Novel antimicrobial LFampin from lactoferrin released by a one-enzyme strategy*

Antimicrobial peptides have been found throughout living nature, yet antimicrobial sequences may still lie hidden within a wide variety of proteins. Objectives: The objective was to develop a rational strategy to select interesting domains from (glyco)proteins, based on the presumed common features of antimicrobial peptides, and to functionally release these from accessible and safe proteins. Methods: To demonstrate the proof of principle bovine lactoferrin (bLF) was screened for suitable stretches, which encompasses a cationic amphipathic α-helix. Next proteolytic enzymes were selected that theoretically can release the potential antimicrobial sequences. These sequences were chemically synthesized and isolated from the native molecule and subsequently tested for their killing activity. Results: Corresponding the identified antimicrobial domain, designated LFampin, peptides were synthesized which indeed possessed broad spectrum antimicrobial activity. In silico proteolysis simulations with selected endoproteinases predicted the liberation of peptides containing the required LFampin sequence. Three predicted peptides were synthesized and tested for their biological activity, elucidating that one single enzyme (endoproteinase AspN) was sufficient to obtain an antimicrobial peptide. The final proof of principle demonstrated that a 32-mer fragment isolated from the AspN-digestion of bLF possessed antimicrobial activity as strong as that of the synthetic LFampin. Moreover, desalted crude digest had improved activity over native bLF. Conclusions: Hence, selective digestion of bLF increases its antimicrobial activity by release of antimicrobial stretches. In this way it turned out possible to liberate a novel antimicrobial peptide from bLF by a one-enzyme strategy, which, theoretically, opens the road to simple and cheap large-scale production of a safe antimicrobial peptide.