Skip to main content
SpringerLink
Log in

Comparative studies on kinetics of inhibition of protein synthesis in intact cells by ricin and conjugate of ricin B-chain with momordin

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Ribosome inactivating proteins from plants have been widely used for the preparation of immunotoxins and hormonotoxins, which have potential application in the therapy of diseases such as cancer. However, these hybrid toxins have been found to be less cytotoxic than native ribosome inactivating proteins. Therefore, it is important to understand the factors that control the intrinsic toxicity of RIPs and the hybrid toxins prepared using them. Here, a hybrid toxin has been prepared by coupling ricin B-chain to momordin and the cytotoxicity of this hybrid toxin has been compared to that observed in case of native ricin. In the two cell types used here, thymocytes and macrophages, the conjugate was found to be about 40 fold less toxic than native ricin. Kinetics of inhibition of protein synthesis showed that prior to onset of inhibition the conjugate exhibits a longer lag phase than native ricin. The rates of inhibition of protein synthesis by the conjugate were also found to be slower than ricin. Analysis of the results suggests that in addition to cell surface binding, the B-chain of ricin facilitates another step in the transmembrane translocation of ricin A-chain to the cytosol.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Stirpe F, Barbieri L: Ribosome-inactivating proteins up to date. FEBS Lett 195: 1–8, 1986

    PubMed  Google Scholar 

  2. Barbieri L, Battelli MG, Stirpe S: Ribosome-inactivating proteins from plants. Biochem Biophys Acta 1154: 237–282, 1993

    PubMed  Google Scholar 

  3. Olsnes S, Phil A: Molecular action of toxins and viruses. In: P. Cohen and S. van Heyningen (eds). Elsevier, New York. 1982, pp 51–106

    Google Scholar 

  4. Endo Y, Tsurugi K: RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J Biol Chem 262: 8128–8130, 1982

    Google Scholar 

  5. Endo Y, Tsurugi K, Lambert JM: The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes: The RNA N-glycosidase activity of the proteins. Biochem Biophys Res Commun 150: 1032–1036, 1988

    PubMed  Google Scholar 

  6. Ramakrishnan S, Fryxell D, Mohanraj D, Olson M, Li, BY: Cytotoxic conjugates containing translational inhibitory proteins. Ann Rev Pharmacol Toxicol 32: 579–621, 1992

    Google Scholar 

  7. Thrush GR, Lark LR, Clinchy BC, Vitetta ES: Immunotoxins: An update. Ann Rev Immunol 14: 49–71, 1996

    Google Scholar 

  8. Leonard JE, Wang Q-C, Kaplan NO, Royston I: Kinetics of protein synthesis inactivation in human T-lymphocytes by selective monoclonal antibody-ricin conjugates. Cancer Res 45: 5263–5269, 1985

    PubMed  Google Scholar 

  9. Wawrzynczak EJ, Watson GS, Cumber AJ, Henry Parnell GD, Rieber PE, Thorpe PE: Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin. Cancer Immunol Immunother 35: 289–295, 1991

    Google Scholar 

  10. McIntosh DP, Edwards DC, Cumber AJ, Parnell CJ, Ross WCJ, Forrester JA: Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent. FEBS Lett 164: 17–20, 1983

    PubMed  Google Scholar 

  11. Vitetta ES: Synergy between immunotoxins prepared with native ricin A chains and chemically-modified ricin B chains. J Immunol 136: 1880–1887, 1986

    PubMed  Google Scholar 

  12. Manske JM, Buchsbaum DJ, Vallera DA: The role of ricin B chain in the intracellular trafficking of anti-CD5 immunotoxins. J Immunol 142: 1755–1766, 1989

    PubMed  Google Scholar 

  13. Timar J, McIntosh DP, Henry R, Cumber AJ, Parnell GD, Davies AJS: The effect of ricin B chain on the intracellular trafficking of an A chain immunotoxin. Br J Cancer 64: 655–662, 1991

    PubMed  Google Scholar 

  14. Newton D, Wales R, Richardson PT, Walbridge S, Saxena S, Ackerman EJ, Roberts LM, Lord JM, Youle R: Cell surface and intracellular functions for ricin galactose binding. J Biol Chem 267: 11917–11922, 1992

    PubMed  Google Scholar 

  15. Youle RJ, Murray GJ, Neville DM Jr: Studies on the galactose-binding site of ricin and the hybrid toxin Man6P-ricin. Cell 23: 551–559, 1981

    PubMed  Google Scholar 

  16. Lambert J, Goldmacher VS, Collinson AR, Nadler LM, Blattler WA: An immunotoxin prepared with blocked ricin: A natural plant toxin adapted for therapeutic use. Cancer Res 51: 6236–6242, 1991

    PubMed  Google Scholar 

  17. Goldmacher VS, Lambert JM, Blatter WA: Interaction of gelonin with macrophages: Effect of lysosomotropic amines. Biochem Biophys Res Commun 183: 758–766

  18. Houston LL, Ramakrishna S, Hermodson MA: Seasonal variations in different forms of pokeweed antiviral protein, a potent inactivator of ribosomes. J Biol Chem 258: 9601–9604, 1992

    Google Scholar 

  19. Goldmacher VS, Blattler WA, Lambert JM, Mcintyre G, Stewart J: Cytotoxicity of gelonin conjugated to targeting molecules: Effects of weak amines, monensin, adenovirus, and adenoviral capsid proteins penton, hexon, and fiber. Mol Pharmacol 36: 818–822, 1989

    PubMed  Google Scholar 

  20. Hegde R, Podder SK: Studies on the variants of the protein toxins ricin and abrin. Eur J Biochem 204: 155–165, 1992

    PubMed  Google Scholar 

  21. Olsnes S: Ricin and ricinus agglutinin, toxic lectins from castor bean. Meth Enzymol 50: 330–335, 1978

    PubMed  Google Scholar 

  22. Knowles PP, Thorpe PE: Purification of immunotoxins containing ricin A-chain and abrin A-chain using blue sepharose CL-6B. Anal Biochem 160: 440–443, 1988

    Google Scholar 

  23. Barbieri L, Stoppa C, Bolognesi A: Large scale chromatographic purification of ribosome-inactivating proteins. J Chromatogr 408: 235–243, 1987

    Google Scholar 

  24. King TP, Li Y, Kochoumian L: Preparation of protein conjugates via intermolecular disulphide bond formation. Biochemistry 17: 1499–1511, 1978

    PubMed  Google Scholar 

  25. Ellman GL: Tissue sulphydryl groups. Arch Biochem Biophys 82: 70–77, 1959

    PubMed  Google Scholar 

  26. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970

    PubMed  Google Scholar 

  27. Blum H, Brier H, Gross HJ: Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93–99, 1987

    Google Scholar 

  28. Frankel A, Burbage C, Fu T, Tagge E, Chandler J, Willingham M: Ricin toxin contains at least three galactose-binding sites located in B chain subdomains 1 alpha, 1 beta, and 2 gamma. Biochemistry 35: 14749–14756, 1976

    Google Scholar 

  29. Sambrook J, Fritsch EF, Manniatis T: Molecular Cloning: A Laboratory Manual. Vol. 3, 2nd edition. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY

  30. Hegde R, Karande AA, Podder SK: The variants of the protein toxins abrin and ricin. A useful guide to understanding the processing events in the toxin transport. Eur J Biochem 215: 411–415, 1993

    PubMed  Google Scholar 

  31. Mosier DE: Separation of macrophages on plastic and glass surfaces. Meth Enzymol 108: 294–304, 1984

    PubMed  Google Scholar 

  32. Ho WKK, Liu SC, Shaw PC, Yeung HW, Ng TB, Chan WY: Biochem Biophys Acta 1088: 311–314, 1991

    PubMed  Google Scholar 

  33. Simmons BM, Stahl PD, Russel JH: Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation. J Biol Chem 261: 7912–7920, 1986

    PubMed  Google Scholar 

  34. Magnusson S, Berg T, Turpin E, Frenoy JP: Interactions of ricin with sinusoidal endothelial rat liver cells. Different involvement of two distinct carbohydrate-specific mechanisms in surface binding and internalization. Biochem J 277: 855–861, 1991

    PubMed  Google Scholar 

  35. Foxwell BMJ, Donovan TA, Thorpe PE, Wilson G: The removal of carbohydrates from ricin with endoglycosidases H, F and D and alphamannosidase. Biochem Biophys Acta 840: 193–203, 1985

    PubMed  Google Scholar 

  36. Kimura Y, Hase S, Kobayashi Y, Kyogoku Y, Ikenaka T, Funatsu G: Structures of sugar chains of ricin D. J Biochem 103: 944–949, 1988

    PubMed  Google Scholar 

  37. Fiani ML, Blum JS, Stahl PD: Endosomal proteolysis precedes ricin A-chain toxicity in macrophages. Arch Biochem Biophys 307: 225–230, 1993

    PubMed  Google Scholar 

  38. Riccobono F, Fiani ML: Mannose receptor dependent uptake of ricin A1 and A2 chains by macrophages. Carbohydr Res 282: 285–292, 1996

    PubMed  Google Scholar 

  39. Kimura Y, Minami Y, Yokuda T, Nakajma S, Takagi S, Funatsu G: Primary structures of N-linked oligosaccharides of momordin-a, a ribosome-inactivating protein from Momordica charantia seeds. Agric Biol Chem 55: 2031–2036, 1991

    PubMed  Google Scholar 

  40. Madan S, Ghosh PC: Interactions of ricin with sinusoidal endothelial rat liver cells. Different involvement of two distinct carbohydratespecific mechanisms in surface binding and internalization. Exp Cell Res 198: 52–58, 1992

    PubMed  Google Scholar 

  41. Cavallaro U, del Vecchio A, Lappi DA, Soria MR: A conjugate between human urokinase and saporin, a type-1 ribosome-inactivating protein, is selectively cytotoxic to urokinase receptor-expressing cells. J Biol Chem 268: 23186–23190, 1993

    PubMed  Google Scholar 

  42. Cavallaro U, Nykjaer A, Nielsen M, Soria MR: Alpha 2-macroglobulin receptor mediates binding and cytotoxicity of plant ribosomeinactivating proteins. Eur J Biochem 232: 165–171, 1995

    PubMed  Google Scholar 

  43. Moestrup SK, Gliemann J, Pallesen G: Distribution of the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues. Cell Tissue Res 269: 375–382, 1992

    PubMed  Google Scholar 

  44. Marsh JW: Cellular processing of a ricin-antibody conjugate. A kinetic analysis of the rate-limiting step. J Biol Chem 264: 10405–10410, 1989

    PubMed  Google Scholar 

  45. Olsnes S, Sandvig K, Refsnes J, Phil A: Rates of different steps involved in the inhibition of protein synthesis by the toxic lectins abrin and ricin. J Biol Chem 251: 3985–3992, 1976

    PubMed  Google Scholar 

  46. Esworthy RS, Neville DM Jr: A comparative study of ricin and diphtheria toxin-antibody-conjugate: Kinetics on protein synthesis inactivation. J Biol Chem 259: 11496–11505, 1984

    PubMed  Google Scholar 

  47. Simpson JC, Lord JM, Roberts LM: Point mutations in the hydrophobic C-terminal region of ricin A chain indicate that Pro250 plays a key role in membrane translocation. Eur J Biochem 232: 458–463, 1995

    PubMed  Google Scholar 

  48. Rapak A, Falnes P, Olsnes S: Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol. Proc Natl Acad Sci (USA) 94: 3783–3788, 1997

    Google Scholar 

  49. Ippoliti R, Lendaro E, D'Agoatino I, Fiani ML, Guidarini D, Vestri S, Benedetti PA, Brunori M: A chimeric saporin-transferrin conjugate compared to ricin toxin: Role of the carrier in intracellular transport and toxicity. FASEB J 9: 1220–1225, 1995

    PubMed  Google Scholar 

  50. Braham K, Junqua S, Tursz T, Le Pecq J-B, Lipinski M: Kinetic analysis of choriocarcinoma cell intoxication induced by ricin and ricin A chain immunotoxin. Cancer Res 48: 806–811, 1988

    PubMed  Google Scholar 

  51. Ramalingam TS, Das PK, Podder SK: Ricin-membrane interaction: Membrane penetration depth by fluorescence quenching and resonance energy transfer. Biochemistry 33: 12247–12254, 1994

    PubMed  Google Scholar 

  52. Ishida B, Cawley DB, Reue K, Wisnieski BJ: Lipid-protein interactions during ricin toxin insertion into membranes. Evidence for A and B chain penetration. J Biol Chem 258: 5933–5637, 1983

    PubMed  Google Scholar 

  53. Beaumelle B, Alami M, Hopkins CR: ATP-dependent translocation of ricin across the membrane of purified endosomes. J Biol Chem 268: 22661–23669, 1993

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, Indian Institute of Science, Bangalore, India

    Shalini Sharma, Sunil K. Podder & Anjali A. Karande

Authors
  1. Shalini Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sunil K. Podder
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anjali A. Karande
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sharma, S., Podder, S.K. & Karande, A.A. Comparative studies on kinetics of inhibition of protein synthesis in intact cells by ricin and conjugate of ricin B-chain with momordin. Mol Cell Biochem 200, 133–141 (1999). https://doi.org/10.1023/A:1007043218769

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1007043218769

Search

Navigation