N-terminal Residues of Plasmatocyte-spreading Peptide Possess Specific Determinants Required for Biological Activity*

Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. The tertiary structure of PSP consists of an unstructured N terminus (residues 1–6) and a well structured core (residues 7–23). A prior study indicated that deletion of the N terminus from PSP eliminated all biological activity. Alanine substitution of the first three residues (Glu1-Asn2-Phe3) further indicated that only replacement of Phe3 resulted in a loss of activity equal to the N-terminal deletion mutant. Here, we characterized structural determinants of the N terminus. Adding a hydroxyl group to the aromatic ring of Phe3 (making a Tyr) greatly reduced activity, whereas the addition of a fluorine (p-fluoro) did not. Substitutions that changed the chirality or replaced the aromatic ring of Phe3 with a branched aliphatic chain (making a Val) also greatly decreased activity. The addition of a methylene group to Val (making a Leu) partially restored activity, whereas the removal of a methylene group from Phe (phenyl-Gly) eliminated all activity. These results indicated that a branched carbon chain with a methylene spacer at the third residue is the minimal structural motif required for activity. The deletion of Glu1 also eliminated activity. Additional experiments identified the charged N-terminal amine and backbone of Glu1 as key determinants for activity.