Dry Eye and Designer Ophthalmics

Abstract

EST, proteomic, and antibody capture assays are revealing a level of tear film protein complexity far greater than previously appreciated. A systems biology approach will be needed to fully appreciate function as tear protein doses fluctuate in time through different conditions. Although consensus is growing on what fully constitutes the human tear proteome, questions remain about the source and significance of the ∼256 tear proteins designated as ‘intracellular’. Many of these may derive from normal cellular turnover and could therefore be informative. A further >183 are designated as ‘extracellular’. Surprisingly, only 4 – 5% of these appear to be dysregulated in the three forms of dry eye preliminarily examined to date. Some differ and a couple overlap, suggesting that disease-specific signatures could be identified. Future dry eye treatment might include recombinant tear protein rescue as a personalized ophthalmic approach to ocular surface disease.

The Expanding Tear Proteome

Early study of tears by SDS-polyacrylamide gel electrophoresis (PAGE) identified lysozyme (LYZ), lactotransferrin (LTF; lactoferrin), and lipocalin-1 (LCN1; von Ebner gland protein or tear specific prealbumin) that together constitute 70 to 85% of total tear protein.¹ Later, transferrin (TF), albumin (ALB; serum albumin), secretory IgA (CD79A), and lipophilin (PLP1)^1–4 were identified, and more recently via immunoassays of increasing sensitivity and reliability⁵: phospholipid transfer protein (PLTP),⁶ growth factors,^7,8 neurotrophic factors,⁹ cytokines,^5,10–13 matrix metalloproteinases,^11,14–16 bradykinins,¹⁷ tachykinins (e.g., substance P),^18,19 fibronectin (FN1),²⁰ plasminogen activator (PLAU),²¹ defensins,²² aquaporins,²³ phospholipase,²⁴ immunoglobulins,²⁵ lactate dehydrogenase,²⁶ proline-rich 4 (lacrimal; PRR4)²⁷ and insulin (INS).⁸ Continuing efforts are underway to document the complete human tear film proteome by mass spectrometry.^28–30 De Souza and coworkers identified almost 500 proteins in human closed eye tears using a ultra-high resolution hybrid linear trap – Fourier Transform (LTQ-FT) and a linear ion trap – orbitrap (LTQ-Orbitrap) approach.²⁹ A surprising number (∼256) are designated as ‘intracellular’ proteins by Gene Ontology, suggesting that the process of normal epithelial turnover is a significant source.³¹ Others may be contaminants of the method of collection. A further >183 are designated as ‘extracellular’ (Table 1). Preliminary indications primarily by 2-D PAGE are that a surprisingly small fraction (4 – 5%) of these are downregulated in dry eye (*, Δ, † in Table 1).^28,32,33 This observation could set the stage for assays defining what is healthy, what is diseased and possibly when disease has been initiated.

TABLE 1.

Proteins in the normal human human tear ‘proteome’ that are predicted to be extracellular according to Gene Ontology (GO). The list is derived from published tear²²,²⁹,⁸⁵, Meibomian gland secretion³⁰, tear capture ELISA or antibody array⁵,⁸⁶ and lacrimal gland EST⁸⁷ analyses. Not listed are the numerous cytoplasmic proteins that are also detected in tears²⁹.

Gene Symbol	Protein	Function (as per Locust Link, OMIM or Source)
		(i) Angiogenesis
ANG	angiogenin, ribonuclease, RNase A family, 55	promotes angiogenesis
BAI3	brain-specific angiogenesis inhibitor 385	possible angiogenesis inhibitor
ECGF1	endothelial cell growth factor 129	promotes angiogenesis
SERPINF1	serp. pep. inhib., cl. F (α-2 antiplas., PEDF), mem. 129	promotes neurodifferent. and inhibits angiogenesis
		(ii) Biosynthesis
ATP5B	ATP synth, H+ transp., mitoch. F1 complex, ppolypep.29	catalyzes ATP synthesis in mitochondrion
B4GALT1	UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase29	polypep,, glycoconjugate and lactose biosynthesis
PDIA6	protein disulfide isomerase family A, member 629	predicted electron transport and protein folding roles
PPIC	peptidylprolyl isomerase C (cyclophilin C)29	protein folding, binds cyclosporin A
		(iii) Calcium
AHSG	alpha-2-HS-glycoprotein29	calcification inhibitor
ANXA2	annexin A2 Ca2+ depend. phospholip. binding prot.29	osteoclast formation and bone resorption
ANXA5	annexin A5 Ca2+ depend. phospholip. binding prot.29,87	promotes Ca2+ channel activity
CALR	calreticulin29	Ca2+ binding protein in ER and nucleus; SS assoc.
CALU	calumenin29	Ca2+ binding protein in ER, protein folding/sorting
CANT1	CANT129	Ca2+ activated nucleotidase
NUCB1	nucleobindin 129	Golgi and peripheral membrane Ca2+ binding protein
NUCB2	nucleobindin 229	peripheral membrane Ca2+ binding protein
		(iv) Carbohydrate
AGL	amylo-1, 6-glucosidase, 4-alphaglucanotransferase29	glycogen degradation
CHI3L2	chitinase 3-like 229	glycan but not heparin binding
ENO1	enolase 1, (alpha)29	glycolytic enzyme
GAPDH	glyceraldehyde-3-phosphate dehydrogenase29,87	carbohydrate metabolism
LGALS3	lectin, galactoside-binding, soluble, 3, galectin29,87	galactose-specific lectin
LGALS3BP	lectin, galactoside-binding, soluble, 3 binding protein29	binds Mac-2 and galectin 1
MANBA	mannosidase, beta A29	lysosomal N-linked oligosaccharide catabolism
PKM2	pyruvate kinase, muscle29	carbohydrate degradat., binds bacterial Opa protein
		(v) Carrier/Binding Protein Steroid Assoc.
ALB	albumin*22,29,85	carrier protein
ARTS-1	type 1 TNFR shedding aminopeptidase regulator29	binds TNFR1 to promote shedding
AZGP1	alpha-2-glycoprotein 122,29,87	zinc-binding, lipid degrad., cell adhesion
CD14	CD14 molecule29	binds LPS binding protein (LBP) and apoptotic cells
DMBT1	deleted in malignant brain tumors 122,29,85,87	scavenger receptor, binds surfactant protein D
DSP	desmoplakin29	key component of desmosomes
GC	group-specific component (vitamin D binding protein)29	carrier protein for vitamin D and metabolites
HP	haptoglobin22,29	hemoglobin binding, turnover to diminish iron loss
HPX	hemopexin22,29	heme binding, turnover
HSPG2	heparan sulfate proteoglycan 2 (perlecan)29,87	growth factor binding, filtration, matrix polymerization
IGFBP1	insulin-like growth factor binding protein 15	slows turnover of IGF’s
IGFBP2	insulin-like growth factor binding protein 25	slows turnover of IGF’s
KPNB1	karyopherin (importin) beta 129	nuclear transport
LCN1	lipocalin 1 (tear prealbumin) Δ22,29–31, 62	hydrophobic prot. binding, cyst. proteinase. Inhibitor
M6PRBP1	mannose-6-phosphate receptor binding protein 129	endosome-to-Golgi transport
PEBP4	phosphatidylethanolamine-binding protein 429	?
SCGB1D1	secretoglobin, family 1D, member 1†22,29,85,87	in complex that binds steroids, including androgen
SCGB2A1	secretoglobin, family 2A, member 122,29,85,87	possibly binds steroids, including androgen
SCGB2A2	secretoglobin, family 2A, member 2 (mammaglobin 2)88	?
TCN1	transcobalamin I (vitamin B12 binding, R bind. family)29,87	binds and helps move vitamin B12 into cells
TF	transferrin22,29,85	iron binding and transport to proliferating cells
TTR	transthyretin (prealbumin, amyloidosis type I)22,29	thyroxine binding and transport
		(vi) Cell Adhesion/Motility/Structure
ACTB	actin, beta22	cell structure, motility
ACTA1	actin, alpha 1, skeletal muscle88	formation of filaments
FGA	fibrinogen alpha chain22,29	cell adhesion, spreading, mitogenic, chemotactic
FGG	fibrinogen gamma chain29	cell adhesion, spreading, mitogenic, chemotactic
FLRT3	fibronectin leucine rich transmembrane protein 385	possibly cell adhesion, receptor signaling
FN1	fibronectin 129,87	cell adhesion, migration, blood coagulation
GSN	gelsolin (amyloidosis, Finnish type)29	blocks actin monom. exchange or promotes nucleat.
LAMA3	laminin, alpha 330	cell adhesion, differentiation
MFGE8	milk fat globule-EGF factor 8 protein29	cell adhesion, rotavirus binding/inhibition
MSLN	mesothelin29,85	possible cell adhesion activity
PFN1	profilin 129	regulator of actin polymerization and cytoskeleton
SLIT3	slit homolog 3 (Drosophila)29	cell migration
THBS1	thrombospondin 129	cell-cell and cell-matrix adhesion
TLN1	talin 129	actin filament assembly and cell spreading
VIM	vimentin29,29	cytoskeletal intermediate filaments
		(vii) Cell Growth
ANGPTL1	angiopoietin-like 129,87	may inhibit cell growth
EGF	epidermal growth factor (beta-urogastrone)5	prosecretory mitogen
GDNF	glial cell derived neurotrophic factor5	dopaminergic neuron survival, differentiation
HGF	hepatocyte growth factor (hepapoietin A; scatter factor)5	serine protease-activated mitogen
LACRT	lacritin *Δ†22,29,30,85,87	prosecretory mitogen
MUC4	mucin 429	epithelial cell proliferation and differentiation
NTF3	neurotrophin 35	sensory neuron survival
NTF5	neurotrophin 55	peripheral sensory sympathetic neuron survival
QSOX1	(QSCN6) quiescin Q6 sulfhydryl oxidase 129	growth regulation
SERPINB5	serpin peptidase inhibit., clade B (ovalbum), member 529	blocks mammary tumor growth
		(viii) Cytoprotective/Anti-Apoptotic
CLU	clusterin22, 29,85,87	inhibits apoptosis
MUC16	mucin 1629	cytoprotective, hydrophilic
MUC5AC	mucin 5AC29	mucus/gel-forming, cytoprotective, hydrophilic
PIP*	prolactin-induced protein22, 29,85,87	inhibitor of T-cell apoptosis, aspartyl proteinase (?)
PRB1	proline-rich protein BstNI subfamily 188	?
PROL1	proline rich, lacrimal 122, 29,30,85,87	possible ocular protective function
PRR4	proline rich 4 (lacrimal)Δ†22, 29,30,85,87	possible ocular protective function
		(ix) Extracellular Matrix
COL6A1	collagen, type VI, alpha 129	microfibril component
MUCL1	mucin-like 185	?
SPARCL1	SPARC-like 1 (mast9, hevin)29,87	reg. of collagen assembly and decorin secretion
		(x) Immune
ATRN	attractin29	receptor or clustering of immune cells
C3	complement component 322,29,87	complement activation
C4A	complement component 4A (Rodgers blood group)29	cleaved to a trimer for complement activation
CCL2	chemokine (C-C motif) ligand 25	monocyte, basophil specific chemotaxis
CCL4	chemokine (C-C motif) ligand 45	Inflammatory, chemokinetic
CCL8	chemokine (C-C motif) ligand 85	monocyte, basophil, eosinphil, lympho. chemotaxis
CCL11	chemokine (C-C motif) ligand 115	eosinophil specific chemotaxis
CCL22	chemokine (C-C motif) ligand 225	NK cell, dendritic, monocyte chemotaxis
CCL24	chemokine (C-C motif) ligand 245	resting T cell chemotaxis
CFB	complement factor B22,29	CFD cleaved to: prolif. serine protease & antiprolif.
CFH	complement factor H22,29,87	restricts complement activation to microbial defense
CSF1	colony stimulating factor 1 (macrophage)5	prod’n, different, function of macrophages
CSF2	colony stimulating factor 2 (granulocyte-macrophage)5	prod’n, different, function of granulocytes, macroph.
CSF3	colony stimulating factor 3 (granulocyte)5	prod’n, different, function of granulocytes, macroph.
CXCL5	chemokine (C-X-C motif) ligand 55	inflammatory cytokine, neutrophil activation
CXCL10	chemokine (C-X-C motif) ligand 105	T cell, monocyte chemotaxis
FCRL5	Fc receptor-like 530	possible mature B cell inhibitory co-receptor
CXCL1	chemokine (C-X-C motif) ligand 1 (MGS activity, alpha)5	neutrophil chemotaxis
CXCL9	chemokine (C-X-C motif) ligand 95	T cell chemotaxis
IL6	interleukin 6 (interferon, beta 2)86	B cell, nerve cell differentiation
IL8	interleukin 85,86	inflammatory response mediator, angiogenic
IL10	interleukin 105	inhibit activated macrophage cytokine synthesis
IL12	interleukin 125	activated T, NK cell mitogen
IL15	interleukin 155	T cell proliferation
IL16	interleukin 16 (lymphocyte chemoattractant factor)5	CD4+ lymphocyte, monocyte,eosinophil migration
IFNG	interferon, gamma5	immunoregulatory, antiviral
ORM1	orosomucoid 122,29	apparent modulator of acute-phase immune activity
PPBP	pro-platelet basic prot. (chemokine (C-X-C motif) lig. 7)5	neutrophil chemoattractant, prosecretory
SERPING1	serpin peptid. inhibitor, clade G (C1 inhib), member 129	complement activation regulator
TGFB2	transforming growth factor, beta 25	suppress IL-2 T cell growth
TNF	tumor necrosis factor (TNF superfamily, member 2)5	autoimmune, pyrogen, mitogen, differentiation
		(xi) Lipid/cholesterol
ANPEP	alanyl (membrane) aminopeptidase29	aminopeptidase
ANXA1	annexin A122	regulate phospholipase a2 activity
APOA1	apolipoprotein A-I22	cholesterol transport
APOA2	apolipoprotein A-II29	in high density lipoprotein particles
APOA4	apolipoprotein A-IV22	in HDL and chylomicrons
APOB	apolipoprotein B29	in chylomicrons and low density lipoproteins
APOH	apolipoprotein H22,29	lipoprotein metabolism, coagulation
PLA2G2A	phospholipase A2, group IIA85	membrane phospholipid metabolism
PLTP	phospholipid transfer protein29,85	cholesterol metabolism
PSAP	prosaposin29,87	enzyme stimulator in glycosphingolipid metabolism
		(xii) Other/Uknown
AGT	angiotensinogen29	cleaved to angiotensin I, blood pressure
B2M	beta-2-microglobulin22,29,85,87	loss assoc. with hypercatabolic hypoproteinemia
EVC2	Ellis van Creveld syndrome 2 (limbin)30	mutation assoc. wdith Ellis-van Creveld syndrome
HSPA4	heat shock 70kDa protein 429	heat shock protein
LRG1	leucine-rich alpha-2-glycoprotein 129	?
SFRP1	secreted frizzled-related protein 129	possible role in polarity of retinal photoreceptor cells
SMR3A	submax. gland androg. reg. prot. 3 homol. A (mouse)22,85	?
SMR3B	submax. gland androg. reg. prot. 3 hom. B (mouse)22,85,87	?
		(xiii) Phosphatase/Kinase/GTPase/Other Enzyme
ACPP	acid phosphatase29	phosphatase
ARHGAP1	Rho GTPase activating protein29	GTPase activator of Rho, Rac and Cdc42
CA2	carbonic anhydrase II29	hydration of carbon dioxide
CP	ceruloplasmin (ferroxidase)22,29,87	peroxidation of Fe(II)transferrin, binds copper
F2	coagulation factor II (thrombin)22	fibrinogen to fibrin conversion
F5	coagulation factor V29	prothrombin/thrombin conversion with coag. factor X
GNPTG	N-acetylglucosamine-1-phosphate transf., γsubunit	targeting of lysosomal hydrolases to lysosomes
HTRA1	HtrA serine peptidase 129	cleaves IGF-binding proteins
MMP9	matrix metallopeptidase 929	matrix collagen IV and V degradation
S100A8	S100 calcium binding protein A822,29	possible cytokine and inhibitor of casein kinase
S100A9	S100 calcium binding protein A922,29	possible inhibitor of casein kinase
TGM2	transglutaminase 229	protein crosslinker
TXN	thioredoxin29	catalyzes dithiol-disulfide exchange and redox rxns
USP5	ubiquitin specific peptidase 5 (isopeptidase T)29	cellular protein degradation
		(xiv) Proteinase/Inhibitor/Antimicrobial
A2M	alpha-2-macroglobulin29,87	proteinase inhibitor, cytokine transporter
AMBP	alpha-1-microglobulin/bikunin precursor29	inhibits trypsin, plasmin, elastase
AZU1	azurocidin 1 (cationic antimicrobial protein 37)29	antibacterial and monocyte chemoattractant
CST1	cystatin SN22,29,85	cysteine proteinase inhibitor
CST2	cystatin SA85	thiol protease inhibitor
CST3	cystatin C29,85	abundant cysteine proteinase inhibitor
CST4	cystatin S22,29,85,87	cysteine proteinase inhibitor
CST5	cystatin D85	cysteine proteinase inhibitor
CTSB	cathepsin B29	lysosomal cysteine proteinase
CTSD	cathepsin D29	lysosomal aspartyl proteinase
CTSG	cathepsin G29	chymotrypsin C-like proteinase, antimicrobial
DCD	dermcidin29	C-terminal antibacterial, N-terminal prosurvival
DEFA3	defensin, alpha 3, neutrophil-specific22	anti-bacterial, -viral, -fungal
DPP4	dipeptidyl-peptidase 4 (CD26)29	intrinsic membrane serine exopeptidase
ELA2	elastase 2, neutrophil22	matrix hydrolysis, antibacterial
IGHA1	immunoglobulin heavy constant alpha 122,29,30,85,87	microbial and foreign antigen defense
IGHA2	immunoglob. heavy constant alpha 2 (A2m marker)31,85	microbial and foreign antigen defense
IGHM	immunoglobulin heavy constant mu22,29,85	microbial and foreign antigen defense
IGJ	immunoglobulin J polypeptide22,85	microbial and foreign antigen defense
IGKC	immunoglobulin kappa constant 85	microbial and foreign antigen defense
IGLC2	immunoglobulin lambda constant 285	microbial and foreign antigen defense
IGLV1-40	immunoglobulin lambda variable 1–4029	microbial and foreign antigen defense
ITIH1	inter-alpha (globulin) inhibitor H129	hyaluronan bp/carrier, pred. serine proteinase inhib.
ITIH2	inter-alpha (globulin) inhibitor H229	hyaluronan bp/carrier, pred. serine proteinase inhib.
ITIH4	inter-alpha (globulin) inhibitor H429	predicted serine proteinase inhibitor
LCN2	lipocalin 2 (oncogene 24p3)29	MMP9 binding, bacteriostatic, growth factor-like
LPO	lactoperoxidase29,87	antibacterial
LTF	lactotransferrin22,29,30,85,87	iron metabolism, antibacterial
LYZ*	lysozyme (renal amyloidosis) *22,29,30,85,87	hydrolase, antibacterial
MPO	myeloperoxidase29	antimicrobial
MUC7	mucin 7, secreted22	antibacterial, antifungal
PIGR	polymeric immunoglobulin receptor22,29,85,87	antibacterial, polymeric Ig transcellular transport
PLG	plasminogen29	activates urokin.-type plasmin. activat., collagenas.,
PRB4	proline-rich protein BstNI subfamily 485	possible bacterial binding (lost in point mutant)
SERPINA1	serpin peptid. inhibit., clade A (alpha-1), member 1*22,29	serine proteinase inhibitor, anti-inflammatory
SERPINA3	serpin peptidase inhibit., clade A (alpha-1), member 329	serine proteinase inhibitor
SERPINC1	serpin peptid. inhibit., clade C (antithrom.), member 122,29	blood coagulation cascade regulator
SLPI	secretory leukocyte peptidase inhibitor22,85,87	acid-stable proteinase inhib., antibacterial
TIMP1	TIMP metallopeptidase inhibitor 15,29	metalloproteinase inhibitor
TIMP2	TIMP metallopeptidase inhibitor 15	metalloproteinase inhibitor, inhib. endothelial prolif.
		(xv) Receptor/Channel/Transport
CLIC2	chloride intracellular channel 285	potential chloride ion channel
SLC7A4	solute carrier family 7, member 485	cationic amino acid transport

^*,Δ,†suggested to be less than normal in tears from patients suffering from *blepharitis²⁸ ΔSjögren’s syndrome³² or †contact lens related dry eye³³.

Functional Checks and Balances

Tear proteins contribute to the anti-microbial and anti-inflammatory defense of the exposed ocular surface. Some form heterocomplexes, and may function in normal epithelial growth, protein/fluid/electrolyte secretion and other aspects of normal ocular physiology. Few have been actually tested on ocular surface cells. Appreciating the true range of functional capacity and synergies will require a systems biology approach that integrates tear protein doses in time through different conditions. Following is a brief commentary on a selection of constituents from each Table 1 functional category with attention to ocular surface biology and dry eye when known.

The capacity for ‘Angiogenesis’ is highly regulated. Normal human tears contain the angiogenic promoters angiogenin (ANG) and endothelial growth factor 1 (ECGF1). Tears also contain the angiogenic inhibitors serpin peptidase inhibitor SERPINF1, brain-specific angiogenesis inhibitor 3 (BAI3) and the multifunctional cell adhesion protein thrombospondin 1 (THBS1; ‘CellAdhesion/Motility/Structure/). Lactotransferrin (LTF; ‘Proteinase/Inhibitor/Antimicrobial’) is anti-angiogenic when fragmented. The primary inhibitor of corneal angiogenesis is soluble fms-related tyrosine kinase 1 (FLT1) that competes with VEGR for angiogenic VEGF. FLT1 is expressed thoughout the corneal epithelium.³⁴

Most tear proteins in the ‘Biosynthetic’ category are primarily intracellular. For example, mitochondrial ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) is a subunit of mitochondrial ATP synthase. The suggestion that ATP5B has an alternative plasmalemmal/extracellular location is interesting because it implicates a potential source of tear ATP. ATP and UTP target ocular surface P2Y purinergic receptors to stimulate the production and release of tear mucins and noninflammatory heparanase.^35,36 Beta1,4-galactosyltransferase (B4GALT1) is primarily associated with the Golgi apparatus, but its alternative plasmalemmal location is well-established. Here it promotes cell adhesion by binding galactosylated proteins such as laminins.³⁷ Numerous tear proteins are heavily galactosylated (ie. mucins and proteoglycans) and are capable of binding B4GALT1. Peptidylprolyl isomerase (PPIC; cyclophilin C) is most commonly associated with the endoplasmic reticulum where it binds cyclosporine A for immunsuppresive signaling. Its alternative extracellular location in tears may have relevance to the topical application of cyclosporine A for dry eye.

Intracellular ‘Calcium’ signaling is fundamental to the normal function of ocular surface epithelia. Corneal or conjunctival calcification is respectively associated with phosphate in artificial tears³⁸ and elevated serum calcium in chronic renal failure.³⁹ Tear alpha-2-HS-glycoprotein (AHSG) is an inhibitor of calcification. Annexin A5 (ANXA5) is a calcium-dependent phospholipid binding protein that promotes corneal wound healing.⁴⁰ Although primarily cytoplasmic, ANXA5 has an alternative plasmalemmal/extracellular location. Also in tears is the calcium binding protein calreticulin (CALR). CALR ligation of Sjogren’s syndrome autoantigen Ro60 kD enhances anti-Ro60 kD autoantibody recognition.⁴¹ CALR is alternatively plasmalemmal/extracellular, and primarily in the endoplasmic reticulum.

A number of ‘Carbohydrate Modifying Proteins’ are found in tears. Almost all are listed as primarily intracellular and alternatively extracellular or plasmalemmal. Two exceptions are the extracellular proteins chitinase 3-like 2 (CHI3L2) and lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has a newly identified extracellular role in the eye as a binding partner for transferrin (TF). Interestingly GAPDH expression is iron regulated.⁴² Galectin-3 (LGALS3) is a beta-galactoside binding protein that plays a key role in corneal re-epithelialization after wounding, as revealed by use of galectin-3 null mice.⁴³ Enolase 1 (alpha; ENO1) is strongly expressed in basal cells of the limbus⁴⁴ and is upregulated in keratoconus. AGL has hydrolase and transferase activity. CHI3L2 has a particular affinity for glycans but has no apparent chitinase activity. ENO1 is glycolytic. Carbohydrate modification or binding can substantially alter or regulate function. One example is tear heparanase cleavage of heparan sulfate on cell surface syndecan-1 for lacritin binding and signaling.⁴⁵ The extent to which carbohydrate modifying proteins regulate ocular surface physiology is not fully explored.

Tears are rich in ‘Carrier/Binding Protein/Steroid Assoc.’ proteins. Albumin (ALB) is abundant in tears as a binding protein for free fatty acids and other elements of the tear film. Alpha-2-glycoprotein 1 (AZGP1) functions in cell adhesion, antigen processing/presentation, immune response, and binds polyunsaturated fatty acids.⁴⁶ CD14 is an important cell surface co-receptor for bacterial lipopolysaccharide in innate immunity. CD14 on macrophages helps clear apoptotic cells. Deleted in Malignant Brain Tumors (DMBT1) is a secreted scavenger receptor cysteine-rich protein that plays a key role in terminal epithelial differentiation and when deleted in mice is embryonic lethal.⁴⁷ Two forms of the predicted twelve different DMBT1 isoforms have been detected in human tears.⁴⁸ Heparan sulfate proteoglycan 2 (HSPG2 or perlecan) is a binding partner for FGF’s and several extracellular matrix molecules. HSPG2 mutations in Schwartz-Jampel Syndrome are associated with myopia.⁴⁹ Lipocalin 1 (LCN1) scavenges a wide variety of lipids and potentially other hydrophobic compounds from tears. Some of this lipid content may derive from exudated ocular surface epithelia as part of the normal course of ocular surface epithelial renewal. LCN1 also has magnesium-dependent endonuclease activity.⁵⁰ Transferrin (TF) delivers iron to cells via transferrin receptor uptake. By depleting extracellular iron, transferrin can limit bacterial growth. Thus these proteins contribute to the health and turnover of ocular surface epithelia.

Tear ‘Cell Adhesion/Motility/Structure’ proteins are surprisingly diverse. Presence of the ubiquitous extracellular matrix/serum constituent fibronectin 1 (FN1; ‘Cell Adhesion/Motility/Structure’) brings a considerable cell adhesive capacity to tear film reflected in FN1’s contribution to corneal wound healing.⁵¹ Gesolin (GSN) is a calcium-regulated protein more commonly associated with amyloidosis. Although its ocular surface role is unknown, it is differentially increased in keratoconus.⁵² Laminin alpha 3 (LAMA3) chain is part of heterotrimeric laminin-5. Mutation in LAMA3 is associated with scarring of the conjunctiva. Laminin mediates corneal epithelial cell adhesion⁵³ and is primarily concentrated in basement membranes. Another tear constituent shared by extracellular matrices is the previously noted thrombospondin 1 (THBS1). THBS1 is highly expressed by corneal epithelial cells, particularly basal cells, is anti-angiogenic, and binds FN1 and heparan sulfate. Thus, the tear film has the compositional attributes of a highly disperse extracellular matrix.

The tear film is enriched by mitogens (‘Cell Growth’), some with prosecretory activity. Angiopoietin-1 (ANGPT1), of the VEGF family, signals through Tie2 in bone marrow to help develop the hemopoietic stem cell niche.⁵⁴ Neovascularization following removal of limbal epithelia in mice involves ANGPT1 expression.⁵⁵ Epidermal growth factor (EGF) promotes tear secretion⁵⁶ and cell growth, and is expressed by both lacrimal acinar cells and basal cells of the corneal epithelium. Corneal epithelial cells respond to hepatocyte growth factor (HGF) in cell proliferation assays suggesting partially shared signaling pathways with EGF.⁵⁷ Lacritin (LACRT)⁵⁸ promotes human corneal epithelial cell growth⁵⁹ and MUC16 production (Wang and Laurie, unpublished), and appears to auto-stimulate tear protein secretion⁶⁰ by lacrimal acinar cells via ERK1/2-independent signaling. Lacritin employs a previously noted heparanase ‘on-switch’ to bind syndecan-1 as a precursor to mitogenic signaling.⁴⁵ Lacritin is largely ocular-specific. Heparanase is constitutively expressed by all layers of the normal corneal epithelium in mice, and has been implicated in glandular morphogenesis, epidermal stem cell migration and cell survival. The transmembrane mucin 4 (MUC4) signals via its EGF domains and interacts with ERBB2 to promote cell differentiation and block apoptosis.⁶¹ Several tear proteins promote neuronal survival including: glial cell derived neurotrophic factor (GDNF), neurotropin 3 (NTF3) and neurotropin 5 (NTF5). This mitogenic/prosecretory capacity compensates for the lack of corneal blood supply and may help serve as a master growth regulator of the ocular surface and associated accessory organs.

The level of tear ‘Cytoprotective/Antiapoptotic’ activity appears to be substantial. In addition to MUC4, GDNF, NTF3 and NTF5 noted above, EGF (porcine photoreceptor)⁶², HGF (human corneal epithelial)⁶³ and lacritin (human corneal epithelial; Wang et al, unpublished) are each cytoprotective. Clusterin (CLU) may help maintain the cornea as a non-keratinizing epithelium,⁶⁴ and is thought to act as an extracellular chaperone. CLU deficiency in the anterior segment of the eye is associated with pseudoexfoliation syndrome/glaucoma.⁶⁵ Mucin 16 (MUC16) on the surface of corneal epithelial cells is protective against S. aureus bacterial binding.⁶⁶ Gel forming mucin 5AC (MUC5AC) is thought to help sweep the ocular surface of pathogens during blinking. Very little is known about putative protection from prolactin-inducible protein (PIP), proline-rich 1 (PROL1) and proline rich 4 (PRR4). A peptide from the N-terminus of PROL1 is an analgesic⁶⁷. PROL1 binds the cell growth/maintenance protein statherin.

A number of tear proteins common to ‘Extracellular Matrix’ have been noted above including: HSPG2, FN1, LAMA3 and THBS1. Others are: the collagen type VI alpha 1 chain (COL6A1), mucin-like 1 (MUCL1) and SPARC-like 1 (SPARCL1). COL6A1 polymerizes with COL6A2 and COL6A2 to form the microfibrillar collagen VI triple helix. Little is known about MUCL1. SPARCL1 influences extracellular matrix formation and is a negative regulator of cell proliferation.⁶⁸

Normal human tear chemokines and cytokines (‘Immune’) are extensive and varied. Chemokines include: chemokine (C-C motif) ligand 2, 4, 8, 11, 22 and 24 (CCL2 – 24), chemokine (CXC motif) ligand 5, 9, 10 (CXCL5, 9, 10) and pro-platelet basic protein ligand 7 (PPBP). Cytokines include: interleukin 6, 8, 10, 12, 15, 16 (IL6 – 16), interferon gamma (IFNG) and tumor necrosis factor (TNF). How the eye handles this normal chemokine/cytokine load is an important question. TNF in the presence of INFG is for example inflammatory for the ocular surface and promotes epithelial death. Both IL6 and TNF are increased in tears of Sjögren’s syndrome dry eye patients.⁶⁹ A full answer will require attention to dose and time, and the level of cytoprotection from cell growth and cytoprotective/anti-apoptotic factors. Helping in innate defense is complement component 3 (C3), a key player in activation of the complement activation pathways. Individuals deficient in C3 have increased risk of bacterial infection. C3 polymorphism is associated with age-related macular degeneration. Knockout of the C3 gene in C57BL/6.NOD-Aec1Aec2 mice reduced the onset of primary Sjögren’s syndrome.⁷⁰ Complement factor H (CFH) regulates the alternative complement pathway. CFH polymorphism leading to decreased CFH activity is associated with increased risk of age-related macular degeneration. Oxidative stress decreases retinal CFH expression.⁷¹ Oxidative stress is a consequence of dry eye.⁷² The ocular innate ‘immune’ defense mechanism is therefore primed for action.

The inclusion of ‘Lipid/Cholesterol’ associated proteins is in line with the lipid nature of the tear film. Tears contain a wide selection of apolipoproteins (A-I [APOA1], A-II [APOA2], A-IV [APOA4], B [APOB], H [APOH]). Tear APOA1 is reportedly upregulated in diabetic retinopathy.⁷³ Phospholipid transfer protein (PLTP) binds heparin, is catalytically active in tears for phospholipid transfer and is thought to be involved in lipid clearance from the ocular surface, much like LCN1.⁷⁴ Prosaposin (PSAP) is a widely expressed lipid binding glycoprotein found in extracellular and lysosomal locations that is cleaved into four saposins involved in antigen presentation.

Among ‘Other/Unknown’ proteins are angiotensinogen (AGT), Beta-2-microglobulin (B2M), three intriguing proteins with no known function and several others. B2M is associated with the MHC class-I heavy chain on cell surfaces. Mice lacking beta-2 microglobulin reject orthotopic corneal allografts like their C57BL/6 controls.⁷⁵

Several enzymes are highlighted in the ‘Phosphatase/Kinase/GTPase/Other Enzyme’ category. Ceruloplasmin (CP) is a copper binding protein involved in peroxidation of the iron metalloprotein transferrin. Deficiency is associated with retinal pigment degeneration linked with increase iron. Matrix metalloproteinase 9 (MMP9) is upregulated in dry eye tears leading to increased corneal epithelial permeability and turnover. MMP9^−/− dry eye mice lack increased permeability and turnover.⁷⁶ Transglutaminase 2 (TGM2) is upregulated in UVB stimulated apoptosis of human corneal epithelial cells. Inhibition of TGM2 suppresses apoptosis.⁷⁷

Tears contain a wide variety of ‘Proteinase/Inhibitor/Antimicrobial’ proteins that play a key role in innate immune defense. Alpha-2-macroglobulin (A2M) is a broad-acting protease inhibitor. It also binds tear proteins B2M, cathepsin B (CTSB; lysosomal cysteine protease) and IL10; and the growth factors nerve growth factor beta polypeptide, platelet-derived growth factor betal polypeptide and transforming growth factor beta 1.⁷⁸ This latter property, via interaction of A2M with its receptor LPR1, is thought to be involved in inhibiting proliferation of retinal glial cells.⁷⁹ Cystatin 4 (CST4) is a cysteine proteinase inhibitor, likely serving a protective role on the ocular surface. Lactotransferrin (LTF) appears to have multiple roles. It reduces UV-B oxidative damage of human corneal epithelial cells,⁸⁰ it may be a general regulator of epithelial cell growth, it is antimicrobial and as previously noted when fragmented has anti-angiogenic activity. A Glu561Asp polymorphism is associated with corneal amyloidosis with trichiasis.⁸¹ LTF binds tear proteins CD14, CP, lipocalin-1 (LCN1), and lysozyme (LYZ). Also anti-bacterial are LCN1 (binds microbial iron chelating siderophores),⁸² lactoperoxidase (LPO), LYZ (a hydrolase that cleaves bacterial cell wall beta(1–4) glycosidic linkages)), and the polymeric immunoglobulin receptor (PIGR) that binds to Streptococcus pneumoniae. This overlapping mechanism of proteinases and inhibitors appears to become unbalanced in dry eye.

Chloride intracellular channel 2 (CLIC2; ‘Receptor/Channel/Transport’) regulates intracellular Cl⁻ concentration in the trabecular meshwork cells. Its role on the ocular surface and in tears appears to be unknown.

Selective Tear Protein Loss in Dry Eye

Although >183 ‘extracellular’ and ∼256 ‘intracellular’ proteins populate the tear proteome surprisingly few appear to change in dry eye (*, Δ, † in Table 1).^28,32,33 A discovery phase comparison of dry eye and normal tears is perhaps best done by sensitive silver- or fluorescent-stained 2-D PAGE. This has advantages to an initial mass spectrometry step. Thus using 2-D PAGE, Koo et al²⁸ detected eight distinct proteins downregulated at least 50% in blepharitis vs normal tears. Each downregulated protein was identified by ESI-Q-TOF MS/MS (Table 1 *). Downregulation for some was verified by Western blotting. Similarly using 2-D differential gel electrophoresis of tears from contact lens-related dry eye versus normals, Green-Church et al³³ found only three tear proteins significantly decreased (Table 1 †). Several others were increased.³³ Each was identified by nano-LC-MS/MS. Current problems with the use of mass spectrometry in the first step include differential ionization efficiency of analytes and a relatively narrow dynamic range. However, a SELDI study suggesting differences between Sjögren’s syndrome and normal tears⁸³ was recently pursued in more depth by LC-MS/MS. Only seven proteins were downregulated (Table 1 Δ).³² One common link between the three studies is the downregulation of lacritin, that as noted above functions as a prosecretory mitogen.⁵⁸ This intriguing pattern invites further study with larger sample numbers and sensitive immunoassays capable of quantitatively detecting low abundant tear proteins.

Recombinant Tear Protein Rescue?

Is downregulation of some tear proteins disease provoking? If so, tear proteins can be generated by recombinant DNA technology.⁵⁹ When mixed with vehicle, topical application could prove effective. Over the past 34 years since the discovery of recombinant DNA technology, an increasing number of human recombinant proteins have been successfully developed into therapeutics for the treatment of anemia, cancer, and renal, cardiovascular, rheumatologic and neurological diseases. Human recombinants include: erythropoietin, G-CSF, plasminogen activator, interferons, interleukins and humanized monoclonal antibodies. In age-related macular degeneration (AMD) or rubeosis iridis, recombinant Avastin and the ocular-reformulated Lucentis have proven effective. Each is a humanized fragment of recombinant anti-VEGF monoclonal antibody. Similarly, uveitis can be treated by the recombinant Infliximab (humanized anti-TNF monoclonal antibody). All three are administered by intravitreal injection. Topically applied human recombinant interferon-gamma has shown efficacy for herpetic keratitis.

Recombinant human proteins are the most rapidly expanding class of therapeutics in human health care with an expected 2010 market value of $52 billion⁸⁴. Each demands a strong translational research effort, and only a fraction of recombinant proteins make it to market. Issues include economy of manufacture and stability in an appropriate vehicle, purity/reproducibility and compliance with current Good Manufacturing Practices, thorough preclinical animal testing of dose efficacy in dry eye models, demonstration of minimal topical and systemic adverse reactions or toxic effects, and definition of basic parameters of absorption, distribution and excretion. For an effective recombinant dry eye therapeutic, there should be evidence of active and preferably prolonged induction of basal tears. Tears should be compositionally similar to normal tears and there should be no toxicity to ocular tissues. Administration would be topical and optimally coupled to an immunoassay. With a selection of human recombinant tear proteins or synthetic peptides available for reconstitution, one might have available a ‘designer’ or personalized ophthalmic treatment regimen for dry eye. Although developing recombinant protein manufacturing processes are time-consuming and much remains to be learned about the cell biology of tear proteins, inducing or reconstituting a natural tear film may be the most satisfactory approach to dry eye.