Lacritin is a 12.3 kDa
glycoprotein
Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycos ...
encoded in humans by the ''LACRT''
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
.
Lacritin's discovery emerged from a screen for factors that stimulate tear protein secretion.
Lacritin is a secreted protein found in
tears
Tears are a clear liquid secreted by the lacrimal glands (tear gland) found in the eyes of all land mammals. Tears are made up of water, electrolytes, proteins, lipids, and mucins that form layers on the surface of eyes. The different types of ...
and
saliva
Saliva (commonly referred to as spit) is an extracellular fluid produced and secreted by salivary glands in the mouth. In humans, saliva is around 99% water, plus electrolytes, mucus, white blood cells, epithelial cells (from which DNA can be ...
. Lacritin also promotes tear secretion,
the
proliferation and survival of
epithelial
Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellula ...
cells,
and corneal wound healing
Lacritin is thus a multifunctional prosecretory
mitogen
A mitogen is a small bioactive protein or peptide that induces a cell to begin cell division, or enhances the rate of division (mitosis). Mitogenesis is the induction (triggering) of mitosis, typically via a mitogen. The mechanism of action of a ...
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
The lacrimal glands are paired exocrine glands, one for each eye, found in most terrestrial vertebrates and some marine mammals, that secrete the aqueous layer of the tear film. In humans, they are situated in the upper lateral region of each or ...
,
including the accessory lacrimal gland of Wolfring.
Some lacritin is produced by the
meibomian gland
Meibomian glands (also called tarsal glands, palpebral glands, and tarsoconjunctival glands) are sebaceous glands along the rims of the eyelid inside the tarsal plate. They produce meibum, an oily substance that prevents evaporation of the eye ...
, and by
epithelial
Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellula ...
cells of the
conjunctiva
The conjunctiva is a thin mucous membrane that lines the inside of the eyelids and covers the sclera (the white of the eye). It is composed of non-keratinized, stratified squamous epithelium with goblet cells, stratified columnar epithelium ...
and
cornea
The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. Along with the anterior chamber and lens, the cornea refracts light, accounting for approximately two-thirds of the eye's total optical power ...
.
Together these epithelia comprise much of the lacrimal functional unit (LFU).
Dry eye
Dry or dryness most often refers to:
* Lack of rainfall, which may refer to
**Arid regions
**Drought
* Dry or dry area, relating to legal prohibition of selling, serving, or imbibing alcoholic beverages
* Dry humor, deadpan
* Dryness (medical)
* ...
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
Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (HSPG, i.e. Heparan Sulfate ProteoGlycan) in which two or three HS chains are attached in close proximity to cell surface or extracellular ma ...
proteoglycan
Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan (GAG) chain(s). The point of attachment is a serine (Ser) residue to whic ...
syndecan-1 (SDC1).
[ubMed - indexed for MEDLINE>] Binding utilizes an enzyme-regulated 'off-on' switch in which active epithelial
heparanase
Heparanase, also known as HPSE, is an enzyme that acts both at the cell-surface and within the extracellular matrix to degrade polymeric heparan sulfate molecules into shorter chain length oligosaccharides.
Synthesis and structure
The protei ...
(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
G protein-coupled receptors (GPCRs), also known as seven-(pass)-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors (GPLR), form a large group of evolutionarily-related p ...
(GPCR) then appears to be ligated.
Targeted cells signal to
NFAT
Nuclear factor of activated T-cells (NFAT) is a family of transcription factors shown to be important in immune response. One or more members of the NFAT family is expressed in most cells of the immune system. NFAT is also involved in the developme ...
and
mTOR
The mammalian target of sirolimus, rapamycin (mTOR), also referred to as the mechanistic target of rapamycin, and sometimes called FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1), is a kinase that in humans is encoded by the ''MT ...
if conditions are suitable for proliferation, or to AKT and FOXO3 under conditions of stress.
Structure
Lacritin consists of 119
amino acids
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
after cleavage of the N-terminal
signal peptide
A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16-30 amino acids long) present at the N-ter ...
and displays several predicted
alpha helices
The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
, 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
An amphiphile (from the Greek αμφις amphis, both, and φιλíα philia, love, friendship), or amphipath, is a chemical compound possessing both hydrophilic (''water-loving'', polar) and lipophilic (''fat-loving'') properties. Such a compoun ...
with
hydrophobic
In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water.
Hydrophobic molecules tend to be nonpolar and, th ...
and
hydrophilic
A hydrophile is a molecule or other molecular entity that is attracted to water molecules and tends to be dissolved by water.Liddell, H.G. & Scott, R. (1940). ''A Greek-English Lexicon'' Oxford: Clarendon Press.
In contrast, hydrophobes are no ...
residues on opposite faces. The hydrophobic face is an important
syndecan-1 binding element.
[ubMed - indexed for MEDLINE /> 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
''O''-linked glycosylation is the attachment of a sugar molecule to the oxygen atom of serine (Ser) or threonine (Thr) residues in a protein. ''O''-glycosylation is a post-translational modification that occurs after the protein has been synthes ...
sites populate the N-terminal half. The C-terminal amphipathic alpha helix is also the site of lacritin's only N-glycosylation
''N''-linked glycosylation, is the attachment of an oligosaccharide, a carbohydrate consisting of several sugar molecules, sometimes also referred to as glycan, to a nitrogen atom (the amide nitrogen of an asparagine (Asn) residue of a protein), ...
site. In ' climatic droplet keratopathy' this site is not glycosylated
Glycosylation is the reaction in which a carbohydrate (or 'glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not alw ...
. 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 p''I'' of lacritin's core protein is 5.
Lacritin is subject to crosslinking by tissue transglutaminase
Tissue transglutaminase (abbreviated as tTG or TG2) is a 78-kDa, calcium-dependent enzyme () of the protein-glutamine γ-glutamyltransferases family (or simply transglutaminase family). Like other transglutaminases, it crosslinks proteins between ...
, thereby giving rise to lacritin multimers including dimers and trimers.[ubMed - indexed for MEDLINE>] Crosslinking is initiated within 1 min in vitro, requiring as little as 0.1 nM lacritin.[ubMed - indexed for MEDLINE /> The ~0.6 micro molar level of tissue transglutaminase estimated in human tears is sufficient to promote crosslinking.][ubMed - indexed for MEDLINE /> Crosslinking involves the donors lysine 82 and 85 and the acceptor glutamine 106.][ubMed - indexed for MEDLINE /> Glutamine 106 resides within the ]amphipathic
An amphiphile (from the Greek αμφις amphis, both, and φιλíα philia, love, friendship), or amphipath, is a chemical compound possessing both hydrophilic (''water-loving'', polar) and lipophilic (''fat-loving'') properties. Such a compoun ...
alpha helix near the C-terminus responsible for binding the N-terminus of syndecan-1.[ubMed - indexed for MEDLINE /> Accordingly, crosslinked lacritin binds syndecan-1 poorly][ubMed - indexed for MEDLINE /> and is inactive.
Several lacritin splice variants have been detected in Aceview,] from NEIBank EST data. Lacritin-b (11.1 kDa; p''I'' 5.3) lacks the sequence SIVEKSILTE. Lacritin-c (10.7 kDa; p''I'' 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
Cathepsin B belongs to a family of lysosomal cysteine proteases known as the cysteine cathepsins and plays an important role in intracellular proteolysis. In humans, cathepsin B is encoded by the ''CTSB'' gene. Cathepsin B is upregulated in ce ...
, calpain
A calpain (; , ) is a protein belonging to the family of calcium-dependent, non-lysosomal cysteine proteases (protease, proteolytic enzymes) expressed ubiquitously in mammals and many other organisms. Calpains constitute the C2 family of proteas ...
, alanyl amino peptidase, arginyl aminopeptidase
Aminopeptidase B (, ''arylamidase II'', ''arginine aminopeptidase'', ''arginyl aminopeptidase'', ''Cl—activated arginine aminopeptidase'', ''cytosol aminopeptidase IV'', ''L-arginine aminopeptidase'') is an enzyme. This enzyme catalyses the fo ...
, MMP9
Matrix metallopeptidase 9 (MMP-9), also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB), is a matrixin, a class of enzymes that belong to the zinc-metalloproteinases family involved in the degradation of the extrace ...
, MMP10
Stromelysin-2 also known as matrix metalloproteinase-10 (MMP-10) or transin-2 is an enzyme that in humans is encoded by the ''MMP10'' gene.
Function
Proteins of the matrix metalloproteinase ( MMP) family are involved in the breakdown of extrace ...
, cathepsin G
Cathepsin G is a protein that in humans is encoded by the ''CTSG'' gene. It is one of the three serine proteases of the chymotrypsin family that are stored in the azurophil granules, and also a member of the peptidase S1 protein family. Cathepsin ...
, plasma kallikrein
Plasma kallikrein (, ''serum kallikrein'', ''kininogenin'', ''kallikrein I'', ''kallikrein II'', ''kininogenase'', ''kallikrein'', ''callicrein'', ''glumorin'', ''padreatin'', ''padutin'', ''kallidinogenase'', ''bradykininogenase'', ''panceatic ka ...
, plasmin
Plasmin is an important enzyme () present in blood that degrades many blood plasma proteins, including fibrin clots. The degradation of fibrin is termed fibrinolysis. In humans, the plasmin protein (in the zymogen form of plasminogen) is encoded ...
, thrombin
Thrombin (, ''fibrinogenase'', ''thrombase'', ''thrombofort'', ''topical'', ''thrombin-C'', ''tropostasin'', ''activated blood-coagulation factor II'', ''blood-coagulation factor IIa'', ''factor IIa'', ''E thrombin'', ''beta-thrombin'', ''gamma- ...
and trypsin
Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the dig ...
. C-terminal proteoforms, like intact lacritin, are selectively deficient in dry eye tears.
Cell targeting
Lacritin targets a restricted group of epithelial cells (including human corneal epithelia), and not fibroblastic, glioma, or lymphoblastic cells. Cell surface proteoglycan syndecan-1 is partly responsible.[ubMed - indexed for MEDLINE />
Biotinylated cell surface proteins from a lacritin-responsive cell were incubated with lacritin under conditions of physiological salt. Those that bound lacritin were sequenced by ]mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
. Few bound. The most prominent was syndecan-1 (SDC1). In confirmatory pull-down assays, binding was not shared with family members syndecan-2 or syndecan-4, indicating that the protein core (and not the negatively charged heparan sulfate side-chains) was the main site of binding. Further analysis narrowed the site to syndecan-1's N-terminal 51 amino acids, and subsequently to the N-terminal sequence GAGAL that is conserved in syndecan-1's from different species.[ubMed - indexed for MEDLINE /> GAGAL promotes the alpha helicity of lacritin's C-terminal amphipathic alpha helix and likely binds to the hydrophobic face.][ubMed - indexed for MEDLINE /> Syndecan-1 binds many ]growth factors
A growth factor is a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. Usually it is a secreted protein or a steroid hormone. Growth factors are important for regu ...
through its long heparan sulfate
Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (HSPG, i.e. Heparan Sulfate ProteoGlycan) in which two or three HS chains are attached in close proximity to cell surface or extracellular ma ...
side-chains. Yet, long heparan sulfate chains interfere with lacritin binding. Since syndecans are always decorated with heparan sulfate, this means that heparanase
Heparanase, also known as HPSE, is an enzyme that acts both at the cell-surface and within the extracellular matrix to degrade polymeric heparan sulfate molecules into shorter chain length oligosaccharides.
Synthesis and structure
The protei ...
must be available to partially or completely cleave off heparan sulfate, allowing lacritin to bind. Indeed, no binding was detected from cells lacking heparanase after siRNA
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to miRNA, and operating wi ...
depletion. Binding was restored by spiking in exogenous heparanase or heparitinase. Thus, heparanase regulates lacritin function as an 'on-switch'. Exposed 3-O sulfated groups on heparanase-cleaved heparan sulfate[ubMed - indexed for MEDLINE /> (that likely interacts with the cationic face of lacritin's C-terminal amphipathic alpha helix), and an N-terminal ]chondroitin sulfate
Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating sugars ( N-acetylgalactosamine and glucuronic acid). It is usually found attached to proteins as part of a proteoglycan. A chondroitin chain can have ove ...
chain (likely also binds to the cationic face) appear to contribute to binding.[ubMed - indexed for MEDLINE /> Point mutagenesis of lacritin has narrowed the ligation site.][ubMed - indexed for MEDLINE /> This novel heparanase mechanism appears at first glance to be poor for ocular health since heparanase release from invading lymphocytes in the corneal stroma is inflammatory. Yet heparanase is a normal secretory product of the corneal epithelium.]
Lacritin-dependent mitogenesis is inhibitable by pertussis toxin
Pertussis toxin (PT) is a protein-based AB5-type exotoxin produced by the bacterium ''Bordetella pertussis'', which causes whooping cough. PT is involved in the colonization of the respiratory tract and the establishment of infection. Res ...
,. The implication is that another key element of lacritin targeting specificity is a G-protein-coupled receptor that would presumably form a cell surface targeting complex with SDC1. Involvement of a G-protein coupled receptor would explain the rapidity of lacritin signaling.
Function
Lacritin is a glycoprotein
Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycos ...
of the human tear film
Tears are a clear liquid secreted by the lacrimal glands (tear gland) found in the eyes of all Mammal, land mammals. Tears are made up of water, electrolytes, proteins, lipids, and mucins that form layers on the surface of eyes. The different ty ...
, and to a lesser extent of saliva, lung lavage and plasma. It is mainly produced by the lacrimal gland
The lacrimal glands are paired exocrine glands, one for each eye, found in most terrestrial vertebrates and some marine mammals, that secrete the aqueous layer of the tear film. In humans, they are situated in the upper lateral region of each or ...
. Some lacritin also is produced by the meibomian gland
Meibomian glands (also called tarsal glands, palpebral glands, and tarsoconjunctival glands) are sebaceous glands along the rims of the eyelid inside the tarsal plate. They produce meibum, an oily substance that prevents evaporation of the eye ...
, and also by epithelial
Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellula ...
cells of the conjunctiva
The conjunctiva is a thin mucous membrane that lines the inside of the eyelids and covers the sclera (the white of the eye). It is composed of non-keratinized, stratified squamous epithelium with goblet cells, stratified columnar epithelium ...
and cornea
The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. Along with the anterior chamber and lens, the cornea refracts light, accounting for approximately two-thirds of the eye's total optical power ...
. The lacritin gene (''LACRT'') is one of the most transcriptionally regulated gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s in the human eye
The human eye is a sensory organ, part of the sensory nervous system, that reacts to visible light and allows humans to use visual information for various purposes including seeing things, keeping balance, and maintaining circadian rhythm.
...
. Functional studies suggest a role in epithelial
Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellula ...
renewal of some non- germative epithelia. By flowing downstream through ducts, it may generate a 'proliferative field'. Lacritin also promotes secretion 440px
Secretion is the movement of material from one point to another, such as a secreted chemical substance from a cell or gland. In contrast, excretion is the removal of certain substances or waste products from a cell or organism. The classical ...
(including that of lipocalin-1 and lactoferrin), cell survival and regeneration of the corneal epithelium after wounding. Three times daily topical treatment with C-terminal lacritin synthetic peptide 'Lacripep' (also known as 'N-94/C-6') at a 4 µM concentration regenerated corneal nerves and the ocular surface epithelium in the mouse Aire-/- dry eye model. This raises the possibility that lacritin may have clinical applications in the treatment of dry eye
Dry or dryness most often refers to:
* Lack of rainfall, which may refer to
**Arid regions
**Drought
* Dry or dry area, relating to legal prohibition of selling, serving, or imbibing alcoholic beverages
* Dry humor, deadpan
* Dryness (medical)
* ...
, the most common eye disease
This is a partial list of human eye diseases and disorders.
The World Health Organization publishes a classification of known diseases and injuries, the International Statistical Classification of Diseases and Related Health Problems, or ICD-10. ...
. It also may be beneficial in promoting healing after LASIK
LASIK or Lasik (''laser-assisted in situ keratomileusis''), commonly referred to as laser eye surgery or laser vision correction, is a type of refractive surgery for the correction of myopia, hyperopia, and an actual cure for astigmatism, since ...
or PRK surgery. Recent studies suggest that lacritin monomer is differentially down regulated in not only in dry eye
Dry or dryness most often refers to:
* Lack of rainfall, which may refer to
**Arid regions
**Drought
* Dry or dry area, relating to legal prohibition of selling, serving, or imbibing alcoholic beverages
* Dry humor, deadpan
* Dryness (medical)
* ...
, but also in blepharitis
Blepharitis is one of the most common ocular conditions characterized by inflammation, scaling, reddening, and crusting of the eyelid. This condition may also cause swelling, burning, itching, or a grainy sensation when introducing foreign objects ...
.
Lacritin is an LFU prosecretory mitogen and survival factor with a biphasic dose response that is optimal at 1 - 10 nM for human recombinant lacritin on human cells. Higher human lacritin concentrations are optimal on rat or mouse cells or on rabbit eyes. In a recent phase I/II clinical trial, a 22 µM topical dose of 'Lacripep' applied three times daily was effective at two weeks in primary Sjogren's Syndrome patients with an eye dryness score greater than 60, a score indicative of moderate to severe dry eye. Both corneal fluorescein staining and the symptom of burning/stinging were reduced. In keeping with a biphasic dose response, the 44 µM dose was largely ineffective. A biphasic dose response has a bell-shaped curve, with doses lower or higher than the dose optimum less effective. Other mitogens share this property. However, in secretion assays using monkey lacritin on monkey lacrimal acinar cells, the dose response appears to be sigmoidal with increasing lipocalin or lactoferrin secretion through a narrow 0.1, 0.3 and 1 μM dose range. Lacritin flows downstream from the lacrimal gland
The lacrimal glands are paired exocrine glands, one for each eye, found in most terrestrial vertebrates and some marine mammals, that secrete the aqueous layer of the tear film. In humans, they are situated in the upper lateral region of each or ...
through ducts onto the eye.
Artificial depletion of lacritin from normal human tears revealed that tears lacking lacritin are unable to promote the survival of ocular surface cells stressed with inflammatory cytokines. Human dry eye tears also lack this activity. However, dry eye tears supplemented with lacritin are fully protective. Similarly, tears artificially depleted of lacritin are deficient in bactericidal activity. The antibody used to deplete lacritin also depletes C-terminal proteoforms. These observations suggest that among all tear proteins, lacritin may be the master protector.
Dry eye tears are subject to premature collapse, as are normal human tears artificially depleted of C-terminal proteoforms. In both cases, stability is largely restored by spiking in synthetic lacritin peptides N-94 or N-94/C-6 as proxy C-terminal proteoforms. Each peptide inserts rapidly into (O-acyl)-omega-hydroxy fatty acid ( OAHFA) thought to reside at the aqueous lipid boundary in tears. OAHFA is the only class of tear lipids apparently downregulated in dry eye.
Signaling
Lacritin mitogenic, survival and secretion signaling have been studied.
Lacritin mitogenic signaling follows two pathways:
* Gαi or Gαo → PKCα/ PLCγ2 → Ca2+ → calcineurin
Calcineurin (CaN) is a calcium and calmodulin dependent serine/threonine protein phosphatase (also known as protein phosphatase 3, and calcium-dependent serine-threonine phosphatase). It activates the T cells of the immune system and can be bloc ...
→ NFATC1
Nuclear factor of activated T-cells, cytoplasmic 1 is a protein that in humans is encoded by the ''NFATC1'' gene.
Function
The product of this gene is a component of the nuclear factor of activated T cells DNA-binding transcription complex. Th ...
* Gαi or Gαo → PKCα/PLCγ2/PLD1
Phospholipase D1 (PLD1) is an enzyme that in humans is encoded by the ''PLD1'' gene, though analogues are found in plants, fungi, prokaryotes, and even viruses.
History
The possibility of PLD1 was first mentioned in 1947 by authors Hanahan and C ...
→ mTOR
Rapid dephosphorylation of PKCα causes it to transiently move from the cytoplasm to the area of the Golgi apparatus and peripheral nucleus. Here, it forms a complex with PKCα and PLCγ2 from which downstream mTOR and NFAT signaling is initiated. The upstream Gαi or Gαo signaling suggests the involvement of a G-protein-coupled receptor
G protein-coupled receptors (GPCRs), also known as seven-(pass)-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors (GPLR), form a large group of evolutionarily-related p ...
(GPCR). A candidate GPCR is under study. Syndecan-1 likely serves as a co-receptor. Binding lacritin may improve its GPCR affinity.
Lacritin survival signaling is observed when cells are stressed. Lacritin promotes survival and homeostasis by transiently stimulating autophagy. The mechanism appears to involve lacritin stimulated acetylation of the transcription factor FOXO3. Acetylated FOXO3 serves as a ligand for the autophagic mediator ATG101. Lacritin also promotes coupling of FOXO1 (that becomes acetylated with stress) with autophagic mediator ATG7. In the absence of lacritin, no coupling is observed. Thus acetylation alone is likely insufficient for FOXO1-ATG7 ligation, unlike an initial claim. Lacritin also restores oxidative phosphorylation and other metabolic events to rescue cells from stress.
Lacritin stimulated secretion of tear proteins lipocalin and lactoferrin from monkey lacrimal acinar cells does not appear to be mediated by Ca2+, unlike the agonist carbachol. When monkey lacrimal acinar cells are stressed with inflammatory cytokines (as occurs in dry eye), carbachol loses its capacity to promote the secretion of lipocalin. However, lacritin stimulates lipocalin secretion even in the presence of stress.
Distribution
Species
Genomic sequencing assembled by Ensembl
Ensembl genome database project is a scientific project at the European Bioinformatics Institute, which provides a centralized resource for geneticists, molecular biologists and other researchers studying the genomes of our own species and other v ...
reveals the existence of putative lacritin orthologues in other species. Comparative genomic alignment suggests that horse lacritin is most similar to human lacritin among all non-primate sequences examined. Moreover, it is detectable in horse tears by immunoblotting or by ELISA. Antibodies directed to the C-, but not N-, terminus of human lacritin are most effective - in keeping with the predicted conservation of the C-terminal amphipathic alpha helix necessary for cell targeting.
Tissue
Tissue distribution has been examined in humans and monkeys. Lacritin is most highly expressed in the lacrimal gland
The lacrimal glands are paired exocrine glands, one for each eye, found in most terrestrial vertebrates and some marine mammals, that secrete the aqueous layer of the tear film. In humans, they are situated in the upper lateral region of each or ...
, including the accessory lacrimal gland of Wolfring. Expression is moderate in salivary gland
The salivary glands in mammals are exocrine glands that produce saliva through a system of ducts. Humans have three paired major salivary glands (parotid, submandibular, and sublingual), as well as hundreds of minor salivary glands. Salivary gla ...
s and slight in mammary
A mammary gland is an exocrine gland in humans and other mammals that produces milk to feed young offspring. Mammals get their name from the Latin word ''mamma'', "breast". The mammary glands are arranged in organs such as the breasts in prima ...
(cancer but not or rarely normal), and thyroid gland
The thyroid, or thyroid gland, is an endocrine gland in vertebrates. In humans it is in the neck and consists of two connected lobe (anatomy), lobes. The lower two thirds of the lobes are connected by a thin band of Connective tissue, tissue cal ...
s. The salivary gland
The salivary glands in mammals are exocrine glands that produce saliva through a system of ducts. Humans have three paired major salivary glands (parotid, submandibular, and sublingual), as well as hundreds of minor salivary glands. Salivary gla ...
expression appears to be attributable to a discrete group of unidentified ductal-like cells. Some lacritin was reported in lung bronchoalveolar lavage and plasma. In lacrimal gland, polarized lacrimal acinar cells appear to be the most prolific lacritin producers, as evidenced by strong staining of secretory granules in keeping with lacritin release after carbachol stimulation. Carbachol-dependent release involves PKC and calcium signaling. Some lacritin is produced by the meibomian gland
Meibomian glands (also called tarsal glands, palpebral glands, and tarsoconjunctival glands) are sebaceous glands along the rims of the eyelid inside the tarsal plate. They produce meibum, an oily substance that prevents evaporation of the eye ...
, and also by epithelial
Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellula ...
cells of the conjunctiva
The conjunctiva is a thin mucous membrane that lines the inside of the eyelids and covers the sclera (the white of the eye). It is composed of non-keratinized, stratified squamous epithelium with goblet cells, stratified columnar epithelium ...
and cornea
The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. Along with the anterior chamber and lens, the cornea refracts light, accounting for approximately two-thirds of the eye's total optical power ...
that together with lacrimal gland comprise much of the lacrimal functional unit (LFU). Viewed collectively, the LFU is the primary source of lacritin in the body, and the eye the main target.
Disease
Dry eye
Dry or dryness most often refers to:
* Lack of rainfall, which may refer to
**Arid regions
**Drought
* Dry or dry area, relating to legal prohibition of selling, serving, or imbibing alcoholic beverages
* Dry humor, deadpan
* Dryness (medical)
* ...
is the most common eye disease
This is a partial list of human eye diseases and disorders.
The World Health Organization publishes a classification of known diseases and injuries, the International Statistical Classification of Diseases and Related Health Problems, or ICD-10. ...
, affecting 5 - 6% of the population. Prevalence rises to 6 - 9.8% in postmenopausal women, and as high as 34% in the elderly. Tears lubricate the lid and are important for the refraction of light. Tears also promote epithelial health. Only a small fraction of the estimated 1543 proteins in tears are differentially deficient or upregulated in dry eye. Lacritin monomer is differentially downregulated in mild to severe aqueous deficient dry eye, and in contact lens-related dry eye. In a larger trial, 95% of tears from patients with aqueous deficient dry eye were lacritin monomer deficient. Two studies that did not differentiate monomer from multimer did note any change of lacritin in dry eye. Topical treatment of eyes of dry eye mice (Aire knockout mouse model of dry eye) restored tearing, and suppressed both corneal staining and the size of inflammatory foci in lacrimal glands. Lacritin monomer deficiency in tears of patients with blepharitis
Blepharitis is one of the most common ocular conditions characterized by inflammation, scaling, reddening, and crusting of the eyelid. This condition may also cause swelling, burning, itching, or a grainy sensation when introducing foreign objects ...
was also reported. Blepharitis is an inflammation of the eyelid often associated with dry eye. In '' climatic droplet keratopathy'', N119 appears to be un-glycosylated
Glycosylation is the reaction in which a carbohydrate (or 'glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not alw ...
. Also a normal breast cancer
Breast cancer is cancer that develops from breast tissue. Signs of breast cancer may include a lump in the breast, a change in breast shape, dimpling of the skin, milk rejection, fluid coming from the nipple, a newly inverted nipple, or a re ...
localization reported by some has not been replicated in Unigene
UniGene was a NCBI database of the transcriptome and thus, despite the name, not primarily a database for genes. Each entry is a set of transcripts that appear to stem from the same transcription locus (i.e. gene or expressed pseudogene). Inform ...
(the 'mammary gland
A mammary gland is an exocrine gland in humans and other mammals that produces milk to feed young offspring. Mammals get their name from the Latin word ''mamma'', "breast". The mammary glands are arranged in organs such as the breasts in primat ...
' hit is for breast cancer) and gene array studies, but some breast cancers appear to display elevated expression or LACRT gene amplification. iTRAQ analysis of tears from diabetics at different stages of disease detected relatively more lacritin, lysozyme, lipophilin A, lipocalin 1, immunoglobulin lambda chain and lactotransferrin in tears of patients with diabetic retinopathy. The analysis did not distinguish lacritin monomer from polymer, and proposed the application of all as biomarkers. Tear lacritin monomer is barely detectable in the initial stage of infection by ''Fusarium solani'' in fungal keratitis. Also down regulated are tear lipocalin-1 and cystatin S. Fungal keratitis accounts for half of all corneal ulcers in Africa and India - the primary source of blindness in these countries. Phase II clinical trial of 'Lacripep™ in Subjects With Dry Eye Associated With Primary Sjögren's Syndrome' (NCT03226444) is complete. Lacripep™ is lacritin synthetic peptide 'N-94/C-6'.
References
Further reading
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{{Refend
Glycoproteins