Lacritin, a Novel Human Tear Glycoprotein, Promotes Sustained Basal Tearing and Is Well Tolerated

Samudre, S. S.; Lattanzio, Frank A.; Lossen, Victoria; Hosseini, Alireza; Sheppard, John D.; McKown, Robert L.; Laurie, Gordon W.; Williams, Patricia B.

doi:10.1167/iovs.10-6220

Cited by 53 publications

(59 citation statements)

References 41 publications

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“…Recombinant proteins were expressed in E. coli, affinity purified on chitin beads (New England BioLabs, Inc., Ipswich, MA), eluted without intein tag, 28 and then further purified on DEAE-Sepharose (GE Healthcare, Little Chalfont, UK), as previously described. 2 Purified proteins were freezedried and stored at À808C until use.…”

Section: Methodsmentioning

confidence: 99%

“…2012;53:6610-6616) DOI:10.1167/iovs.11-8729 L acritin is a 12.3 kDa human tear glycoprotein that is prosecretory, mitogenic, 1 and antimicrobial (McKown RL, et al IOVS 2010;51:ARVO E-Abstract 4181) and that promotes sustained basal tearing in rabbits. 2 Lacritin acutely augments constitutive but not stimulated lacrimal acinar cell secretion, even when prior treatment with interferon-c and TNF has neutralized the response to carbachol (Fujii, et al IOVS 2011;52:ARVO E-Abstract 3714).…”

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confidence: 99%

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Tear Lacritin Levels by Age, Sex, and Time of Day in Healthy Adults

Seifert¹,

Gandia²,

Wilburn³

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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PURPOSE.Several small proteomic studies suggest that the prosecretory tear protein lacritin may be selectively downregulated in dry eye syndrome and in blepharitis, yet little information is available about normal baseline levels. This study assessed lacritin levels in tears from healthy individuals and addressed whether they differ according to sex, age, or time of day.METHODS. Rabbit antibodies against lacritin N-terminal peptide EDASSDSTGADPAQEAGTS (Pep Lac N-Term) were generated and characterized against human recombinant lacritin and N-65 truncation mutant. Basal tears were collected from 66 healthy individuals ranging in age from 18 to 52 years, and at four times during one 24-hour period from 34 other individuals. Lacritin levels were then analyzed by ELISA and Western blotting.RESULTS. Anti-Pep Lac N-Term bound lacritin, but not truncation mutant N-65 that lacks the N-terminal antigenic site. Tear lacritin levels followed a normal distribution with a mean of 4.2 6 1.17 ng/100 ng total tear protein. Levels differed little by age or sex, and decreased slightly between 4 and 8 hours in a 24-hour cycle. Tear-blocking effects were minimal, as suggested by spiking of tears with recombinant lacritin.CONCLUSIONS. Anti-Pep Lac N-Term-detectable lacritin comprises~4.2 ng/100 ng total tear protein in healthy individuals, with no significant differences between males and females or among individuals between 18 and 52 years old. Levels decrease slightly in the late afternoon. These findings provide a baseline for future immunodiagnostic studies of lacritin in dry eye and other ocular diseases. (Invest Ophthalmol Vis Sci. 2012;53:6610-6616 Lacritin's ability to stimulate tear production makes it an interesting protein to study for its potential involvement in dry eye syndrome and other eye-related diseases. Dry eye affects the lives of over 25 million Americans, yet it is poorly understood and lacks sensitive early-stage diagnostics. Current tests are more appropriate for later disease stages, making difficult the diagnosis of patients with mild to moderate symptoms. 3 Moreover, tests such as Schirmer strips, ocular surface staining, and tear film breakup time are still not uniformly applied 4 (although standardization has improved with publication of the International Dry Eye Workshop report 5 ), and new devices to assess tear osmolarity show promise, 6 although not in isolation. 7 Development of an assay to help diagnose both early-onset and later dry eye, recognizing that there may be different etiologies, would be of great benefit.Dry eye syndrome and other associated conditions are believed to correlate with changes in specific protein content of the ocular surface. 8 Some small proteomic studies suggest that lacritin is one of only 4% to 5% of the tear proteome that is downregulated in dry eye or dry eye-related conditions. Lacritin levels measured by mass spectroscopy analysis of tear samples were 7-fold less from 11 individuals with contact lensrelated dry eye than from 10 users of contact lenses with normal ...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Tear Lacritin Levels by Age, Sex, and Time of Day in Healthy Adults

Seifert¹,

Gandia²,

Wilburn³

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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“…17,18 Furthermore, in vivo instillation of recombinant human lacritin stimulated basal tear flow in rabbits. 19 However, no data are available on lacritin-induced secretion of tear proteins in the more relevant monkey model. Thus, the purpose of the present study was to determine if lacritin induces protein secretion from monkey lacrimal acinar cells cultured under physiologic and inflammatory conditions.…”

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confidence: 99%

Lacritin-Induced Secretion of Tear Proteins From Cultured Monkey Lacrimal Acinar Cells

Fujii

Morimoto-Tochigi

Walkup

et al. 2013

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. During inflammation of the ocular surface, increased proinflammatory cytokines depress tear protein secretion, suggesting that a decline in lacrimal cell function contributes to dry eye. Lacritin, a glycoprotein secreted from lacrimal acinar cells, may function as an autocrine factor to stimulate tear protein secretion. The purpose of the present experiment was to investigate lacritin-induced protein secretion in normal and cytokine-pretreated (inflammation model) monkey acinar cells.METHODS. Acinar cells from monkey lacrimal glands were cultured with or without tumor necrosis factor alpha (TNF-a) plus interferon gamma (IFN-c). Protein secretion was induced by lacritin or carbachol (Cch, positive control). Proteins were detected and identified by immunoblotting and immunocytochemistry. Intracellular Ca 2þ was measured with the fluorophore Calcium-4, and cell damage was determined by LDH leakage into the culture medium. RESULTS. In cultured monkey acinar cells, lacritin stimulated tear protein secretion in normal cells without elevating intracellular Ca 2þ. In contrast, Cch elevated intracellular Ca 2þ and release of tear proteins. This contrast suggested an alternate, calcium-independent mechanism for lacritin-induced protein secretion. TNF-a plus IFN-c caused LDH leakage from sensitive human corneal epithelial cells, but even higher doses of TNF-a plus IFN-c did not cause LDH leakage from monkey acinar cells, suggesting a higher tolerance against these cytokines. In cytokine-treated acinar cells, lacritin stimulated protein secretion as much as that in normal cells. In contrast, Cch-induced elevation of Ca 2þ and release of proteins were depressed by cytokines.CONCLUSIONS. Lacritin might be a useful biotechnology-based treatment agent against ocular surface diseases where endogenous lacritin is inadequate.

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“…Proteins that have been considered include LCN1 (lipocalin), a multifunctional protein that serves as the predominant lipid carrier in human tears and which is critical to functions involving lipids in protection of the ocular surface [203][204][205]. Another example is LACRT (lacritin), a glycoprotein discovered in an unbiased screen for novel factors that stimulate tear secretion [206,207]. LACRT has prosecretory activity in the lacrimal gland and mitogenic activity at the corneal epithelium.…”

Section: Clu As a Possible Biotherapeutic For Dry Eyementioning

confidence: 99%

Clusterin in the eye: An old dog with new tricks at the ocular surface

Fini

Bauskar

Jeong

et al. 2016

Experimental Eye Research

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, M. R. (2016). Clusterin in the eye: an old dog with new tricks at the ocular surface. Experimental Eye Research, Clusterin in the eye: an old dog with new tricks at the ocular surface AbstractThe multifunctional protein clusterin (CLU) was first described in 1983 as a secreted glycoprotein present in ram rete testis fluid that enhanced aggregation ('clustering') of a variety of cells in vitro. It was also independently discovered in a number of other systems. By the early 1990s, CLU was known under many names and its expression had been demonstrated throughout the body, including in the eye. Its homeostatic activities in proteostasis, cytoprotection, and anti-inflammation have been well documented, however its roles in health and disease are still not well understood. CLU is prominent at fluid-tissue interfaces, and in 1996 it was demonstrated to be the most highly expressed transcript in the human cornea, the protein product being localized to the apical layers of the mucosal epithelia of the cornea and conjunctiva. CLU protein is also present in human tears. Using a preclinical mouse model for desiccating stress that mimics human dry eye disease, the authors recently demonstrated that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration in the tears. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to LGALS3 (galectin-3), a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. CLU depletion from the ocular surface epithelia is seen in a variety of inflammatory conditions in humans and mice that lead to squamous metaplasia and a keratinized epithelium. This suggests that CLU might have a specific role in maintaining mucosal epithelial differentiation, an idea that can now be tested using the mouse model for desiccating stress. Most excitingly, the new findings suggest that CLU could serve as a novel biotherapeutic for dry eye disease. Competing Interests: US patent 9,241,974 B2 entitled "Clusterin pharmaceuticals and treatment methods using the same" (inventors: MEF and SJ), assigned to the University of Southern California, is connected with this work. MEF holds a management position with Proteris Biotech, Inc., Pasadena, CA, which is developing Protearin for dry eye based on clusterin. MRW declares that he has no competing interests. Page 3 of 65 AbstractThe multifunctional protein clusterin (CLU) was first described in 1983 as a secreted glycoprotein present in ram rete testis fluid that enhanced aggregation ('clustering') of a variety of cells in vitro. It was also independently discovered in a number of other systems. By the early 1990s, CLU was known under many names and its expression had bee...

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Lacritin, a Novel Human Tear Glycoprotein, Promotes Sustained Basal Tearing and Is Well Tolerated

Cited by 53 publications

References 41 publications

Tear Lacritin Levels by Age, Sex, and Time of Day in Healthy Adults

Tear Lacritin Levels by Age, Sex, and Time of Day in Healthy Adults

Lacritin-Induced Secretion of Tear Proteins From Cultured Monkey Lacrimal Acinar Cells

Clusterin in the eye: An old dog with new tricks at the ocular surface

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