Defensins promote fusion and lysis of negatively charged membranes

Abstract

Defensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike a-helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of 0-sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated primarily by electrostatic forces and to a lesser extent, by hydrophobic interactions.Circular dichroism and fluorescence spectroscopy of native defensins indicate that the amphiphilic 0-sheet structure is maintained throughout the fusion process. Taken together, these results support the idea that proteinmediated membrane fusion depends not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form a three-dimensional amphiphilic structure, which promotes the efficient mixing of the lipids between membranes. A molecular model for membrane fusion by defensins is presented, which takes into account the contributions of electrostatic forces, hydrophobic interactions, and structural amphiphilicity.

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

“…These observations suggest that cell membrane permeabilization is the main source of defensin activity in vivo. This is supported by the observations that defensins form voltage-dependent ion channels in planar bilayers (Kagan et al, 1990) and induce lysis of small sonicated lipid vesicles (Fujii et al, 1993). The work presented here investigates the mechanism of the permeabilization of large unilamellar vesicles by the human defensin HNP-2.…”

“…0.2 (SD) pM (18 experiments), whereas for rHNP-2, 50% leakage occurs at 0.07 pM rHNP-2. Fujii et al (1993) have also observed that reduced human and rabbit defensins are more active than the native forms. The gen- era1 features of defensin-induced leakage described here have also been observed for melittin (Schwarz et al, 1992) and the channel-forming peptide GALA (Parente et al, 1990).…”

“…The graded leakage induced by reduced defensin suggests some sort of destabilizing effect on all of the vesicles in the system, acting, perhaps, in the manner of a detergent. Fujii et al (1993) have suggested that the conformational changes they observed for reduced human and rabbit defensins induced amphipathic structures that transiently destabilized bilayers. Consistent with this, we have observed that rHNP-2, unlike the native form, induces leakage in vesicles formed from charge-neutral POPC alone (data not shown).…”

“…The GALA peptide, on the other hand, was shown only to release molecules smaller than approximately 800 Da, leading Parente et al (1990) to conclude that GALA forms pores that are 10-20 A in diameter. Fujii et al (1993) reported that the human defensin HNP-I induced 20-50% fusion of sonicated vesicles of 3: 1 DPPC:DOPS. Similarly, we find that native HNP-2 induces intervesicular lipid mixing of POPG LUV (Figs.…”

“…The structural models show that defensins are compact, globular proteins composed mainly of 3-stranded anti-parallel &sheet without 0-helices. The 3 conserved disulfide bonds per molecule impart a high degree of structural rigidity, based on NMR (Pardi et al, 1988(Pardi et al, , 1992, circular dichroism (Fujii et al, 1993), and fluorescence spectroscopy (Fujii et al, 1993) measurements. HNP-3 in crystals exists as tight homodimers (Hill et al, 1991), and HNP-I appears to form dimers or higher multimers in solution (Pardi et al, 1988(Pardi et al, , 1992.…”