June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Lacritin Bactericidal Peptide N-104 Binds Surface-Exposed Lipoprotein YaiW, a Known Peptide Transporter
Author Affiliations & Notes
  • Mohammad Sharifian Gh.
    Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, United States
  • Mihaela G Gadjeva
    Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Gordon W Laurie
    Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, United States
    Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, Virginia, United States
  • Footnotes
    Commercial Relationships   Mohammad Sharifian Gh., None; Mihaela Gadjeva, None; Gordon Laurie, None
  • Footnotes
    Support  NIH RO1 EY0024327; NIH R01 EY026171; NIH RO1 EY022054; An unrestricted gift from TearSolutions Inc
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 407. doi:
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    • Get Citation

      Mohammad Sharifian Gh., Mihaela G Gadjeva, Gordon W Laurie; Lacritin Bactericidal Peptide N-104 Binds Surface-Exposed Lipoprotein YaiW, a Known Peptide Transporter. Invest. Ophthalmol. Vis. Sci. 2021;62(8):407.

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

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Abstract

Purpose : Lacritin C-terminal proteoform 'N-104' is a main source of tear bactericidal activity. By screening for N-104 resistance, we recently discovered the importance of respective putrescine and iron transporters PotH and FeoB as likely virulence factors for P. aeruginosa (PA14) ocular pathogenesis. PotH and FeoB are constituents of the inner bacterial membrane, whereas N-104 is in tears and not membrane disruptive. How then does N-104 bactericidal activity involve PotH and FeoB? Here we search for N-104 binding proteins in the outer membrane of PA14.

Methods : N-104, and negative control lacritin peptide C-95, synthesized with cysteine added to the N-terminus, were coupled to beads of SulfoLink® Coupling Resin with an efficiency of 0.7 mmole peptide to ml resin, and then blocked with L-cysteine. Overnight cultures of PA14, either without or with prior surface biotinylation, were washed in PBS, resuspended in lysis buffer containing 200 mM octyl β-D-glucopyranoside ('OG') with rocking overnight at 4°C to allow membrane proteins to insert into OG vesicles, and centrifuged. Supernatant was passed through PierceTM Agarose Resin precolumns and then onto N-104 or C-95 columns with rocking overnight at 4°C. After washing with 20 ml of running buffer containing 20 mM KCl, proteins were eluted by progressive addition of 50, 75, 100, 125, 150, 300, 500 or 1000 mM KCl. 500 mM KCl eluted proteins from N-104 and C-95 columns were subjected to mass spectrometry.

Results : 'YaiW' was the sole outer membrane protein hit with 7.4x higher enrichment from N-104 vs negative control C-95 columns, and mutants lacking YaiW resistant to N-104 with a slope ratio threshold of 0.7. YaiW contributes to the transport of proline-rich Bac7 peptides across the outer membrane. No hits were obtained from biotinylated cells, in keeping with amine-reactive interference.

Conclusions : YaiW is a candidate outer membrane receptor for N-104 with the potential capability to transport N-104 into the periplasm, and thus in proximity to inner membrane PotH and FeoB.

This is a 2021 ARVO Annual Meeting abstract.

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