INTERACTIONS BETWEEN HUMAN DEFENSINS AND LIPID BILAYERS - EVIDENCE FOR FORMATION OF MULTIMERIC PORES

Defensins comprise a family of broad-spectrum antimicrobial peptides t hat are stored in the cytoplasmic granules of mammalian neutrophils an d Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human n eutrophil defensins, permeabilizes large unilamellar vesicles formed f rom the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As o bserved by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmito yloleoylphosphatidylcholine (POPC) demonstrates that binding is initia ted through electrostatic interactions. Because vesicle aggregation an d fusion can confound studies of the interaction of HNP-2 with vesicle s, those processes were explored systematically by varying the concent rations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 mu M POPG) readily aggregated at HNP-2 concentrations above 1 mu M, bu t no mixing of vesicle contents could be detected for concentrations a s high as 2 mu M despite the fact that intervesicular lipid mixing cou ld be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer c ontact sites. Under conditions of limited aggregation and intervesicul ar lipid mixing, the fractional leakage of small solutes is a sigmoida l function of peptide concentration. For 300 mu M POPG vesicles, 50% o f entrapped solute is released by 0.7 mu M HNP-2. We introduce a simpl e method for determining whether leakage from vesicles is graded or al l-or-none. We show by means of this fluorescence ''requenching'' metho d that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle c ontents. At HNP-2 concentrations that release 100% of small (similar t o 400 Da) markers, a fluorescent dextran of 4,400 Da is partially reta ined in the vesicles, and a 18,900-Da dextran is mostly retained. Thes e results suggest that HNP-2 can form pores that have a maximum diamet er of approximately 25 Angstrom. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.