Characterisation of DEFB107 by mass spectrometry: Lessons from an anti-antimicrobial defensin

Mammalian defensins are small endogenous cationic proteins which form a class of antimicrobial peptides that is part of the innate immune response of all mammalian species [R. Lehrer, Nat. Rev. Microbiol. 2 (9) (2004) 727; T. Ganz, R.I. Lehrer, Curr. Opin. Inummol. 6 (4) (1994) 584] [1,2]. We have developed mass spectrometry based strategies for characterising the structure-activity relationship of defensins [D.J. Campopiano, D.J. Clarke, N.C. Polfer, P.E. Barran, R.J. Langley, J.R.W. Govan, A. Maxwell, J.R. Dorin, J. Biol. Chem. 279 (47) (2004) 48671; P.E. Barran, N.C. Polfer, D.J. Campopiano, D.J. Clarke, P.R.R. Langridge-Smith, R.J. Langley, J.R.W. Govan, A. Maxwell, JR. Dorin, R.P. Millar, M.T. Bowers, Int. J. Mass Spectrom. 240 (2005) 2731 [3,4], and here we present data obtained from a five cysteine containing P-defensin, DEFB 107. The synthetic product of this human defensin exists with a glutathione capping group, its oxidation state and disulphide connectivity have been determined via accurate mass measurements and peptide mass mapping respectively, and despite possessing three disulphide bridges, it does not fit the beta-defensin canonical motif. With the use of molecular modelling, we have generated candidate geometries to discern the influence of disulphide bridging on the overall tertiary structure of DEFB 107. These are compared with experimental results from ion mobility measurements. Defensins display activity against a wide variety of pathogens including both gram-negative and gram-positive bacteria. Their mechanism of mode of action is unknown, but is believed to involve defensin aggregation at cell surfaces, followed by cell permeabilisation and hence death. To probe this mechanism, the localisation of DEFB 107 in synthetic vesicles was studied using H/D exchange and mass spectrometry. The results obtained are used to analyse the antimicrobial activity of DEFB 107. (C) 2006 Elsevier B.V. All rights reserved.