Aggregatation, fusion and aqueous content release from liposomes induced by lysozyme derivatives: Effect on the lytic activity

Abstract

Chemically modified lysozymes, namely: N-succinyl lysozyme, glycine methyl ester of N-succinyl lysozyme and oxoindole lysozyme have been prepared. Aggregation, fusion and leakage of phospholipid vesicles induced by these derivatives have been studied in comparison with the effect of the unmodified protein. The experiments were carried out with negatively charges 9PC/PA, 9:1) and uncharged (PC and PC/DOPE/Chol (10:5:5)) lipid vesicles of different packing. Fusion and aggregation of negatively charged phospholipid vesicles in induced by proteins positively charged at pH 7.0 involving electrostatic interactions, a similar pattern on fusion and aggregation of the least stably packed lipid vesicles points also to hydrophobic forces playing a role in the lipid-protein interaction. A conformational change of the protein involved increasing beta-turns, loops and unordered structure at the expenses of beta-sheet without affecting alpha helix content. The conformational effect is necessary to provoke the effects studied, since one of the derivatives (N-succinyl lysozyme) neither changes conformation nor causes aggregation and fusion of vesicles. However, there is no relationship between lysozyme activity and fusion or aggregation of lipid vesicles that catalytic and fusogenci sites of, indicating lysozyme are topographically different.

Lysozyme interaction with negatively charged lipid bilayers: protein aggregation and membrane fusion

“…[4][5][6] HEWL is structurally homologous to human lysozyme, involved in several destabilizing mutations-related to protein deposition into amyloid plaques found in the liver, kidneys, and spleen of patients affected by non-neuropathic hereditary amyloidosis. [7][8][9][10][11] Because of its homology to human lysozyme, HEWL has been extensively used as a model in studies for a number of investigations: protein adsorption at interfaces, 12,13 membrane fusion, [14][15][16][17] protein folding and aggregation into amyloids. [18][19][20][21] Human lysozyme forms amyloid fibrils in vitro upon incubation at low pH and elevated temperature, 22,23 whereas HEWL forms amyloid fibrils when incubated in organic solvents 24 or in the presence of negatively charged lipid membranes.…”

Interactions of lysozyme with phospholipid vesicles: effects of vesicle biophysical features on protein misfolding and aggregation

“…23 Furthermore, many studies have shown the ability of lysozyme to bind lipid bilayers with a high affinity to negatively charged phospholipid vesicles favoured by its net positive charge over a broad pH range. 20,21,[24][25][26][27][28][29][30] The data presently available indicate that lysozyme association with phospholipid bilayers involves both electrostatic and hydrophobic interactions (reviewed in ref. 31) and modulates its aggregation propensity.…”

“…27 The model, subsequently adapted for Annexin V-induced aggregation and fusion of PS vesicles, 28 envisages a change in solvent structure in the contact region between the protein and membrane surface upon lysozyme binding. Actually, at low pH values the massive presence of bound lysozyme molecules induces a strong dehydration of the lipid membrane surface; the latter most likely results from disruption of membrane integrity due to pH-and ionic strengthdependent protein penetration inside the bilayer, 20 with changes in liposome permeability 29 and liposome fusion. 30,32 These data have been confirmed and extended by a recent RET-based study; 33 the latter showed that both lysozyme location at the lipid-water interface and its penetration inside the bilayer depend on protein coverage of the liposome surface and that lysozyme can induce local lipid demixing in POPC/POPG model membranes with formation of lateral domains enriched in negative phospholipids.…”

Towards efficient biocatalysts: photo-immobilization of a lipase on novel lysozyme amyloid-like nanofibrils

“…Liposomal leakage test. The assay was performed according to Vechetti et al 65 Briey to prepare DOPC : DOPA (8 : 2) SUV containing calcein trapped within, suitable amount of lipids were dried under nitrogen onto the wall of a Corex glass tube and sonicated with probe-type sonier under nitrogen and controlled temperature. In order to separate calcein-loaded SUV from free dye a Sephadex G-75 was used.…”

“…HEWL is also referred as muramidase due to its activity on bacterial cell wall, 45 as well as a membrane disrupter in cell-free system. 46 In this way, it has proved essential to learn whether after the formation of amyloid aggregate, the muramidase and the membrane permeabilization activities are prevalent in the protein. Figure 1e shows the muramidase activity of 1/10 dilution of 1 mg/ml HEWL on Micrococcus lysodecticus alone or in the presence of 0.750 mg/ml heparin.…”