June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Constitutive Release of Lacritin's Latent Bactericidal Activity Explored by MALDI-TOF Mass Spectrometry
Author Affiliations & Notes
  • Jeff Romano
    Cell Biology, University of Virginia, Charlottesville, Virginia, United States
  • Nick Sherman
    Cell Biology, University of Virginia, Charlottesville, Virginia, United States
  • Robert L McKown
    Integrated Science and Technology, JMU, Harrisonburg, Virginia, United States
  • Tadayuki Iwase
    Bacteriology, The Jikei University School of Medicine, Minato, Japan
  • Gordon W Laurie
    Cell Biology, University of Virginia, Charlottesville, Virginia, United States
  • Footnotes
    Commercial Relationships   Jeff Romano, None; Nick Sherman, None; Robert McKown, None; Tadayuki Iwase, None; Gordon Laurie, TearSolutions LLC. P (F), UVa Patent Foundation (P)
  • Footnotes
    Support  NIH Grant EY024327
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2646. doi:
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    • Get Citation

      Jeff Romano, Nick Sherman, Robert L McKown, Tadayuki Iwase, Gordon W Laurie; Constitutive Release of Lacritin's Latent Bactericidal Activity Explored by MALDI-TOF Mass Spectrometry
      . Invest. Ophthalmol. Vis. Sci. 2017;58(8):2646.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Lacritin is a pluripotent basal tear promoting cytokine in tears with latent C-terminal bactericidal activity representable by the synthetic peptide N-104, although larger C-terminal fragments are also bactericidal (McKown et al, J Biol Chem '14). Recently Azkargorta et al (J Prot Res '15) validated this observation, and interestingly detected 51 different lacritin fragments in normal tears - all but 5 C-terminal. Thus, a natural lacritin processing mechanism is continuously in play as a substantial contributor to the sterility of tears. Processing of recombinant lacritin in vitro can be blocked by chymostatin or leupeptin, suggesting a serine protease.

Methods : Recombinant lacritin (5 µM) was incubated at different times (1, 3, 5, 10, 20, 40 or 90 min) with increasing concentrations of serine proteases ESP (0.1, 1, 10, 100, 1000, or 2000 nM) or cathepsin G (0.1, 0.5, 1, 2 or 5 mU). Cleavage analysis of fragments >4 kDa was monitored by MALDI-TOF mass spectrometry and Western blotting.

Results : MALDI-TOF mass spectrometry revealed cleavage sites on the carboxy side of amino acids 14, 24, 70, 76 and 77 for ESP, and 15, 24, 59, 65, 71, 72 and 75 for cathepsin G. Fragments released at amino acids >59 could potentially be bactericidal. In agreement, some Azkargorta et al '15 fragments begin at amino acids 71, 72, 76 and 78, although over >17 other cleavage sites are apparent suggesting that other tear proteases may thus contribute - much like the processing of skin antibiotic dermcidin. Some bactericidal fragments appear to be resistant to further digestion, as per MALDI-TOF mass spectrometry of lacritin N-94 and N-94/C-6 synthetic peptides.

Conclusions : Both ESP and cathepsin G may contribute to lacritin C-terminal processing, for which it is likely that multiple proteases may be involved.

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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