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Mohammad Sharifian Gh., Margaret Ryan, Mirco Sorci, Georges Belfort, Georgi Georgiev, Gordon W Laurie; Putative Carpet Accumulation of Bactericidal Peptide N-104 on Model Gram-Negative and Positive Bacterial Membranes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5183.
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© ARVO (1962-2015); The Authors (2016-present)
Tears covering the surface of the eye comprise a remarkably sterile medium inclusive of various antimicrobial compounds. A C-terminal fragment of the prosecretory mitogen lacritin known as 'N-104', together with larger N-104 containing fragments, appear to play a key role in the innate protection of the eye. Exactly how they are bactericidal is not known. Here we studied real-time interactions of N-104 and ten single serine-substituted analogs with supported lipid bilayers modeling membranes of Gram-negative and positive bacteria.
N-104, and analogs in which serine was individually substituted for leucine ('L108S', 'L109S', 'L114S', 'L115S'), lysine ('K107S', 'K110S', 'K111S', 'K116S'), phenylalanine ('F112S'), and tryptophan ('W118S') were synthesized with N-terminus acetylated. Lipid bilayers of PC:PG:PE (59:21:20; Gram-negative) and PC:PG (75:25; Gram-positive) were formed on lipophilic surface plasmon resonance (SPR) sensor chips (L1). Time-resolved SPR responses of the peptides were then examined at a flow rate of 30 μl/min. Collected data were used as constraints to obtain the peptides adsorption, desorption, and disruption rates by fitting them to a newly derived model that was applicable to N-104 and all analogs.
In both model membranes, two adsorption phases (an initial fast followed by a slow rise in SPR) were observed for N-104 and all L/S substitutions except L114S, plus the K107S peptide. In all other K/S, F/S, and W/S substitutions, the first adsorption phase was followed by a release phase (a decay in SPR). A subsequent buffer wash initiated a relatively fast followed by slow SPR decay. For F/S and W/S substitutions and all K/S substitutions (particularly at 110 and 111 in the α-helical region and 116 in the C-terminal coiled coil), the decay dropped to negative values suggesting membrane disruption. Comparison to colony forming unit assays in E. coli and P. aeruginosa suggests that slow release rate is largely, although not perfectly, predictive of bactericidal activity.
These data are suggestive of a 'carpet' accumulation of N-104 onto both model membranes apparently mediated by electrophilic (largely lysines 110, 111, 116) and hydrophobic (phenylalanine 112, tryptophan 118) interactions between the peptide and membrane phospholipids. Mutating N-104 at those amino acid residues would result in higher disruption of the membrane.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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