Structure-activity studies of AtPep1, a plant peptide signal involved in the innate immune response.
AtPep1, a 23-amino acid peptide recently isolated from Arabidopsis leaves, induces the expression of the genes encoding defense proteins against pathogens. We investigated the structure-activity relationship of AtPep1 with its receptor, a 170 kDa leucine-rich repeat receptor kinase (AtPEPR1) by utilizing a suspension cell assay (the alkalinization assay). Binding of AtPep1 to AtPEPR1 on the cell surface is accompanied by an increase in the pH of Arabidopsis suspension cell media by 1 pH unit in 15 min with a half-maximal response of 0.25 nM. Sequential removal of N-terminal amino acids had little effect on activity until the peptide was reduced to 15 amino acids [AtPep1(9-23)], which decreased the activity by less than one order of magnitude. Activity was completely abolished when nine C-terminal amino acids remained. Removal of the C-terminal asparagine from AtPep1(9-23), resulted in a decrease in activity (12 max approximately 100 nM). AtPep1(9-23) was used for alanine-substitution analysis and revealed two important residues for activity, a serine, [A(15)]AtPep1(9-23) (12 max approximately 10nM), and a glycine, [A(17)]AtPep1(9-23) (12 max approximately 1000 nM). Neither [A(17)]AtPep1(9-23) nor the C-terminal truncated AtPep1, AtPep1(9-22), were able to compete with AtPep1(9-23) in the alkalinization assay. The importance of the glycine residue for binding to the AtPep receptor was also confirmed by competition assays using radiolabeled AtPep1. d-Alanine or 2-methylalanine substituted at the glycine position displayed only a slight decrease in activity whereas l- and d-proline substitution caused a loss of activity. Homologs of AtPep1 identified in Arabidopsis and other species revealed a strict conservation of the glycine residue.
Pearce
Gregory
G
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA. pearce@wsu.du
Yamaguchi
Yube
Y
Munske
Gerhard
G
Ryan
Clarence A
CA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007113:Immunity, Innate / Q000187:drug effects*; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000494:pharmacology*; D018515:Plant Leaves / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D000596:Amino Acids; D029681:Arabidopsis Proteins; C512592:PEPR1 protein, Arabidopsis; D010455:Peptides; D011956:Receptors, Cell Surface