Abstract
Purpose: :
Lacritin is a small glycoprotein largely secreted by lacrimal acinar cells to be released into tears where it promotes basal tear secretion, selective epithelial cell proliferation, and plays a cytoprotective role. Several studies suggest that 23 kDa (monomeric) lacritin may be selectively downregulated in dry eye. While characterizing a new generation of anti-lacritin N- vs C-domain-specific antibodies (including mab 1F5-C9-F4) with N- and C-truncation mutants, immunoreactive 75 to 83kDa material was noted in human tear, and lacrimal gland and salivary gland blots. Here, we report that lacritin serves as a substrate for tissue transglutaminase (TGM2) as a possible mechanism for diminishing levels of monomeric lacritin.
Methods: :
1F5-C9-F4 N-terminal specific anti-lacritin monoclonal antibody was generated and tested in blots against lacritin and lacritin truncation mutants, or of human tears or human lacrimal and salivary gland lysates. New N- and C-terminal polyclonal antibodies were also tested. In cross-linking studies, 3 µM lacritin was incubated in HEPES (37°C) containing 10 mM Ca2+ and 1.5 µM TGM2 with or without 30 µM of competitive inhibitor. The reaction was terminated by adding 50 mM EDTA, and blotted with 1F5-C9-F4.
Results: :
Greater than 90% of lacrimal and salivary gland lacritin appear to be cross-linked in samples of the individuals tested. Monomeric lacritin is modified by TGM2 within 1 min and converted into 73 to 250 kDa multimers within 40 min. The N- and C-terminal halves are both subject to cross-linking activity. Candidate amine acceptors include Gln14, Gln42, Gln50, or Gln60 in the N-terminal half and Gln96 or Gln106 in the C-terminus.
Conclusions: :
Human tear, lacrimal and salivary gland lacritin is subject to multimerization by TGM2, a component of human tears. Bioactivity could thereby be affected.
Keywords: cornea: tears/tear film/dry eye