title: Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.
abstract: OBJECTIVE
  Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears.
  
  
  METHODS
  Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed.
  
  
  RESULTS
  Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA.
  
  
  CONCLUSIONS
  Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.
authors.affiliation: Department of Animal and Poultry Sciences, Virginia Polytechnic and State University, Blacksburg, Virginia, USA. glaurie@virginia.edu
authors.forename: Diane E
authors.initials: DE
authors.lastname: Laurie
authors.forename: Rebecca K
authors.initials: RK
authors.lastname: Splan
authors.forename: Kari
authors.initials: K
authors.lastname: Green
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Still
authors.forename: Robert L
authors.initials: RL
authors.lastname: McKown
authors.forename: Gordon W
authors.initials: GW
authors.lastname: Laurie
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000032:analysis* / Q000235:genetics; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics; D006736:Horses; D013058:Mass Spectrometry; D008934:Mitogens; D010446:Peptide Fragments / Q000737:chemistry; D013666:Tears / Q000737:chemistry*
chemical_list: D005136:Eye Proteins; D006023:Glycoproteins; D008934:Mitogens; D010446:Peptide Fragments