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R. L. McKown, E. V. Coleman, R. W. Raab, A. M. Deleault, M. E. Kimberly, G. W. Laurie; Mutational Analysis of Antimicrobial Activity in Recombinant Human Lacritin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4264.
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© ARVO (1962-2015); The Authors (2016-present)
Recombinant lacritin promotes human corneal epithelial cell proliferation with a biphasic 1-10 nM dose optimum, stimulates prolonged basal tearing in rabbits, and is antimicrobial in uM concentrations. Computational analysis predicts an ordered C-terminal half with an amphipathic alpha helical region. Here we define the functional domain of antimicrobial activity in lacritin and ask if the amphipathic alpha helix is required for activity.
Lacritin deletion mutants and site-directed point mutants were created from mature lacritin, confirmed by DNA sequencing, expressed in E. coli, and purified. Deletion variants were generated with amino acid residues removed from the ends of lacritin and selected amino acid residues in the amphipathic helix were targeted for site-directed mutagenesis. A colony forming unit assay was used to assay antimicrobial activity. Circular Dichroism conformational analysis of recombinant lacritin and mutant variants was studied using a Jasco J-810 spectroplarimeter with CD Pro Software.
The antimicrobial activity of lacritin is localized to a domain between amino acids 80 and 119 of mature lacritin; a region that encompasses the C-terminal amphipathic alpha helix. The Minimum Inhibitory Concentration (MIC) of lacritin which results in greater than 90% bacterial cell death against E. coli ranged from 0.6µM to 10µM for mature lacritin and the deletion mutants. Both gram negative and gram positive pathogenic bacteria (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa) were sensitive at physiological conditions. Individual point mutations on the hydrophobic surface of the amphipathic alpha helix altered antimicrobial activity and produced conformational changes.
Recombinant lacritin is antimicrobial and bactericidal with an MIC in the uM range for gram positive and gram negative bacteria. The antimicrobial domain in lacritin is localized in the C-terminal 39 amino acids. The amphipathic alpha helix in the C-terminus of lacritin appears to play an important role in antimicrobial activity.
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