title: Tissue transglutaminase is a negative regulator of monomeric lacritin bioactivity.
abstract: OBJECTIVE
  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.
authors: Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908-0732, USA.
authors: Francisco
authors: F
authors: Velez V
authors: Jeffrey A
authors: JA
authors: Romano
authors: Robert L
authors: RL
authors: McKown
authors: Kari
authors: K
authors: Green
authors: Liwen
authors: L
authors: Zhang
authors: Ronald W
authors: RW
authors: Raab
authors: Denise S
authors: DS
authors: Ryan
authors: Cindy M L
authors: CM
authors: Hutnik
authors: Henry F
authors: HF
authors: Frierson
authors: Gordon W
authors: GW
authors: Laurie
mesh_terms: D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000032:analysis* / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000494:pharmacology*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012463:Saliva / Q000737:chemistry; D013666:Tears / Q000737:chemistry*; D011503:Transglutaminases / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D005136:Eye Proteins; D006023:Glycoproteins; C431764:LACRT protein, human; C000717608:TGM2 protein, human; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins