Genomic clones encoding two isoforms of pokeweed antiviral protein in seeds (PAP-S1 and S2) and the N-glycosidase activities of their recombinant proteins on ribosomes and DNA in comparison with other isoforms.
Pokeweed antiviral proteins (PAPs) are single-chain ribosome-inactivating proteins (RIPs) isolated from several organs of Phytolacca americana (Pokeweed) that are characterized by their ability to depurinate not only ribosomes but also various nucleic acids. PAP-S is one of the isoforms found in seeds. In this study, we obtained three different genomic clones encoding two forms of PAP-S (here designated as PAP-S1 and PAP-S2) and alpha-PAP after PCR using a pair of degenerated primers based on the known N- and C-terminal amino acid sequences of PAP-S. The nucleotide sequences of the genomic clones contained no introns. The deduced amino acid sequences of PAP-S1 and PAP-S2, which showed 83% identity to each other, were found to correspond to sequences reported independently for PAP-S protein and cDNA, respectively, demonstrating that at least two forms of PAP-S actually exist in seeds of the same plant. The recombinant PAP-S1, PAP-S2, alpha-PAP, and PAP I (a form appearing in spring leaves) exhibit the same level of depurinating activity on rat ribosomes, while their efficiencies on Escherichia coli ribosomes and salmon sperm DNA differ substantially from one another in the order of PAP I > alpha-PAP > PAP-S1 > PAP-S2 and alpha-PAP > PAP I > PAP-S1 > PAP-S2. Structural comparisons suggest that the large difference in ribosome recognition between PAP-S1 (or S2) and PAP I is caused by the alteration of residues adjacent to the adenine-binding site.
Honjo
Eijiro
E
Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan. watakei@cc.saga-u.ac.jp
Dong
Danghong
D
Motoshima
Hiroyuki
H
Watanabe
Keiichi
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D045647:DNA Glycosylases; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000201:enzymology*; D012639:Seeds / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D010940:Plant Proteins; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein