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 (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.
Clark
K D
KD
Department of Entomology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Volkman
B F
BF
Thoetkiattikul
H
H
Hayakawa
Y
Y
Strand
M R
MR
D000588:Amines / Q000378:metabolism; D000818:Animals; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006434:Hemocytes / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009036:Moths; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary
D000588:Amines; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C108084:plasmatocyte-spreading peptide 1; D018698:Glutamic Acid; D010649:Phenylalanine