Modification of the carbohydrate in ricin with metaperiodate-cyanoborohydride mixtures. Effects on toxicity and in vivo distribution.
Attempts to target antibody-ricin conjugates (immunotoxins) to designated cell types in vivo may be thwarted by their rapid clearance by hepatic reticuloendothelial cells which have receptors that recognise oligosaccharide side chains on the toxin. The B-chain of ricin contains high mannose type oligosaccharides and the A-chain contains a complex unit (GlcNAc)2-Fuc-Xyl-(Man)4-6, all of which potentially could be recognised by the reticuloendothelial system. Treatment of ricin with a mixture of sodium metaperiodate and sodium cyanoborohydride at pH 3.5 resulted in oxidative cleavage of the carbohydrates and reduction of the aldehyde groups thus formed to primary alcohols. By conducting the modification procedure at acidic pH, both the possibility of Schiff's base formation between the aldehyde groups and amino groups in the protein and the possibility of non-specific oxidation of amino acids were minimised. The extent of the carbohydrate modification depended on the duration of treatment, resulting maximally in the destruction of 13 of the 18 mannose residues and of all xylose and fucose. The toxicity of the modified toxin to cells in culture declined by up to 90% as the carbohydrate was destroyed. This was not due to a reduced ability of the B-chain to bind to cells or of the A-chain to inactivate ribosomes. In contrast to the in vitro results, the toxicity of the modified toxin to mice and rats was elevated by up to fourfold. The modification greatly reduced the clearance of the toxin by non-parenchymal cells in the liver and prevented the damage to hepatic Kupffer and sinusoidal cells and to the red pulp of the spleen that is inflicted by the native toxin. The elevated toxicity to animals appears to be because the modified toxin evades the reticuloendothelial system and persists in the bloodstream for longer periods, thus resulting in lethal damage to vital tissues in the animal at lower dosage. The results suggest that immunotoxins prepared from modified ricin would not be readily cleared by the reticuloendothelial system and so be more effective at killing their target cells.
Thorpe
P E
PE
Detre
S I
SI
Foxwell
B M
BM
Brown
A N
AN
Skilleter
D N
DN
Wilson
G
G
Forrester
J A
JA
Stirpe
F
F
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001894:Borohydrides; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D003208:Concanavalin A / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008806:Mice, Inbred AKR; D008808:Mice, Inbred CBA; D010084:Oxidation-Reduction; D010504:Periodic Acid; D051381:Rats; D011919:Rats, Inbred Strains; D012276:Ricin / Q000378:metabolism / Q000633:toxicity*; D014018:Tissue Distribution
D000596:Amino Acids; D001894:Borohydrides; D002241:Carbohydrates; D010504:Periodic Acid; D003208:Concanavalin A; D012276:Ricin; C009288:metaperiodate; C009282:sodium cyanoborohydride; D007930:Leucine