Ricin B chain-containing immunotoxins prepared with heat-denatured B chain lacking galactose-binding ability potentiate the cytotoxicity of a cell-reactive ricin A chain immunotoxin.
Ricin B chain incubated at 37 degrees C in the absence of lactose loses its ability to bind the galactose-containing protein, asialofetuin. Circular dichroism analysis of the B chain during thermal denaturation indicates that the loss of galactose-binding ability by the B chain correlates with limited unfolding of the molecule. As a result of this conformational change, disulfide bonds that are shielded from the solvent by the compact folded structure of the B chain become exposed and the chitobiosyl cores of both N-linked oligomannose chains become susceptible to cleavage by endoglycosidases. The heat-denatured B chain does not enhance the toxicity of a ricin A chain-containing rabbit anti-human immunoglobulin (RAHIg-A) to Daudi cells. However, when heat-denatured B chain is coupled to goat anti-rabbit immunoglobulin (GARIg), the resulting immunotoxin, GARIg-hdB, potentiates the killing of RAHIg-A-treated Daudi cells to an extent similar to that of an immunotoxin prepared with GARIg and native B chain. These results indicate that the native, galactose-binding structure of the B chain is not necessary to enhance the cytotoxicity of the cell-reactive A chain immunotoxin (IT-A) and suggests that regions of the B chain exposed by unfolding the molecule may mediate potentiation of cytotoxicity.
Wawrzynczak
E J
EJ
Drug Targeting Laboratory, Imperial Cancer Research Fund, London, U.K.
Drake
A F
AF
Watson
G J
GJ
Thorpe
P E
PE
Vitetta
E S
ES
D000276:Adjuvants, Immunologic / Q000633:toxicity*; D002460:Cell Line; D003603:Cytotoxins / Q000633:toxicity*; D004220:Disulfides; D004357:Drug Synergism; D005690:Galactose / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D011487:Protein Conformation; D011489:Protein Denaturation; D011956:Receptors, Cell Surface / Q000502:physiology; D012276:Ricin / Q000633:toxicity*; D013329:Structure-Activity Relationship
D000276:Adjuvants, Immunologic; D003603:Cytotoxins; D004220:Disulfides; D011956:Receptors, Cell Surface; C039707:galactose receptor; D012276:Ricin; D005690:Galactose