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Gordon W Laurie, Margaret Ryan, Amber Watkins, Kevin-Jude Sankoorikal; Contribution of Nonpolar Side Chains to Lacritin N-104 Bactericidal Activity. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2647.
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© ARVO (1962-2015); The Authors (2016-present)
Lacritin is a multifunctional tear protein with latent bactericidal activity, important in normal human tears, that is concentrated in its C-terminal fifteen amino acids represented by the synthetic peptide 'N-104' (McKown et al, JBC ’14). This activity is largely insensitive to physiological salt, unlike most cationic antimicrobial proteins including cathelicidins and most defensins for which electrostatic binding is key. How does N-104 interact with bacterial membranes? Here we explored the role of residues with nonpolar side chains.
Eleven different nonpolar to polar (serine) mutant N-104 peptides with acetylated N-termini were synthesized: (i) N-104/L108S, (ii) N-104/L109S, (iii) N-104/F112S, (iv) N-104/L114S, (v) N-104/L115S, (vi) N-104/W118S, (vii) N-104/L108S/L109S, (viii) N-104/L114S/L115S, (ix) N-104/L108S/L109S/F112S/W118S, (x) N-104/F112S/L114S/L115S/W118S, (xi) N-104/L108S/L109S/F112S/L114S/L115S/W118S. Each was tested at 1, 10 and 100 micromolar concentrations in an E. coli CFU assay in which peptide was incubated for 1.5 hrs (37°C) with cells, and then diluted for overnight growth on LB agar.
Mutation of leucine-109 significantly reduced activity, as reflected in single mutant (L109S) and all other L109S mutants, particularly L108S/L109S/F112S/W118S and L108S/L109S/F112S/L114S/L115S/W118S. Mutation of tryptophan-118 gave equivocal results.
Mutual hydrophobic interactions, particularly those involving leucine-109, appear to be important for lacritin N-104 bactericidal activity. Pores are formed, but death appears to be regulated.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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