June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Unbiased Screening of Meibomian Lipids for Affinity to Natural C-Terminal Lacritin Proteoforms Necessary for the Prevention of Tear Film Collapse.
Author Affiliations & Notes
  • Jeff Romano
    Cell Biology, University of Virginia, Charlottesville, Virginia, United States
  • Georgi Georgiev
    Universidade de Lisboa, Lisboa, Lisboa, Portugal
  • Jianzhong Chen
    Cell Biology, University of Virginia, Charlottesville, Virginia, United States
  • Marc Odrich
    Opthamology, University of Virginia, Charlottesville, Virginia, United States
  • Gordon W Laurie
    Cell Biology, University of Virginia, Charlottesville, Virginia, United States
  • Footnotes
    Commercial Relationships   Jeff Romano None; Georgi Georgiev None; Jianzhong Chen None; Marc Odrich None; Gordon Laurie TearSolutions, Inc, Code F (Financial Support), TearSolutions, Inc, Code I (Personal Financial Interest), TearSolutions, Inc, Code O (Owner), UVA Licensing and Ventures Group, Code P (Patent), TearSolutions, Inc, Code S (non-remunerative)
  • Footnotes
    Support   EY026171 and EY032956 to GWL, and an unrestricted gift to the Department of Cell Biology from TearSolutions, Inc.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 195. doi:
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      Jeff Romano, Georgi Georgiev, Jianzhong Chen, Marc Odrich, Gordon W Laurie; Unbiased Screening of Meibomian Lipids for Affinity to Natural C-Terminal Lacritin Proteoforms Necessary for the Prevention of Tear Film Collapse.. Invest. Ophthalmol. Vis. Sci. 2023;64(8):195.

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

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Abstract

Purpose : Tear film collapse is a hallmark of dry eye disease whereby breakdown of the protective lipid layer at the air/liquid interface leads to reduced surface tension, coupled with chronic inflammation and tear loss affecting 7-10% of the world population. Protein/lipid complexes stabilize the tear film and play an essential role at the aqueous interface. The mitogenic, basal tear promoting, homeostasis restorative cytokine lacritin rapidly binds and stabilizes tear lipids via its enzymatically processed C-terminal proteoforms, that unlike N-proteoforms are necessary to prevent collapse. Previously, our focus was on N-94 and N-94/C-6 proteoforms that interact with meibum lipids, including OAHFA and when absent promote premature collapse. Spiking each into dry eye tears restored stability. Here we focus on the slightly smaller lacritin C-terminal proteoform N-104 in an unbiased screen for bound meibum lipids. As a negative control, we use a more N-terminal lacritin peptide N-80/C-25 of similar length and pI.

Methods : Lacritin Cys-N-104 or negative control peptide Cys-N-80/C-25 peptides were synthesized as acetate salts with termini appropriately blocked. Each were coupled to agarose beads using SulfoLink (Thermo), and incubated with mixing for 2 hr at 35 C in 500 µl of PBS with 60 mg pooled meibum resuspended in acetone, initially collected from 6 donors with expression forceps (first dissolved in CHCl3 and flash frozen at -80 C, then evaporated under N2 gas). Washed beads were stained with fluorescent lipid dye Nile Red, and beads were eluted with chloroform for lipid identification by LC/MS/MS.

Results : N-104-, but not N-80/C-25-, coated beads displayed strong meibum lipid binding as visualized with Nile Red. Lipid species detected on N-104 at a level two or more-fold greater than N-80/C-25 include: CE, DG, FA, FAHFA, LPC, LPS (Lysophosphatidylserine), PG, and PI. More precise identification of lipid species is being developed by coupling thin layer chromatography with subsequent LC/MS/MS.

Conclusions : Lacritin C-terminal proteoform interaction with meibomian lipids is much more extensive than with OAHFA's. The capability of N-104 to preferentially and specifically capture both polar and non-polar lipids points to the complexity of the tear film and the role of lacritin proteoforms in the prevention of premature tear film collapse in dry eye disease.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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