ReviewLacritin and the tear proteome as natural replacement therapy for dry eye☆
Introduction
Tears accumulate on the avascular corneal epithelium, and vascularized conjunctiva, as a translucent film rich in proteins, lipids and metabolites. The tear proteome is estimated to comprise 1543 proteins (Zhou et al., 2012), with over half designated as ‘intracellular’ (Table 1) by Gene Ontology, implying that cell death from normal epithelial renewal may be a contributor. Beyond its capacity to lubricate the lid, tears are essential for the refraction of light (Montés-Micó, 2007). Equally important and irreplaceable by drugs or drops is the role of tears in promoting corneal epithelial health for when tears are chronically insufficient, the epithelium becomes stressed and releases inflammatory cytokines that further exacerbate the situation (Massingale et al., 2009). Dry eye affects 5–6% of the general population, rising to 6–9.8% and as high as 34%, respectively in postmenopausal women (Schaumberg et al., 2003) and the elderly (Lin et al., 2005).
Relatively few tear proteins appear to be selectively down- or upregulated in dry eye (Table 1). Appreciating which are bioactive and at what molar levels would be insightful. The only growth factor-like molecule downregulated in mild to severe aqueous deficiency was lacritin (Srinivasan et al., 2012). Lacritin promotes basal tearing when added topically in rabbits (Samudre et al., 2011). Also decreased was lipocalin-1 (Srinivasan et al., 2012), that cleanses the ocular surface of lipids that would otherwise interfere with ocular surface wetting (Glasgow and Gasymov, 2011). Lacritin was the most severely downregulated protein in contact lens-related dry eye (Nichols and Green-Church, 2009) – perhaps in part because it is readily adsorbed on contact lenses (Green-Church and Nichols, 2008). It is also deficient in blepharitis (Koo et al., 2005), a common inflammation of the eyelid, associated with evaporative dry eye (Mathers, 1993). Two studies did not note any lacritin change in dry eye using mass spectrometry coupled with liquid chromatographic cationic separation followed by reverse phase separation (Zhou et al., 2009, Boehm et al., 2013), although one observed a decrease of lipocalin-1 (Zhou et al., 2009). 2-D SDS PAGE prior to mass spectrometry (Koo et al., 2005, Nichols and Green-Church, 2009, Srinivasan et al., 2012) is necessary to distinguish monomeric lacritin from inactive multimeric lacritin (Velez et al., 2013), and likely also the inactive lacritin-c splice variant (McKown et al., 2009). Exploration of lacritin cell targeting and signaling mechanisms has revealed a network of interdependent molecules, each necessary for lacritin activity. New evidence is suggesting that some of these are also decreased in dry eye. Here we review recent advances in our understanding of lacritin, and provide an overview of lipocalin-1 whose eye specific expression parallels that of lacritin. We also update our current understanding of the tear proteome.
Section snippets
Structure and expression
The discovery of lacritin indirectly emerged from a screen for novel factors capable of promoting tear protein secretion, with cDNA cloning out of a human lacrimal gland library (Sanghi et al., 2001). The lacritin gene, LACRT, is one of the most eye specific (Sanghi et al., 2001) and resides on 12q13, within ∼1.24 Mb of the AAAS gene associated with alacrima (Kumar et al., 2002). Human lacritin is coded by a 417 bp open reading frame that translates as a 14.3 kDa hydrophilic protein with a 19
Structure and expression
Tear lipocalin (LCN1; recently reviewed by Glasgow and Gasymov, 2011, Dartt, 2011) was originally noted as an unknown band in early electrophoretic separations of human tears and named tear pre-albumin (Erickson et al., 1956), based on its paper electrophoretic mobility near serum albumin proximal to the anode. Lack of immunological cross-reactivity in normal human tears suggested that the two were distinct (Josephson and Lockwood, 1964), in keeping with earlier studies by Fleming suggesting
New additions to the tear proteome
We previously assembled all tear proteomic data into a single table, restricting entry to proteins designated as ‘extracellular’ or ‘plasma membrane’ in their primary or alternative location (Laurie et al., 2008). Now updated with 139 new entries from Zhou et al. (2012), the additions supplement tears with proangiogenic, anti-angiogenic, retinal survival, epithelial repair, cysteine protease inhibitor, immunosuppressive, and immunostimulatory activities (Table 1). Thirteen are highlighted below.
Conclusions
Advantage should be taken of tear proteins as potential biomarkers, drug targets, and biotherapeutics. Tear-based biotherapeutics have considerable potential, particularly with the relatively small number of tear proteins that appear to be selectively downregulated in dry eye. Rather than simply alleviating symptoms, causes of ocular surface diseases may be addressable. Lacritin- and lipocalin-1-based therapeutics offer a platform to initiate this approach. Newly identified members of the tear
Notes added in proof
- 1.
One tear proteomic article was overlooked. Aluru et al (PLoS One 7, e51979, 2012) compared tears from 73 normals to 129 individuals suffering from aqueous deficient dry eye. 2-D SDS PAGE with mass spectrometry identified seven downregulated proteins: AZGP1, CST_ [cystatin type not specified], IGJ, LACRT, LTF, PRR4 and SCGB2A2. As per Table 1, all except IGJ have been previously noted as downregulated in dry eye. Lacritin was downregulated in 95% of cases.
- 2.
Low et al, 2013 (J. Proteomics [Epub
Acknowledgments
GWL is supported by R01 EY013143 and EY018222. RK is supported by SR/FT/LS-157/2012 (RK). The authors acknowledge the multi-institutional Lacritin Consortium for help with much of the lacritin work reviewed, particularly the development of lacritin and syndecan-1 constructs by Ron Raab and Robert McKown at James Madison University, the supply of human tears by Denise Ryan (Walter Reed Army Medical Center), animal studies by Pat Williams' group (Eastern Virginia Medical School), and mechanistic
References (108)
- et al.
Comparative proteomics of human male and female tears by two-dimensional electrophoresis
Exp. Eye Res.
(2011) - et al.
Extracellular angio-associated migratory cell protein plays a positive role in angiogenesis and is regulated by astrocytes in coculture
Microvasc. Res.
(2002) - et al.
The 1.8-A crystal structure of human tear lipocalin reveals an extended branched cavity with capacity for multiple ligands
J. Biol. Chem.
(2005) Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases
Prog. Retin. Eye Res.
(2009)Tear lipocalin: structure and function
Ocul. Surf.
(2011)- et al.
Lipocalin-interacting-membrane-receptor (LIMR) mediates cellular internalization of beta-lactoglobulin
Biochim. Biophys. Acta
(2008) - et al.
Interaction of tear lipocalin with lysozyme and lactoferrin
Biochem. Biophys. Res. Commun.
(1999) - et al.
Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity
Biochim. Biophys. Acta
(1999) - et al.
Focus on molecules: tear lipocalin
Exp. Eye Res.
(2011) - et al.
Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6
Exp. Eye Res.
(2001)
Expression of the gene for tear lipocalin/von Ebner's gland protein in human prostate
FEBS Lett.
Comparative binding studies of cyclophilins to cyclosporin A and derivatives by fluorescence measurements
Anal. Biochem.
Endogenous Gas6 and Ca2+-channel activation modulate phagocytosis by retinal pigment epithelium
Cell. Signal.
Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a binding partner of epithelial growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1). Implications for macular degenerations
J. Biol. Chem.
Core protein structure and sequence determine the site and presence of heparan sulfate and chondroitin sulfate on syndecan-1
J. Biol. Chem.
Ocular evaporation in meibomian gland dysfunction and dry eye
Ophthalmology
Lacritin and other new proteins of the lacrimal functional unit
Exp. Eye Res.
Visfatin/pre-B-cell colony-enhancing factor (PBEF), a proinflammatory and cell motility-changing factor in rheumatoid arthritis
J. Biol. Chem.
Role of the tear film in the optical quality of the human eye
J. Cataract Refract. Surg.
Establishment of an appropriate animal model for lacritin studies: cloning and characterization of lacritin in monkey eyes
Exp. Eye Res.
Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds
J. Biol. Chem.
The epithelin precursor encodes two proteins with opposing activities on epithelial cell growth
J. Biol. Chem.
cDNA cloning and sequencing reveals human tear prealbumin to be a member of the lipophilic-ligand carrier protein superfamily
J. Biol. Chem.
Antibody array characterization of inflammatory mediators in allergic and normal tears in the open and closed eye environments
Exp. Eye Res.
cDNA and genomic cloning of lacritin, a novel secretion enhancing factor from the human lacrimal gland
J. Mol. Biol.
Prevalence of dry eye syndrome among US women
Am. J. Ophthalmol.
Tear proteome and protein network analyses reveal a novel pentamarker panel for tear film characterization in dry eye and meibomian gland dysfunction
J. Proteomics
Lacritin Rescues Stressed Epithelia via Rapid Forkhead Box O3 (FOXO3)-associated Autophagy That Restores Metabolism
J. Biol. Chem.
Molecular cloning of a novel lipocalin-1 interacting human cell membrane receptor using phage display
J. Biol. Chem.
Antisense down-regulation of lipocalin-interacting membrane receptor expression inhibits cellular internalization of lipocalin-1 in human NT2 cells
J. Biol. Chem.
Functional cavity dimensions of tear lipocalin
Curr. Eye Res.
Fibulins 3 and 5 antagonize tumor angiogenesis in vivo
Cancer Res.
Corneal avascularity is due to soluble VEGF receptor-1
Nature
Pathogen induced changes in the protein profile of human tears from Fusarium keratitis patients
PLoS One
s-cyclophilin is retained intracellularly via a unique COOH-terminal sequence and colocalizes with the calcium storage protein calreticulin
J. Cell Biol.
Alterations in the tear proteome of dry-eye patients – a matter of the clinical phenotype
Invest. Ophthalmol. Vis. Sci.
Specific tear prealbumin: a unique lachrymal protein absent from serum and other secretions
Nature
Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican
J. Cell Biol.
Role of laminin-1, collagen IV, and an autocrine factor(s) in regulated secretion by lacrimal acinar cells
Am. J. Physiol.
Hyperosmolarity-induced cornification of human corneal epithelial cells is regulated by JNK MAPK
Invest. Ophthalmol. Vis. Sci.
Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors
Genome Biol.
Filter-paper electrophoresis of tears. II. Animal tears and the presence of a slow-moving lysozyme
AMA Arch. Ophthalmol.
Lysozyme tear level in patients with herpes simplex virus eye infection
Invest. Ophthalmol. Vis. Sci.
On specificity of the protein of human tears
Brit. J. Exp. Path.
Human tear lipocalin exhibits antimicrobial activity by scavenging microbial siderophores
Antimicrob. Agents Chemother.
Lacritin-induced secretion of tear proteins from cultured monkey lacrimal acinar cells
Invest. Ophthalmol. Vis. Sci.
Purification of the isoforms of tear specific prealbumin
Curr. Eye Res.
Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: evidence for features that confer promiscuity in ligand binding
Biochemistry
Relaxation of beta-structure in tear lipocalin and enhancement of retinoid binding
Invest. Ophthalmol. Vis. Sci.
Tear lipocalins bind a broad array of lipid ligands
Curr. Eye Res.
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Grant information: NIH RO1EY013143, RO1EY018222 (GWL); SR/FT/LS-157/2012 RK.