Alanine-scanning mutagenesis of plasmatocyte spreading peptide identifies critical residues for biological activity

Plasmatocyte spreading peptide (PSP) is a 23-amino acid cytokine that induc es a class of insect immune cells called plasmatocytes to spread on foreign surfaces. The structure of PSP consists of a disordered N terminus (residu es 1-6) and a well-defined core (residues 7-23) stabilized by a disulfide b ridge between Cys(7) and Cys(19), hydrophobic interactions, and a short bet a -hairpin, Structural comparisons also indicate that the core region of PS P adopts an epidermal growth factor (EGF)-like fold very similar to the C-t erminal subdomain of EGF-like module 5 of thrombomodulin, To identify resid ues important for plasmatocyte spreading activity, we bioassayed PSP mutant s in which amino acids were either replaced with alanine or deleted. Within the well-defined core of PSP, alanine replacement of Cys(7) and Cys(19) (C 7.19A) eliminated all activity. Alanine replacement of Arg(13) reduced acti vity similar to 1000-fold in comparison to wildtype PSP, whereas replacemen t of the other charged residues (Asp(16), Arg(18), Lys(20)) surrounding Cys (19) diminished activity to a lesser degree. The point mutants Y11A, T14A, T22A, and F23A had activity identical or only slightly reduced to that of w ild-type PSP, The mutant PSP-(7-23) lacked the entire unstructured domain o f PSP and was found to have no plasmatocyte spreading activity. Surprisingl y, E1A and N2A had higher activity than wild-type PSP, but F3A had almost n o activity. We thus concluded that the lack of activity for PSP-(7-23) was largely due to the critical importance of Phe(3). To determine whether redu ctions in activity correlated with alterations in tertiary structure, we co mpared the C7.19A, R13A, R18A, and F3A mutants to wildtype PSP by NMR spect roscopy. As expected, the simultaneous replacement of Cys(7) and Cys(19) pr ofoundly affected tertiary structure, but the R13A, R18A, and F3A mutants d id not differ from wild-type PSP, Collectively, these results indicate that residues in both the unstructured and structured domains of PSP are requir ed for plasmatocyte-spreading activity.