Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity.
Ricin is a potent plant toxin consisting of two disulfide-bonded subunits. The A chain of ricin is an N-glycosidase which inactivates 28 S RNA and inhibits protein synthesis. The B chain is a galactose-specific lectin with two galactose-binding sites. The genes encoding preproricin and its A and B chains have been cloned and expressed. In addition, X-ray crystallographic studies have identified the galactose-contact residues in both the high- and low-affinity galactose-binding sites of the B chain. In this study, the high-affinity galactose-contact residue of the B chain was changed from Asn-255 to Ala-255 by oligonucleotide-directed mutagenesis. The resulting mutant was sequenced to confirm the presence of a single mutation and was expressed in Cos-M6 cells. Both wild-type and mutant recombinant B chain could be immunoprecipitated with a heterologous anti-B chain antibody and both could form A-B heterodimers. However, as compared to the wild-type, the mutant B chain lacked more than 99% of its lectin activity and cytotoxicity as an A-B dimer. In conclusion, altering the contact residue of the high-affinity galactose-binding site of ricin B chain from Asn-255 to Ala-255 abrogates more than 99% of its lectin activity and the cytotoxicity of the A-B heterodimer to ricin-sensitive cells.
Vitetta
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Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Yen
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D001212:Asialoglycoproteins; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000502:physiology; D004252:DNA Mutational Analysis; D060748:Fetuins; D015870:Gene Expression; D006023:Glycoproteins / Q000502:physiology; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D009841:Oligonucleotides; D033902:Periplasmic Binding Proteins; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012276:Ricin / Q000032:analysis / Q000096:biosynthesis*; D013329:Structure-Activity Relationship; D014162:Transfection; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D060748:Fetuins; D006023:Glycoproteins; D009004:Monosaccharide Transport Proteins; D009841:Oligonucleotides; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; C023550:galactose-binding protein; D012276:Ricin