Primary structure and function analysis of the Abrus precatorius agglutinin A chain by site-directed mutagenesis. Pro(199) Of amphiphilic alpha-helix H impairs protein synthesis inhibitory activity.
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.
Liu
C L
CL
Institute of Biochemistry, College of Medicine, National Taiwan University, Nankang, Taipei, Taiwan, Republic of China.
Tsai
C C
CC
Lin
S C
SC
Wang
L I
LI
Hsu
C I
CI
Hwang
M J
MJ
Lin
J Y
JY
D000036:Abrin / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D037102:Lectins / Q000737:chemistry* / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D037121:Plant Lectins; D011500:Protein Synthesis Inhibitors / Q000737:chemistry; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000187:drug effects; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D037102:Lectins; D037121:Plant Lectins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C016940:abrus agglutinin; D000036:Abrin