Mutational analysis of the carbohydrate-binding activity of the NeuAc(alpha-2,6)Gal/GalNAc-specific type 2 ribosome-inactivating protein from elderberry (Sambucus nigra) fruits.
Sambucus nigra agglutinin I (SNA-I) is a type 2 ribosome-inactivating protein. Site-directed mutagenesis was used to mimic the conversion of the highly active B-chain of fruit-specific SNA (SNA-If) into the completely inactive B-chain of the closely related and naturally occurring loss-of-activity mutant called S. nigra agglutinin lectin-related protein. In the first mutant SNA-If-M1 the high-affinity site 2 of SNA-If was disrupted by replacing the presumed critical residue Asp231 with Glu231. In the double mutant SNA-If-M2, site 1 of SNA-If-M1 was also disrupted by substituting the presumed critical residue Asn48 with Ser48. The parent type 2 ribosome-inactivating protein and both mutants were expressed in Nicotiana tabacum Samsun NN and the recombinant proteins were purified and analysed. Recombinant SNA-If agglutinated rabbit erythrocytes equally well as SNA-If, but both mutants were completely inactive in this test. Binding assays to immobilized galactose and fetuin revealed that the mutation Asp231-->Glu231 reduces the affinity of the B-chain for galactose and fetuin by more than 50%. Furthermore, the introduction of the second mutation Asn48-->Ser48 reduces the binding activity to less than 20% of the original activity.
Chen
Ying
Y
Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Leuven, Belgium.
Rouge
Pierre
P
Peumans
Willy J
WJ
van Damme
Els J M
EJ
D000116:Acetylgalactosamine / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D037121:Plant Lectins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D027924:Sambucus nigra / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D037102:Lectins; D037121:Plant Lectins; D011994:Recombinant Proteins; D000116:Acetylgalactosamine