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Primary Structure and Function Analysis of the Abrus precatorius Agglutinin A Chain by Site-directed Mutagenesis

2000; Elsevier BV; Volume: 275; Issue: 3 Linguagem: Inglês

10.1074/jbc.275.3.1897

1083-351X

Chao‐Lin Liu, Chia-Chu Tsai, Su‐Chang Lin, Li-In Wang, Chong-Ing Hsu, Ming‐Jing Hwang, Jung‐Yaw Lin,

Biochemical and Structural Characterization

Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius , is less lethal than abrina (ABRa) in mice (LD 50 = 5 mg/kg versus 20 μg/kg of body weight). Nucleotide sequence analysis of a cDNA clone encoding full-length AAG showed an open reading frame with 1641 base pairs, corresponding to a 547-amino acid residue preproprotein containing a signal peptide and a linker region (two amino acid residues) between the AAG-A and AAG-B subunits. AAG had high homology to ABRa (77.8%). The 13 amino acid residues involved in catalytic function, which are highly conserved among abrins and ricins, were also conserved within AAG-A. The protein synthesis inhibitory activity of AAG-A (IC 50 = 3.5 nm) was weaker than that of ABRa-A (0.05 nm). Molecular modeling followed by site-directed mutagenesis showed that Pro 199 of AAG-A, located in amphiphilic helix H and corresponding to Asn 200 of ABRa-A, can induce bending of helix H. This bending would presumably affect the binding of AAG-A to its target sequence, GpApGpAp, in the tetraloop structure of the 28 S rRNA subunit and could be one of the major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of AAG.