Alanine-scanning Mutagenesis of Plasmatocyte Spreading Peptide Identifies Critical Residues for Biological Activity

Abstract

diminished activity to a lesser degree. The point mutants Y11A, T14A, T22A, and F23A had activity identical or only slightly reduced to that of wild-type PSP. The mutant PSP-(7-23) lacked the entire unstructured domain of PSP and was found to have no plasmatocyte spreading activity. Surprisingly, E1A and N2A had higher activity than wild-type PSP, but F3A had almost no 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 reductions in activity correlated with alterations in tertiary structure, we compared the C7.19A, R13A, R18A, and F3A mutants to wildtype PSP by NMR spectroscopy. As expected, the simultaneous replacement of Cys 7 and Cys 19 profoundly affected tertiary structure, but the R13A, R18A, and F3A mutants did not differ from wild-type PSP. Collectively, these results indicate that residues in both the unstructured and structured domains of PSP are required for plasmatocyte-spreading activity.

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

“…Binding Constants of Representative Peptides-Prior analysis indicated that several residues are invariant among ENF peptide family members (8,16). These include Cys 7 and Cys 19 , Thr 14 and Thr 22 , all of the charged amino acids in the structured C terminus (Arg 13 , Asp 16 , Arg 18 , and Lys 20 ), and the three N-terminal residues Glu 1 , Asn 2 , and Phe 3 .…”

“…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.…”

“…Plasmatocytes and granular cells account for 30 and 65%, respectively, of the total hemocyte population in P. includens (19 -20). Plasmatocytes were isolated to high purity on Percoll step gradients as previously described (16). On average, 1.2 ϫ 10 6 plasmatocytes were collected per gradient.…”

“…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.…”

“…Deletion mutants in the unstructured N terminus also eliminate all biological activity. 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).…”