Differential toxicity profile of ricin isoforms correlates with their glycosylation levels.

Ricin is one of the most potent and deadly plant toxins from the seeds of Ricinus communis. In view of its high toxicity, ricin is being used as an immunotoxin in cancer therapy. Ricin also has several isoforms with differential glycosylation depending on the seed variety. Our study shows three isoforms designated 1, 2 and 3, which differed in their surface charge, resulting in a different behavior on cation exchange chromatography, two dimensional (pI 5.5-8.7) and native PAGE. The molecular masses of isoform-1, 2 and 3 were measured as 63.55 kDa, 64.03 kDa and 62.8 kDa, respectively, by MALDI-TOF/MS. In vitro studies with monkey kidney (Vero) cells showed a time dependent increase in cytotoxicity of the isoforms evaluated by extracellular lactate dehydrogenase activity and mitochondrial dehydrogenase assay. These isoforms also induce oxidative stress and DNA damage. Among the isoforms, isoform-3 was quick to generate reactive oxygen species (ROS), (in 90 min) and exhibited maximum cytotoxicity. Morphological changes, catalase activity and DNA fragmentation were significantly higher with isoform-3 treatment compared to others. The glycosylation studies by MALDI-TOF/MS showed that isoform-3 is highly glycosylated with high sugar levels containing more of hybrid/complex type glycopeptides with mannose as hexose units. These experimental evidences clearly suggest that isoform-3 is superior in its early ROS generation, potency to induce oxidative stress and cytotoxicity, that could be due to it's higher glycosylation levels which make isoform-3 as an ideal candidate for immunotoxin studies.

Purification, characterization and toxicity profile of ricin isoforms from castor beans.

The castor seed contains the toxin ricin, one of the most poisonous naturally occurring toxins. The whole of the plant is poisonous, however the seeds are considered the major source of ricin. Ricin exists in different forms in beans of different origin. We investigated the presence of ricin in different isoforms and elucidate some of their structural and biological features isolated from the castor seeds. The isoforms were sub fractionated into ricin I, II and III by chromatography. Their molecular weights lie between 60-65 kDa with difference in their relative electrophoretic mobility. An acidic native PAGE of ricin isoforms at pH 2.9 was performed. Ricin I, II and III are highly cytotoxic against Vero cell line with IC(50) values of 60, 30 and 8 ng/ml respectively. Difference in cytotoxicity of isoforms was confirmed through hemagglutination assay, ricin III caused high degree of hemolysis. The preliminary in vivo toxicity studies showed that ricin III is highly toxic. Immunological studies revealed that anti-ricin I and II antibodies are cross reactive with all the ricin variants, whereas the anti-ricin III antibody is highly specific. The present study shows that anti-ricin I and II antibodies can be used for detection of entire ricin isoforms.

Denatured ricin can be detected as native ricin by immunological methods, but nontoxic in vivo.

Ricin is a glycoprotein from Ricinus communis seeds. It is known to have diverse toxic effects on cells of different visceral organs. In the present study, we purified and denatured ricin in a boiling water bath for different time intervals. We further made an attempt to identify native and denatured ricin by immunobased detection systems. All the antigen/antibody-based assays identified native and denatured ricin. On SDS-PAGE, only native ricin was observed. In western blotting, ricin boiled for 3.75 min gave a strong band on X-ray film. On native polyacryl amide gel electrophoresis, native and denatured ricin gave ricin band in 60-kDa region. The denatured ricin did not [corrected] cause mortality up to 25 mg/kg, while 5 and 10 microg/kg of native ricin caused 50% and 100% mortality, respectively. Detection of native and denatured ricin is very difficult, and the investigating agencies, forensic scientists, and analysts should be very careful while interpreting the results.