Chen Ying, Rouge Pierre, Peumans Willy J, van Damme Els J M
Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Leuven, Belgium.
Biochem J. 2002 Jun 1;364(Pt 2):587-92. doi: 10.1042/BJ20020006.
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.
黑接骨木凝集素I（SNA-I）是一种2型核糖体失活蛋白。采用定点诱变来模拟将果实特异性SNA（SNA-If）的高活性B链转化为密切相关的天然存在的失活突变体黑接骨木凝集素相关蛋白的完全无活性B链。在第一个突变体SNA-If-M1中，通过将假定的关键残基天冬氨酸231替换为谷氨酸231，破坏了SNA-If的高亲和力位点2。在双突变体SNA-If-M2中，SNA-If-M1的位点1也通过将假定的关键残基天冬酰胺48替换为丝氨酸48而被破坏。亲本2型核糖体失活蛋白和两个突变体均在烟草品种三生NN中表达，对重组蛋白进行了纯化和分析。重组SNA-If凝集兔红细胞的效果与SNA-If相当，但两个突变体在此试验中均完全无活性。与固定化半乳糖和胎球蛋白的结合试验表明，天冬氨酸231→谷氨酸231的突变使B链对半乳糖和胎球蛋白的亲和力降低了50%以上。此外，第二个突变天冬酰胺48→丝氨酸48的引入使结合活性降低至原始活性的20%以下。
