June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Canine Tear Lacritin is Down Regulated in Clinical Dry Eye
Author Affiliations & Notes
  • Robert L McKown
    Integrated Science & Technology, James Madison University, Harrisonburg, VA
  • Alan C. Tate
    Integrated Science & Technology, James Madison University, Harrisonburg, VA
  • Alison M. Enghauser
    Integrated Science & Technology, James Madison University, Harrisonburg, VA
  • Cara L. Soyars
    Integrated Science & Technology, James Madison University, Harrisonburg, VA
  • Ronald W. Raab
    Integrated Science & Technology, James Madison University, Harrisonburg, VA
  • Gordon W Laurie
    Cell Biology, University of Virginia, Charlottesville, VA
    Ophthalmology, University of Virginia, Charlottesville, VA
  • Ian P. Herring
    Small Animal Clinical Studies, VA-MD Regional College of Veterinary Medicine, Blacksburg, VA
  • Footnotes
    Commercial Relationships Robert McKown, None; Alan Tate, None; Alison Enghauser, None; Cara Soyars, None; Ronald Raab, None; Gordon Laurie, None; Ian Herring, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 288. doi:
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      Robert L McKown, Alan C. Tate, Alison M. Enghauser, Cara L. Soyars, Ronald W. Raab, Gordon W Laurie, Ian P. Herring; Canine Tear Lacritin is Down Regulated in Clinical Dry Eye. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):288.

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

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Abstract

Purpose: Lacritin is a human tear glycoprotein first characterized as a secretory agonist for isolated rat lacrimal acinar cells. Later, it was found to stimulate basal tearing in normal rabbits, and Aire-/- dry eye mice, and to restore health to human corneal epithelial cells stressed with inflammatory cytokines. The monomeric (active) form of lacritin appears to be selectively deficient in human dry eye. Dry eye is naturally occurring in some dogs on which topical cyclosporine was originally tested. As a first, we now report the cDNA cloning and recombinant protein purification of a canine ortholog of lacritin, development of an ELISA immunoassay to monitor levels in tears, and analysis of canine lacritin levels in normal and dry eye animals.

Methods: Total RNA extracted from normal dog lacrimal gland was reverse transcribed and then PCR amplified using primers developed from predicted lacritin coding sequences and cloned into the bacterial expression vector pTYB2. Recombinant canine lacritin was purified by chitin affinity and DEAE chromatography. Using a synthetic peptide corresponding to N-terminal amino acids EGDSSDPAPGAAAADPGGL, antibody cLACRT NT was generated in rabbits and used for assay against tears by ELISA and Western blotting from normal and dry eye dogs.

Results: Dog and human lacritin share 35% amino acid identity, although canine lacritin is 20 amino acids shorter. Recombinant canine lacritin was purified to a single band on SDS PAGE that migrated at approximately 18 kDa. ELISA detected an average of 4.11 ng lacritin in normal tears and an average of 0.67 ng lacritin in tears from dogs diagnosed with dry eye. Western blot analysis detected prominent bands in healthy tears at approximately 18 kDa corresponding to monomeric lacritin and higher molecular weight bands predicted to be cross-linked monomers of lacritin. In tears from dry eye dogs, bands were absent or faintly observed.

Conclusions: Canine lacritin has been cDNA cloned, sequenced, expressed in E. coli, and purified. An antibody specific for canine lacritin has been developed and utilized for ELISA and Western blot analysis of canine tears. Canine lacritin showed an approximate 6 fold decrease in tears from dry eye dogs compared to tears from normal dogs by ELISA. In tears from dry eye dogs, the monomer was absent or faintly observed by Western blot analysis.

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