Plasmatocyte Spreading Peptide (PSP) is a 23 amino acid cytokine that induces a class of insect immune cells called plasmatocytes to spread on foreign surfaces. PSP consists of an unstructured N-terminus (residues 1-6) and a well-defined core (residues 7-23) stabilized by a disulfide bond and a short b-hairpin. Mutagenesis studies have identified several domains critical for activity. These include the cysteines used to form the disulfide bond, charged residues within the b-hairpin and, most importantly, residues Glu1 and Phe3. The structure of Phe3 is essential, as adding a hydroxyl (Tyr), or altering the chirality (D-Phe), or replacing the aromatic ring with a branched aliphatic chain (Val) each destroyed the activity. Activity was partially restored by the addition of a methylene group to Val (to make Leu), whereas removal of a methylene group from Phe (phenyl-Gly) destroyed activity, indicating a branched carbon chain with a methylene spacer as the minimum functional structural motif. We also tested alterations of Glu1 and Asn2. Ala substitutions at Glu1 or Asn2 both increased activity, but a Glu1 deletion eliminated activity, indicating that only the charged N-terminal amine is essential. Mutant peptides lacking activity were assayed for antagonism of wild-type PSP activity. Phe3 substitutions were unable to antagonize PSP, whereas mutants lacking the charged N-terminus were effective. These data suggest that Phe3 is essential for PSP/receptor binding. This Phe3-mediated interaction most likely serves to stabilize the normally unstructured N-terminus and therefore facilitates binding and activation of the receptor by the N-terminal amine.
Species 1: Lepidoptera Noctuidae Pseudoplusia includens (soybean looper)
Keywords: peptide cytokine, structure function
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