An investigation of the susceptibility of alpha defensin to proteolysis
Background: Antimicrobial peptides play an important role in host defence, particularly in the oral cavity where there is constant challenge by microorganisms. The defensins comprise a family of cationic antimicrobial peptides that are widely distributed in mammals, insects and plants and are ancient components of the host response to bacterial, fungal and viral pathogens. The alpha or classical defensins (also known as human neutrophil peptides; HNPs), were the first to be isolated and comprise a sub-family of polypeptides found in human and animal neutrophils. Objectives: The current study was designed to determine whether alpha defensin-1 (HNP-1) was susceptible to proteolysis by a range of proteases known to be associated with oral disease. Methods: HNP-1 was incubated with cathepsin B, cathepsin G, elastase and matrix metalloproteinase 2 in an in vitro enzyme assay system. Degradation of HNP-1 was monitored over a range of time points from 0 to 120 minutes by Tris-Tricine SDS-PAGE and by matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS). Results: Both MALDI-MS and Tris-Tricine SDS-PAGE indicated that HNP-1 was not susceptible to degradation when incubated with cathepsin B, cathepsin G, elastase or matrix metalloproteinase 2. The small size and constrained three dimensional structure imposed by the disulphide bridges in HNP-1 could confer resistance to proteolysis within a physiologically relevant time-frame. This suggests that once released, HNP-1 could persist and retain their antimicrobial activity for a relatively long half-life in vivo. Conclusion: This study reports that HNP-1 is resistant to proteolysis which could have an important bearing on future therapeutic interventions with HNP-1.