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Mapping Of Microbicidal Domains Of Thrombocidins And Design Of Thrombocidin-Derived Peptides With Optimized Activity

Thrombocidin-1 (TC-1) and -2 are microbicidal proteins isolated from human blood platelets, and are derivatives of CXC-chemokines neutrophil activating peptide2 (NAP-2) and connective tissue activating peptide-III (CTAP-IH), respectively. The only difference between these chemokines and the TCs is a C-terminal truncation of two amino acids in the latter. The aims of this study were to identify microbicidal domains in TCs by peptide mapping, and to optimize the microbicidal activity of these peptides by amino acid substitutions. Synthetic peptides, 15 amino acids in length and overlapping by 10 residues, covering the entire sequence of CTAP-III were used for initial mapping of microbicidal activity, and finemapping was done using peptides shifted by 1 residue. Testing of microbicidal activity of these peptides against Bacillus subtilis, Escherichia coli and Staphylococcus aureus and against the fungus Cryptococcus neoformans uncovered two functional domains, one near the C-terminus and one around the CXC-motif in the N-terminal part of the protein. The peptide derived from the latter domain (peptide L18) had stronger microbicidal activity than the C-terminal peptide. Cysteines in L18 were shown to be indispensable for microbicidal activity. Successive lysine substitutions in L18 yielded 2 peptides with activity against all test organisms equivalent to that of native TC. C-terminally amidated forms of these 2 L18-derived peptides had further increased bactericidal activity and killed pathogenic gram-positive bacteria at 1-4 uM. The peptides with optimized microbicidal activity showed a decrease in cytotoxic activity for erythrocytes. In conclusion, peptide mapping of TCs allowed the identification of two functional domains and development of peptides with high microbicidal activity. Based on these results a model for the microbicidal action of TCs is proposed.