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.
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Research Article|
June 01 2002
Mutational analysis of the carbohydrate-binding activity of the NeuAc(α-2,6)Gal/GalNAc-specific type 2 ribosome-inactivating protein from elderberry (Sambucus nigra) fruits
Ying CHEN
;
Ying CHEN
1
∗Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Leuven, Belgium
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Pierre ROUGE
;
Pierre ROUGE
†Institut de Pharmacologie et Biologie Structurale, UMR-CNRS 5089, 205 Route de Narbonne, 31077 Toulouse Cedex, France
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Willy J. PEUMANS
;
Willy J. PEUMANS
∗Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Leuven, Belgium
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Els J.M. van DAMME
Els J.M. van DAMME
2
∗Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Leuven, Belgium
2To whom correspondence should be addressed (e-mail Els.VanDamme@agr.kuleuven.ac.be).
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Biochem J (2002) 364 (2): 587–592.
Article history
Received:
January 02 2002
Revision Received:
February 18 2002
Accepted:
March 25 2002
Citation
Ying CHEN, Pierre ROUGE, Willy J. PEUMANS, Els J.M. van DAMME; Mutational analysis of the carbohydrate-binding activity of the NeuAc(α-2,6)Gal/GalNAc-specific type 2 ribosome-inactivating protein from elderberry (Sambucus nigra) fruits. Biochem J 1 June 2002; 364 (2): 587–592. doi: https://doi.org/10.1042/bj20020006
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