Specific residues in plasmatocyte-spreading peptide are required for receptor binding and functional antagonism of insect immune cells.
Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. PSP consists of two regions: an unstructured N terminus (1-6) and a highly structured core (7-23). Prior studies identified specific residues in both the structured and unstructured regions required for biological activity. Most important for function were Arg13, Phe3, Cys7, Cys19, and the N-terminal amine of Glu1. Here we have built on these results by conducting cell binding and functional antagonism studies. Alanine replacement of Met12 (M12A) resulted in a peptide with biological activity indistinguishable from PSP. Competitive binding experiments using unlabeled and 125I-M12A generated an IC50 of 0.71 nm and indicated that unlabeled M12A, at concentrations > or =100 nm, completely blocked binding of label to hemocytes. We then tested the ability of other peptide mutants to displace 125I-M12A at a concentration of 100 nm. In the structured core, we found that Cys7 and Cys19 were essential for cell binding and functional antagonism, but these effects were likely because of the importance of these residues for maintaining the tertiary structure of PSP. Arg13, in contrast, was also essential for binding and activity but is not required for maintenance of structure. In the unstructured N-terminal region, deletion of the phenyl group from Phe3 yielded a peptide that reduced binding of 125I-M12A 326-fold. This and all other mutants of Phe3 we bioassayed were unable to antagonize PSP. Deletion of Glu1 in contrast had almost no effect on binding and was a strong functional antagonist. Experiments using a photoaffinity analog indicated that PSP binds to a single 190-kDa protein.
Clark
Kevin D
KD
Department of Entomology, University of Georgia, Athens, Georgia 30602, USA.
Garczynski
Stephen F
SF
Arora
Aditi
A
Crim
Joe W
JW
Strand
Michael R
MR
D000107:Acetylation; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000166:cytology; D036341:Intercellular Signaling Peptides and Proteins; D008715:Methionine; D008969:Molecular Sequence Data; D009036:Moths / Q000276:immunology* / Q000502:physiology; D009154:Mutation; D010455:Peptides / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D019920:Photoaffinity Labels; D011485:Protein Binding; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D019920:Photoaffinity Labels; C108084:plasmatocyte-spreading peptide 1; D008715:Methionine; D000409:Alanine