Abstract
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 microg/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.
Highlights
Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius, is less lethal than abrina (ABRa) in mice (LD50 ؍5 mg/kg versus 20 g/kg of body weight)
Using molecular modeling and site-directed mutagenesis, we found that Asn200 of ABRa-A and Pro199 of AAG-A are involved in the differences in the protein synthesis inhibition and toxicity of these two type II Ribosome-inactivating proteins (RIPs)
Sequence analysis showed that the AAG cDNA contains 2047 base pairs, with an open reading frame encoding a preproprotein with 547 amino acid residues: a 20-residue signal peptide, a 258-residue polypeptide (AAG-A), a 2-residue linker peptide, and a 267-residue polypeptide (AAG-B)
Summary
Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius, is less lethal than abrina (ABRa) in mice (LD50 ؍5 mg/kg versus 20 g/kg of body weight). We report the determination of the amino acid sequence of AAG by protein techniques and the molecular cloning of the cDNA encoding AAG. Using molecular modeling and site-directed mutagenesis, we found that Asn200 of ABRa-A and Pro199 of AAG-A are involved in the differences in the protein synthesis inhibition and toxicity of these two type II RIPs. Materials—Taq DNA polymerase, the pGEM-T and pGEM-T-easy vectors, and the rabbit reticulocyte lysate system were obtained from Promega (Madison, WI).
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