significance of mucin on the ocular surface

Significance of Mucin on the Ocular Surface

Hitoshi Watanabe, M.D., Ph.D.

Purpose. To review the significance of mucin in the tear film andthe ocular surface epithelium. Methods. Summary of the informa-tion on how mucin derived from the corneal and conjunctivalepithelia and from goblet cells plays a role in the stability of thetear film over the ocular surface. The change in mucin expressionderived from the ocular surface epithelium is also discussed withreference to ocular surface disease. Results. The corneal and con-junctival epithelia produce transmembrane mucins such as MUC1,MUC2, and MUC4. In contrast, goblet cells produce the gel-forming secretory mucin, MUC5AC. The lacrimal gland producesMUC7. On the ocular surface, cooperation between transmem-brane mucin and secretory mucin is necessary for the stability ofthe tear film. The expression of mucin from the ocular surfaceepithelium is coordinated from the time of eyelid opening and isaltered in conditions such as squamous metaplasia and dry eye.This alteration may result in instability of the tear film. Conclu-sion. The induction of mucin from the ocular surface may facilitatethe stability of the tear film, and increased knowledge may lead tothe development of a new modality for the treatment of dry eye.Key Words: Mucin—Tear film—Ocular surface—Cornea—Conjunctiva.

Mucin is a high-molecular-weight glycoprotein with a high pro-portion of carbohydrate content.1 The structure of mucin resemblesa test tube brush (Fig. 1). The carbohydrate is linked by O-glycosidic bonds to serine or threonine residues that are present inthe protein core. Although the core protein of mucin appears to beidentical between the tissues, the carbohydrate portion varies ac-cording to the organ, the individual, and other factors. Therefore,it is difficult to classify mucin based on the carbohydrate portion.Recently, however, it has been shown that the protein core ofmucin contains a tandem repeat of a certain number of aminoacids, and this forms the basis for classification.2 To date, humangenome mapping has identified 13 human mucin genes (MUC1–MUC12), numbered in the order in which their cloning was re-ported. The human mucin genes and some of their characteristicsare shown in Table 1.3–13

MUC 1, 2, and 7 cDNAs have been completely sequenced,while the structures of MUC 3, 4, 5AC, 5B, 6, and 8 have beenpartially determined. The number of tandem repeats per mucinmolecule can vary in each individual. Each mucin has a unique setof tandem repeats, and the only common feature between mucinsis that they have a rich serine or threonine content, providing sitesfor O-glycosidic linkage of carbohydrates.

Based on the full or partial cloning of mucin cDNA, humanmucins are categorized into two types: transmembrane and secre-tory.23 Secretory mucins are further subdivided into gel-formingand soluble types.

MUCIN EXPRESSED BY THE OCULAR SURFACEEPITHELIUM AND TEAR FILM

On the ocular surface, mucin is well known to be secreted fromgoblet cells in the conjunctiva. This mucin is the main componentof the innermost layer of the tear film. However, in addition to themucin derived from the goblet cell, the corneal and conjunctivalepithelia have recently been reported to produce mucin,14,15 inparticular the transmembrane mucin, MUC1.16 MUC1 mRNA hasbeen detected in all cell layers of the corneal epithelium, andimmunohistochemical analysis has shown that MUC1 protein waspresent in the apical membranes of the superficial cells of thecornea and conjunctiva (Fig. 2). The role of MUC1 is not known,but it may facilitate the spread of gel-forming mucin from thegoblet cells. However, MUC1 knockout mice do not apparentlyshow any instability of the tear film.17,18 In contrast, MUC1 hasbeen shown to play a protective role against the adherence ofpathogens.17 There remains, however, debate on this issue,18 andfurther examination is required.

MUC2 has been reported to be produced in the corneal andconjunctival epithelia.19,20 However, the amount of MUC2 issmall, and this mucin is not recognized to play an important rolein the ocular surface epithelium.

MUC4 is expressed in the conjunctival epithelium.19,21 The cor-neal epithelium also expresses MUC4 in humans. Although MUC4is present in the cornea, it would be localized in the limbal regionand not in the central portion of the cornea.19,21 In situ hybridiza-tion studies have shown that the conjunctival epithelium expressesMUC4 mRNA in all layers.21 The goblet cells in the conjunctivaexpress gel-forming secretory mucin, MUC5AC20,21 (Fig. 3).

In addition to these mucins, lacrimal glands have been shown toproduce soluble MUC7 (M.M. Jumblatt, personal communication,2000). MUC7 has not been shown to be present in the tear film.However, MUC7 is supposedly present in the tear film. Overall,there are at least four types of mucin in the ocular surface epithe-lium and tear film (Fig. 4).

Submitted September 24, 2001. Accepted October 3, 2001.From the Department of Ophthalmology, Osaka University Medical

School, Suita, Japan.This work was supported in part by grant-in-aid 1038769 for scientific

research from the Japanese Ministry of Education, Science, Sports, andCulture and by a grant for scientific research from Osaka Eye Bank As-sociation Fund.

Address correspondence and reprint requests to Dr. H. Watanabe, OsakaUniversity Medical School, Department of Ophthalmology, Room E7, 2-2Yamadaoka, Suita, 565-0871, Japan; E-mail: [email protected]

Cornea 21(Suppl. 1): S17–S22, 2002. © 2002 Lippincott Williams & Wilkins, Inc., Philadelphia

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ADDITIONAL MUCINS EXPRESSED IN THEOCULAR SURFACE EPITHELIUM

It is possible that additional mucins are present in the ocu-lar surface epithelium. Mucin is recognized to play a role in themaintenance of the tear film, but because it has not been clari-fied which mucin is important for the tear film spread, other mu-cins than MUC1 and MUC4 may be involved. To date, there areseveral candidates for this, although they have not been fullycharacterized.

In the rat, Gipson et al. have reported that a highly glycosylatedhigh-molecular-weight glycoprotein recognized by monoclonalantibody R339 is present along the glycocalyx of the apical mem-brane of the apical cells (Fig. 5).22 This glycoprotein was particu-larly prominent at the tip of the microplicae (Fig. 6) and was alsofound in the small vesicles in the cytoplasm of two or three celllayers of subapical cells of the corneal and conjunctival epithe-lium. Biochemical analysis showed that this molecule has the clas-sic characteristics of a mucin; 60% of the glycoprotein is carbo-hydrate and it has predominantly O-linked sugars.14 Moreover, theprotein part contains high percentages of serine and threonine.14

The comparable mucin in the human ocular surface epitheliumis the glycoprotein recognized by monoclonal antibody H185.15

This glycoprotein has a high molecular weight and is highly gly-cosylated. Moreover, the glycoprotein is O-linked. The core pro-tein structure has not been determined, and the glycoprotein cannotbe defined as a mucin, but the available data suggest that theglycoprotein recognized by H185 is a mucin-like glycoprotein. Itis present in the glycocalyx of the apical membrane of apical cellsof the cornea and the conjunctiva (Fig. 7), and is prominent at thetips of the microvilli and microplicae. This mucin-like glycopro-

tein is also present in the cells below the apical cells of the cornea,along the small vesicle in the cytoplasm of the subapical cells.15

A definitive comparison between this mucin-like glycoproteinand MUC1 has not been completed, but biochemical analysis sug-gests that it is different from MUC1. Detailed characterizationawaits further investigation.

SIGNIFICANCE OF MUCINS ON THEOCULAR SURFACE

The most important role of mucin is in stabilizing the tear film.The findings to date suggest that the mucin from goblet cells is a

TABLE 1. Human mucin gene

Mucingene

Type if sequenceverified

Chromosomalmapping

Aminoacids

in tandemrepeat Ocular surface

CorneaMUC1 Transmembrane 1q21q24 20 Conjunctiva

CorneaMUC2 Gelforming/secretory 11p15 23 ConjunctivaMUC3 7 17 —MUC4 Transmembrane 3 16 Conjunctiva

CorneaMUC5AC Gelforming/secretory 11p15 8 Goblet cellsMUC5B Gelforming/secretory 11p15 29 —MUC6 Gelforming/secretory 11p15 169 —MUC7 Soluble, monomer/

secretory4 23 Lacrimal gland

MUC8 12 13/41 Not detectedMUC9 1p13 15 Not detectedMUC10 11p14.3 Not detectedMUC11 7q22 28 Not detectedMUC12 7q22 28 Not detected

FIG. 1. Structure of mucin. The sugar portion binds to the proteincore through serine or threonine residues in the protein core.

FIG. 2. Presence of MUC1 in the corneal and conjunctival epithe-lium. A: MUC1 protein in the apical membrane of the superficial cellof the corneal epithelium. B: Phase contrast of Figure 2A. Repro-duced from Inatomi et al.16 courtesy of Investigative Ophthalmologyand Visual Science.

FIG. 3. MUC5AC expression in the conjunctiva. A: MUC5AC mRNAwas observed in goblet cells in the conjunctiva. B: High magnifica-tion of the photograph shows MUC5AC mRNA was intense in thegoblet cell. C: Sense riboprobe showed no binding in the goblet cells.Reproduced from Inatomi et al.21 courtesy of Investigative Ophthal-mology and Visual Science.

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Cornea, Vol. 21, Suppl. 1, 2002

gel-forming mucin that is the main component of the mucouslayer, the innermost layer of the tear film. The mucous layerspreads over the glycocalyx containing the mucin derived from thecorneal epithelium and the conjunctival epithelium. These twotypes of mucin, the transmembrane and the gel-forming secretorymucin, facilitate generation of the overlying aqueous layer of thetear film. In this way, transmembrane mucin derived from theocular surface epithelium and the gel-forming mucin from thegoblet cells are necessary for the spread of the tear film.

There is no direct evidence to show that the mucin produced bythe ocular surface epithelium is important for the spread of the tearfilm. However, clinical data suggest a significant role for thismucin in the spread of the tear film. Anterior specular microscopy(DR-1, Kowa Co., Tokyo, Japan) is used to detect dry eye.23 Thetechnique shows the pattern of the lipid layer of the tear film,which is helpful in determining whether the patient has dry eye.The microscopy shows not only the pattern of the lipid layer, butalso shows how the tear film spreads over the cornea. In normalcircumstances, the lipid layer spreads from the lower to the upperpart of the cornea just after lid opening. Usually, the goblet cell–

derived mucin spreads over the glycocalyx containing the ocularsurface epithelium-derived mucin. The spread of the goblet cell–derived gel-forming secretory mucin over the ocular surface epi-thelium facilitates the spread of the aqueous and lipid layer of thetear film.

When we examined patients with corneal ulcer resulting fromdebridement of the epithelium caused by a foreign body in thecornea using anterior specular microscopy, we observed that thelipid layer moved similarly to the normal stream in the intact

FIG. 4. Structure of the tear film and the origin of mucin in the tearfilm and the ocular surface epithelium.

FIG. 5. Mucin expression recognized by R339 monoclonal antibodyin the rat ocular surface epithelium. A: Immunofluorescence studyhas shown that R339 binding was observed on the several superfi-cial layers of the corneal epithelium. B: R339 binding was also ob-served in the goblet cells and in the several superficial layers of theconjunctival epithelium. Reproduced from Gipson et al.22 courtesy ofInvestigative Ophthalmology and Visual Science.

FIG. 6. Immunoelectron microscopic observation of mucin expres-sion recognized by R339 monoclonal antibody in the ocular surfaceepithelium. A: Immunoelectron microscopy showed the mucin wasobserved along the apical membrane of the apical cell. Arrows indi-cate the membrane of the abutting subapical cell. B: High magnifi-cation electron micrograph shows that the mucin is prominent at themicroplicae in the superficial cell. C: The mucin was observed alongthe cytoplasmic vesicles. Reproduced from Gipson et al.22 courtesyof Investigative Ophthalmology and Visual Science.

FIG. 7. Expression of the mucinlike glycoprotein recognized by H185monoclonal antibody in normal subjects. A: Light micrograph showsa corneal section. B: Immunofluorescence micrograph showed thatthe mucinlike glycoprotein recognized by H185 was localized to onlyflattened superficial cells. Arrows indicate the basal cell of the cor-neal epithelium. C: The mucinlike glycoprotein was also observed inthe apical cells of the conjunctival epithelium and the goblet cells.Inset indicates abrupt ending of H185 binding at the lid edge in theconjunctiva. Reproduced from Watanabe et al.15 courtesy of Inves-tigative Ophthalmology and Visual Science.

MUCIN ON THE OCULAR SURFACE S19


epithelium of the cornea, but that it divided at the edge of thecorneal ulcer and moved to surround the ulcer site (Fig. 8). In thesepatients, the number of goblet cells was normal on impressioncytology, suggesting that the gel-forming mucin was similar tonormal under these circumstances. However, transmembrane mu-cin produced by the corneal epithelium was absent in the lesion ofthe corneal ulcer. As mentioned above, transmembrane mucin andgel-forming mucin are important for the spread of the tear film.

Thus, the tear film cannot spread over the ulcer site because thegoblet cell mucin cannot spread over the site in the absence oftransmembrane mucin. This observation suggests that transmem-brane mucin expression in the ocular surface epithelium is neces-sary for the spread of the tear film.

DEVELOPMENTAL EXPRESSION OF MUCIN INOCULAR SURFACE EPITHELIUM

What happens to mucin produced by the ocular surface duringdevelopment? The mucin produced by the corneal and conjuncti-val epithelia has been investigated in developing rats using anti-body R339.24 The mucin-like glycoprotein recognized by R339 ispresent in adults in the apical membrane of the superficial cells ofthe entire ocular surface epithelium. Newborn rats are born with

FIG. 8. Schema of the tear spread over the ocular surface. The lipidlayer interference pattern shows that the tear moves from the lowerto the upper part of the cornea, but it splits at the lower edge of thecorneal ulcer.

FIG. 9. The appearance of R339 specific mucin in the developing rat.A: After eyelid opening, the mucin is present all along the ocularsurface, the corneal and conjunctival epithelium. B: While the eyelidis closed, the mucin was only localized in the palpebral conjunctivaat the lid edge. Reproduced from Watanabe et al.24 with permissionof Investigative Ophthalmology and Visual Science.

FIG. 10. The expression of H185-specific mucin in the ocular surfaceepithelium in normal individuals. A: Normal superficial cells of theconjunctival epithelium show a cobblestone appearance. Arrow in-dicates goblet cells stained by periodic acid-Schiff. B: The H185-specific mucin expression in each normal cell is graded as high (X),medium (Y), or low (Z). The pattern of H185-specific mucin in theconjunctiva shows a mosaic pattern. Reproduced from Watanabe H,Maeda N, Kiritoshi A, et al. Expression of a mucin-like glycoproteinproduced by ocular surface epithelium in normal and keratinizedcells. Am J Ophthalmol 1997;124:753,27 with permission fromElsevier Science.

FIG. 11. The expression of H185-specific mucin in superior limbickeratoconjunctivitis. A: Localized inflammation was observed in thesuperior limbic conjunctiva. B: Squamous metaplasia also was ob-served in the superior conjunctiva of superior limbic keratoconjunc-tivitis. C: H185-specific mucin was not observed in the conjunctivalsuperficial cells with squamous metaplasia in superior limbic kera-toconjunctivitis. Reproduced from Watanabe H, Maeda N, KiritoshiA, et al. Expression of a mucin-like glycoprotein produced by ocularsurface epithelium in normal and keratinized cells. Am J Ophthalmol1997;124:754,27 with permission from Elsevier Science.

H. WATANABES20


their lids closed and open their eyes approximately 12 to 15 daysafter birth. When the eyelids are closed, the mucin-like glycopro-tein is localized in the palpebral conjunctiva near the lid fusion.The glycoprotein is not found in the corneal epithelium while theeyelid remains closed. When the eyelid opens, the mucin-like gly-coprotein expression in the ocular surface epithelium rapidly ex-tends to all apical squamous epithelium of the entire ocular surfaceepithelium including that of the cornea (Fig. 9). These phenomenahave been confirmed by subsequent experiments in which artificialeyelid opening induced rapid appearance of the mucin-like glyco-protein. This observation corresponds closely to previous reports.Hazlett et al. have shown that mucin levels are not appreciablebefore eyelid opening.25 In contrast, a heavier surface coat pre-sumed to be mucin is present after eyelid opening.25 These datasuggest that mucin derived from the ocular surface epithelium isexpressed along the ocular surface epithelium after eyelid opening.

With respect to the mucin derived from goblet cells in the rat, ithas been shown that Muc5AC was first found in a few cells nearthe fornix 7 days after birth.26 By 14 days after birth, many gobletcells showed rMuc5AC expression not only in the fornix region,but also in the palpebral and bulbar conjunctiva.

These two developmental experiments clearly showed thattransmembrane mucin and gel-forming mucin work together toachieve tear spreading after lid opening, although the two typesappear at different times.

NORMAL EXPRESSION OF MUCIN IN THEOCULAR SURFACE EPITHELIUM

It has been reported that MUC1 is expressed in the superficialcells of the corneal epithelium and conjunctival epithelium.16

However, the pattern of expression in all the superficial cells hasnot been shown. We investigated the expression of the mucinlikeglycoprotein recognized by H185 in each of the superficial cells.27

Our results showed that expression varied from cell to cell, allow-ing individual cells to be distinguished easily. The expression wasgraded as high, medium, and low (Fig. 10), and this produced amosaic pattern. We hypothesized that this pattern could be ex-plained as follows. The mucin-like glycoprotein was expressed atthe tip of the microvilli and microplicae of the apical surface of thecornea and conjunctiva. The apical surface epithelia are dividedinto three types of cell: dark, medium, and light, according to thedegree of scattering they effect during scanning electron micros-copy.28–30 The lighter cells contain a greater density of microvilli,and darker ones contain a lower density. Therefore, the mucinexpression in the superficial cells varies according to the density ofthe microvilli and microplicae. The superficial cells with the fewermicrovilli are thought to be the oldest cells at the ocular surface.

The superior conjunctiva in patients with superior limbic kera-toconjunctivitis shows squamous metaplasia. We found that themucin-like glycoprotein expression is absent or remarkably re-duced in those cells obtained from the superior conjunctiva ofthese patients27 (Fig. 11). These data suggest that the keratinizedapical cells of the ocular surface epithelium are altered in appear-ance and lack the normal mosaic pattern of expression of themucin produced by the ocular surface epithelium. This alterationof the mucin-like glycoprotein expression was restored with nor-malization of the cells.27

We further investigated the expression of the mucin-like glyco-protein in patients with dry eye.31 The mosaic pattern was ob-

served in the conjunctiva of normal eyes, but as dry eye pro-gressed, the pattern was gradually lost and replaced by a starry skypattern in which a lack of apical cell binding (dark areas) andincreased binding to goblet cells (light disks) was observed (datanot shown). Therefore, the expression of the mucin-like glycopro-tein produced by the ocular surface epithelium is diminished orabsent in dry eye as well as superior limbic keratoconjunctivitis.

HOW TO DETECT MUCIN IN THE OCULARSURFACE EPITHELIUM

There is no direct method to detect mucin over the ocular sur-face epithelium. However, rose bengal staining can be used in theclinical setting as an indirect method. As Tseng et al. showed,32,33

gel-forming mucin can form a diffusion barrier to rose bengalstaining of the ocular surface epithelium under the condition thattransmembrane mucin is present. Rose bengal staining does notstain the cell in vivo because these two kinds of mucin harmonizein vivo. In contrast, in vitro, the monolayer of the epithelial celldoes not express the transmembrane mucin and gel-forming mucincannot overlie the transmembrane mucin. In such cases, rose ben-gal actually stains the cell. Therefore, rose bengal staining indi-rectly shows whether both gel-forming mucin and transmembranemucin are intact.

INDUCTION OF MUCIN FOR THE TREATMENTOF DRY EYE

Transmembrane mucin and gel-forming mucin in the ocularsurface are thought to be essential for maintenance of the tear film.In dry eye, the expression of transmembrane mucin in the ocularsurface epithelium has been reported to be altered.31 This changein expression may result from dry eye, but in cases of dry eyecaused by mucin deficiency, it is possible that the induction ofmucin from ocular surface epithelium could be effective as treat-ment. It is important to investigate what could induce the expres-sion of transmembrane mucin and gel-forming mucin. If certainfactors or cytokines could be confirmed to induce these mucins,this could lead to the development of a drug for the treatment ofdry eye. Further investigation is expected.

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MUCIN ON THE OCULAR SURFACE S21


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significance of mucin on the ocular surface

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