Lacritin is a 12.3 kDa glycoprotein encoded in humans by the LACRT gene. Lacritin's discovery emerged from a screen for factors that stimulate tear protein secretion. Lacritin is a secreted protein found in tears and saliva. Lacritin also promotes tear secretion, the proliferation and survival of epithelial cells, and corneal wound healing Lacritin is thus a multifunctional prosecretory mitogen with cell survival activity. Natural or bacterial cleavage of lacritin releases a C-terminal fragment that is bactericidal. Most lacritin is produced by the lacrimal gland, including the accessory lacrimal gland of Wolfring. Some lacritin is produced by the meibomian gland, and by epithelial cells of the conjunctiva and cornea. Together these epithelia comprise much of the lacrimal functional unit (LFU). Dry eye is the most common disease of the LFU. A growing number of studies suggest that lacritin may be differentially downregulated in dry eye, including contact lens-related dry eye. Topical lacritin promotes tearing in rabbit preclinical studies. In the Aire knockout mouse model of dry eye (considered similar to human Sjogren's syndrome), topical lacritin restores pilocarpine-induced tearing, largely eliminates lissamine green staining and reduces the size of inflammatory foci in the lacrimal gland. Lacritin cell targeting is dependent on the cell surface heparan sulfate proteoglycan syndecan-1 (SDC1). Binding utilizes an enzyme-regulated 'off-on' switch in which active epithelial heparanase (HPSE) cleaves off heparan sulfate to expose a binding site in the N-terminal region of syndecan-1's core protein. A G-protein-coupled receptor (GPCR) then appears to be ligated. Targeted cells signal to NFAT and mTOR if conditions are suitable for proliferation, or to AKT and FOXO3 under conditions of stress.
Structure Lacritin consists of 119 amino acids after cleavage of the N-terminal signal peptide and displays several predicted alpha helices, mostly in the C-terminal half. Of these, the two C-terminal ones have been confirmed by circular dichroism. The most C-terminal alpha helix is amphipathic with hydrophobic and hydrophilic residues on opposite faces. The hydrophobic face is an important syndecan-1 binding element. PONDR (Predictor of Naturally Disordered Regions) predicts that the C-terminal and N-terminal halves are respectively 'ordered' and 'disordered'. 11 - 12 predicted O-glycosylation sites populate the N-terminal half. The C-terminal amphipathic alpha helix is also the site of lacritin's only N-glycosylation site. In 'climatic droplet keratopathy' this site is not glycosylated. Lacritin recombinantly generated in E. coli (no glycosylation) and lacritin in tears (glycosylated) differ in size with respective mobilities of ~18 and ~25 kDa by SDS-PAGE. With a predicted protein core molecular weight of 12.3 kDa, it is possible that mobility is partially retarded by lacritin's amphipathic alpha helices. Predicted pI of lacritin's core protein is 5. Lacritin is subject to crosslinking by tissue transglutaminase, thereby giving rise to lacritin multimers including dimers and trimers. Crosslinking is initiated within 1 min in vitro, requiring as little as 0.1 nM lacritin. The ~0.6 micro molar level of tissue transglutaminase estimated in human tears is sufficient to promote crosslinking. Crosslinking involves the donors lysine 82 and 85 and the acceptor glutamine 106. Glutamine 106 resides within the amphipathic alpha helix near the C-terminus responsible for binding the N-terminus of syndecan-1. Accordingly, crosslinked lacritin binds syndecan-1 poorly and is inactive. Several lacritin splice variants have been detected in Aceview, from NEIBank EST data. Lacritin-b (11.1 kDa; pI 5.3) lacks the sequence SIVEKSILTE. Lacritin-c (10.7 kDa; pI 4.6) displays a novel C-terminus that should be incapable of binding syndecan-1, and lacks cell survival activity. Splice variants are proteoforms. Proteoforms include proteolytically processed forms of lacritin. Top down mass spec sequencing revealed that human tears contain five N- and forty-two different C-terminal lacritin-a proteoforms. Some approximate the bioactive lacritin synthetic peptides 'N-104', 'N-94' and 'N-94/C-6' from lacritin's C-terminus. Protease inhibitor studies suggest that processing of lacritin into C-terminal proteoforms requires a variety of tear proteases including cathepsin B, calpain, alanyl amino peptidase, arginyl aminopeptidase, MMP9, MMP10, cathepsin G, plasma kallikrein, plasmin, thrombin and trypsin. C-terminal proteoforms, like intact lacritin, are selectively deficient in dry eye tears.
… excerpt ends here. Continue reading the full article.
