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Published on: December 15, 2011
Tissue transglutaminase is a negative regulator of monomeric lacritin bioactivity
Francisco Velez V1, Jeffrey A Romano, Robert L McKown
1Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908-0732, USA.
Insights
Tissue transglutaminase (TGM2) cross-links lacritin in tears, forming multimers that reduce its bioactivity. This explains how lacritin levels in tears differ from optimal cell culture concentrations, revealing a key regulatory mechanism.
Area of Science:
- Epithelial biology
- Proteomics
- Ocular surface science
Background:
- The tear film is crucial for vision and ocular surface health.
- Lacritin, a tear glycoprotein, plays vital roles in tear secretion, cytoprotection, and mitogenesis.
- Observed lacritin levels in tears (mid-micromolar) differ from optimal cell culture concentrations (1-10 nM).
Purpose of the Study:
- To reconcile the discrepancy between lacritin concentrations in tears and cell culture.
- To investigate potential mechanisms regulating lacritin bioactivity in the tear film.
Main Methods:
- Generation of monoclonal and polyclonal antibodies against lacritin.
- Western blotting of human tears and saliva.
- In vitro analysis of tissue transglutaminase (TGM2) mediated lacritin cross-linking using mass spectrometry.
Main Results:
- Human tears and saliva contain higher molecular weight, cross-linked forms of lacritin.
- TGM2 rapidly cross-links lacritin in vitro, forming multimers.
- Cross-linking involves specific lysine and glutamine residues and reduces syndecan-1 binding.
Conclusions:
- TGM2-mediated cross-linking of lacritin forms multimers in tears.
- This cross-linking negatively regulates lacritin's mitogenic and cytoprotective functions by impairing syndecan-1 binding.
- Identifies a mechanism explaining lacritin's bioactivity regulation in vivo.
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 μM TGM2 in tears. Cross-linking was absent when Tgm2 was inactive, and cross-linked lacritin, unlike recombinant monomer, bound syndecan-1 poorly.
Conclusions:
Since syndecan-1 binding is necessary for lacritin mitogenic and cytoprotective activities, TGM2 cross-linking negatively regulates lacritin bioactivity.
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