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 Arg(13), Phe(3), Cys(7), Cys(19), and the N-terminal amine of Glu(1). Here we have built on these results by conducting cell binding and functional antagonism studies. Alanine replacement of Met(12) (M12A) resulted in a peptide with biological activity indistinguishable from PSP. Competitive binding experiments using unlabeled and I-125-M12A generated an IC50 of 0.71 nM and indicated that unlabeled M12A, at concentrations greater than or equal to100 nM, completely blocked binding of label to hemocytes. We then tested the ability of other peptide mutants to displace I-125-M12A at a concentration of 100 nM. In the structured core, we found that Cys(7) and Cys(19) 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. Arg(13), 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 Phe(3) yielded a peptide that reduced binding of I-125-M12A 326-fold. This and all other mutants of Phe(3) 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.