-ricin B complex was determined by silver staining of SDS-PAGE gels. The putative complex had a Mr of 45,000, indicating a complex containing equimolar amounts of RNase A and ricin B chain. Immunoblotting experiments confirmed that the 45kD species visualised on SDS-PAGE contained antigenic determinants derived from both RNase A and ricin B chain molecules. Two dimensional gel electrophoresis of iodinated RNase A-ricin B complex confirmed the formation of the complex as well as showing that the complex was basic having an isoelectric point in the range pH 8.0-8.5. The RNase A-ricin B complex had to retain the functional activity of both the proteins, so that its effect on Daudi cells in culture could be examined. The enzymatic activity of the RNase A present in the RNase A-ricin B complex was assessed using activity gels. Areas of enzymatic digestion of the yeast RNA present in the activity gel were visualised as clear plaques. Iodination of RNase A then linkage of this to the ricin B polypeptide showed that the ricin B chain was binding to galactose-containing molecules on the cell surface. Unfortunately, when the effects of the RNase A-ricin B complex were examined on cells in culture, no effect on DNA or protein synthesis due to the enzymatic action of RNase A was observed. The results therefore confirmed earlier work which showed that insulin-ricin B conjugate, bound to galactose containing receptors present on the cell surface. The question of the internalisation of the chimeric protein was not answered, although it is known that hybrid proteins formed between abrin A-ricin B or gelonin-ricin B are toxic to cells which could reflect a synergistic mode of action of the toxic molecule and the lectin molecule from plants.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Keywords: Biochemistry
• Date of Award: 1987
• Depositing User: Enlighten Team
• Unique ID: glathesis:1987-76705
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Dec 2019 09:15
• Last Modified: 19 Dec 2019 09:15
• URI: https://theses.gla.ac.uk/id/eprint/76705