PURPOSE. Molar accounting of bioactive fluids can expose new regulatory mechanisms in the growing proteomic focus on epithelial biology. Essential for the viability of the surface epithelium of the eye and for normal vision is the thin, but protein-rich, tear film in which the small tear glycoprotein lacritin appears to play a prominent prosecretory, cytoprotective, and mitogenic role. Although optimal bioactive levels in cell culture are 1 to 10 nM over a biphasic dose optimum, ELISA suggests a sustained tear lacritin concentration in the midmicromolar range in healthy adults. Here we identify a reconciling mechanism.METHODS. Monoclonal anti-lacritin 1F5 antibody was generated, and applied together with a new anti-C-terminal polyclonal antibody to tear and tissue Western blotting. In vitro tissue transglutaminase (Tgm2) cross-linking was monitored and characterized by mass spectrometry.RESULTS. Blotting for lacritin in human tears or saliva surprisingly detected immunoreactive material with a higher molecular weight and prominence equal or exceeding the~23 to 25 kDa band of monomeric glycosylated lacritin. Exogenous Tgm2 initiated lacritin cross-linking within 1 minute and was complete by 90 minutes-even with as little as 0.1 nM lacritin, and involved the donors lysine 82 and 85 and the acceptor glutamine 106 in the syndecan-1 binding domain. Lacritin spiked into lacritin-depleted tears formed multimers, in keeping with~0.6 lM TGM2 in tears. Cross-linking was absent when Tgm2 was inactive, and cross-linked lacritin, unlike recombinant monomer, bound syndecan-1 poorly. Enhanced TGM2 expression correlates with reduced cell viability, caspase activation, TNF receptor clustering, 7 and mitochondrial dysfunction 8 associated with hyperosmolar stress in dry eye. 9 Transglutaminases encompass a multifunctional family of enzymes involved in high-fidelity posttranslational modifications that catalyze Ca 2þ -dependent covalent bond formation between primary amines or e-amino groups of lysine and c-carboxamide groups of glutamine. Cross-linking affects function, both negatively and positively. TGM2 crosslinked collagen is resistant to metalloproteinase digestion and less mitogenic 10 ; and cross-linked IL-2 (unlike IL-2 monomer) is cytotoxic for oligodendrocytes. 11 TGM2 also positively regulates the activity of midkine, a small heparin binding growth factor, 12 and is required for the activation of latent TGF-b 13 and S100A11.14 Could TGM2 in tears regulate ocular surface biology?Lacritin is a 12.3 kDa tear prosecretory mitogen 15 with glutamine and lysine residues suitable for TGM2 catalyzed cross-linking. Lacritin promotes corneal epithelial cell survival (Zimmerman K, et al. IOVS 2012;53:ARVO E-Abstract 4231) and proliferation, 16 and basal tear protein secretion by lacrimal acinar cells. 15 When topically applied to rabbit eyes, lacritin acutely increases basal tear flow. 17 Lacritin is largely restricted to tears and to a lesser extent saliva, through its lacrimal acinar cell, 15

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