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

Abstract

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 (Glu 1 -Asn 2 -Phe 3 ) further indicated that only replacement of Phe 3 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 Phe 3 (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 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 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 Glu 1 also eliminated activity. Additional experiments identified the charged N-terminal amine and backbone of Glu 1 as key determinants for activity.

Specific Residues in Plasmatocyte-spreading Peptide Are Required for Receptor Binding and Functional Antagonism of Insect Immune Cells

“…Peptide Synthesis and Purification-All peptides were synthesized on an Applied Biosystems 433 synthesizer using standard Fmoc chemistry as previously described (16,17). The resin-peptide was cleaved and deprotected for 4 h in reagent K (21), a mixture containing 5% phenol, 1.25% water, 2.5% thioanisole, and 2.5% dithioethane in trifluoroacetic acid.…”

“…In contrast, the consensus sequence for the N terminus of PSP is found solely in the ENF peptide family (11). Using alanine replacement and deletion mutants, we also have identified residues in both the structured and unstructured regions of PSP that are essential for plasmatocyte-spreading activity (16,17). These include Cys 7 and Cys 19 , which form the disulfide bond required for the proper threedimensional structure of PSP, and the charged residue Arg 13 within the ␤-hairpin turn.…”

“…Ala replacement of Phe 3 (F3A) abolishes activity because of the specific requirement of the phenyl side chain (16). In contrast, Ala replacement of Glu 1 (E1A) actually enhances activity (17). This is, in part, because the activity of PSP requires the presence of the N-terminal primary amine but not the side chain of Glu 1 (17).…”

“…In contrast, Ala replacement of Glu 1 (E1A) actually enhances activity (17). This is, in part, because the activity of PSP requires the presence of the N-terminal primary amine but not the side chain of Glu 1 (17). Recent studies of the PSP homolog growth-blocking peptide likewise identify Phe 3 as critical for plasmatocyte activation in Pseudaletia separata (10).…”

“…Each concentration of 125 I-F23Bpa was tested alone or in combination with either N2A (17) or R13A at a final concentration of 1 M. These hemocytes were incubated for 2 h at room temperature, washed two times at 4°C, plated (40 l/well), and exposed to UV light at 366 nm (BlakRay) for 1 h on ice. Cells were lysed and prepared for SDS-PAGE by adding 10 l of 5ϫ sample buffer to each well and mixing rapidly.…”