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 activ ates a class of insect immune cells called plasmatocytes. The tertiary stru cture of PSP consists of an unstructured N terminus (residues 1-6) and a we ll structured core (residues 7-23). A prior study indicated that deletion o f the N terminus from PSP eliminated all biological activity. Alanine subst itution of the first three residues (Glu(1)-Asn(2)-Phe(3)) further indicate d that only replacement of Phe(3) resulted in a loss of activity equal to t he N-terminal deletion mutant. Here, we characterized structural determinan ts of the N terminus. Adding a hydroxyl group to the aromatic ring of Phe(3 ) (making a Tyr) greatly reduced activity, whereas the addition of a fluori ne (p-fluoro) did not. Substitutions that changed the chirality or replaced the aromatic ring of Phe(3) 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 methyl ene group from Phe (phenyl-Gly) eliminated all activity. These results indi cated that a branched carbon chain with a methylene spacer at the third res idue is the minimal structural motif required for activity. The deletion of Glu(1) also eliminated activity. Additional experiments identified the cha rged N-terminal amine and backbone of Glu(1) as key determinants for activi ty.