Mucin genes expressed by the ocular surface Epithelium

PII: S 1350-9462(96)00022-5

Mucin Genes Expressed by the Ocular Surface Epithelium Ilene K. Gipson

and Tsutomu

Inatomi

Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA CONTENTS Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The Mucus Layer of the Tear Film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81 82

2. Mucins, an Emerging Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Transmembrane Mucin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Secretory Mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Gel-fbrming mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Non-gel-forming mucin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Less well-characterized mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83 84 85 85 86 86

3. Mucin Genes Expressed by the Ocular Surface Epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. M U C l - - t h e Transmembrane Mucin of Corneal and Conjunctival Epithelium . . . . . . . . . . . 3.2. M U C 4 a Less Well-characterized Mucin of the Conjunctival Epithelium . . . . . . . . . . . . . . 3.3. M U C 5 - - a Conjunctival Goblet Cell Mucin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Are There Additional and/or Unique Mucins Expressed by the Ocular Surface Epithelium? 3.4.1. Corneal epithelial glycocalyx mucin of rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Ocular surface glycocalyx glycoprotein of humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Role of Specific Mucins in the Tear Film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Mucins and Ocular Surface Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

86 86 88 88 89 89 90 93 94

4. Summary and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95

Acknowledgements: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

96

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract--Mucin,; are the glycoproteins which form the viscous, gel-like mucus layer of the tear film. Molecular characterization of mucins has been slow due to their heavy glycosylation and high molecular weight, but recent cloning of human gut and trachea, mammary gland, and salivary gland mucins has begun to shed light on the primary structure of these important protective molecules. To date nine human mucin genes, designated MUC1-8, have been cloned. The presence in the protein backbone of tandem repeats of series of amino acids rich in serine and/or threonine is a feature common to all. Numbers and sequences of amino acids in each tandem repeat varies with each mucin. Numbers of amino acids per repeat vary from 169 (MUC6) to 8 (MUC5AC). Until recently little has been known regarding the expression of these genes by the ocular surface epithelium. This review summarizes recent work from our laboratory aimed at determining the molecular character of mucins expressed by conjunctival and corneal epithelium. Using northern blot analysis and in situ hybridization techniques, we have demonstrated that the stratified epithelium of both cornea and conjunctiva express the transmembrane mucin, MUCI. This widely expressed mucin is the best characterized of the cloned mucins. Using similar methodologies we demonstrated that the conjunctival goblet cell expresses MUC5AC, a gel-forming mucin, which has a cysteine-rich domain responsible for the disulfhydryl bonding between mucin molecules. Conjunctival stratified epithelium expresses MUC4, a relatiw:ly uncharacterized mucin, whose function is not known. Are there additional and/or unique mucins expressed by the ocular surface epithelium? We summarize our recent work on characterization of a mucin isolated from rat and human corneal epithelium. Antibodies to these mucins localize to the glycocalyx of the corneal epithelial apical cells. Since MUC1 appears to be the only one of the cloned mucins Progress in Retinal and Eye Research Vol. 16, No. 1, pp. 81-98, 1997 Copyright © 1996 Elsevier Science Ltd. All rights reserved. Printed in Great Britain. 1350-9462/97 $17.00 + 0.00

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I. K. Gipson and T. Inatomi expressed by the corneal epithelium,it is a candidatefor comparison to our mucin isolate. Current effortsare directed toward this comparison. Identification of mucin genes expressed by the ocular surface epithelium opens many new avenues for investigation into expression, regulation, and glycosylationof mucins in ocular surface pathologies and into the specific character of these molecules which enhance protection from pathogen invasion. Copyright © 1996 Elsevier Science Ltd.

1. THE MUCUS LAYER OF THE TEAR FILM The tear film on the surface of the eye serves multiple functions. Not only does it provide a smooth and refractive surface of high optical quality (Holly and Lemp, 1977), but it serves to protect, lubricate, and maintain the corneal and conjunctival epithelium. Early studies reported the tear film to be approximately 7-pm thick and to be divided into three layers, an outer lipid layer, a middle aqueous layer and an inner mucus layer adherent to the apical cells of the epithelium. The middle aqueous layer was thought to provide the primary thickness of the film (Mishima, 1965), but recent studies using laser interferometry and confocal microscopy suggest that the tear film is much thicker (36-45 pm) and that the film is composed substantially of mucus, not free aqueous fluid (Prydal and Campbell, 1992; Prydal et al., 1992; Dilly, 1994). It has been suggested that the mucus is present in a gradient distribution, the highest concentration being at the epithelial surface where the mucus is most viscous (Holly, 1973). Mucus is a viscous gel intimately associated with the apical edge of all 'wet-surfaced' epithelia. Much of our current thinking about mucus character is derived from studies of gastrointestinal and respiratory mucus, where the material is more abundant. As described by Gum (1992), mucus "consists of water, ions, and various sloughed substances and cells; however, it is the mucus glycoproteins or mucins that render it viscoelastic and sticky". The major components of mucus, the mucins, are highly glycosylated, high-molecularweight glycoproteins, some types of which associate with one another via disulfide bonds. The viscous or gelled character of the mucus layer is a result of this intermolecular association (Strous and Dekker, 1992). The gel character and its optimal function are influenced by the concert-

tration of mucins and their structure, ionic conditions, and presence of other, non-covalently bound proteins and lipids (Strous and Dekker, 1992). A complete review of the biochemical and physical aspects of mucus is beyond the scope of this review. Excellent reviews on the topic are available elsewhere (Carlstedt et al., 1985; Gum, 1992; Rose, 1992; Strous and Dekker, 1992; Gendler and Spicer, 1995). The major function of the ocular mucus layer is believed to be its role in stabilizing and spreading the tear film (i.e. preventing tear breakup) and in providing protection against microbial invasion and desiccation. Ocular m u c u s , like that found in the gastrointestinal and respiratory tracts, is a heterogeneous viscous substance, which contains water, ions, sloughed cells, secreted antimicrobial agents, and other substances (Holly, 1973; Moore and Tiffany, 1979, 1981; Chao et al., 1983; Dilly, 1994). Mucins, the major glycoprotein of ocular surface mucus, have not been extensively characterized, particularly at the molecular level. Three cellular sources of ocular surface mucins have been proposed. Goblet cells of the conjunctiva are believed to be the primary source of ocular surface mucins (Holly and Lemp, 1977). Goblet cells, potentially derived from stem cells that differentiate into both stratified epithelial and goblet cells (Wei et al., 1995), contain large mucin packets in their cytoplasm. Little information has been available on the molecular character of the conjunctival goblet cell mucins. Several investigators have proposed that the stratified squamous epithelium of the conjunctiva is a 'second' source of mucins for the ocular surface (Srinivasan et al., 1977; Dilly, 1985; Greiner et al., 1985). This proposal was based on the binding to the stratified epithelia of dyes with high affinity for carbohydrate (PAS, Alcian blue) and on ultrastructural characteristics, which showed secretorylike vesicles at the apices of the outer layer of

83

Mucins of the Ocular Surface stratified cells of the conjunctiva. Recent work from our laboratory corroborates and extends the data that a second source of mucin is present at the ocular surface, by showing that the entire ocular surface stratified epithelium, including both cornea and conjunctiva, express mucin genes (see description below). The lacrimal gland has been suggested as a third source of tear film mucus. Studies have demonstrated high sialic acid content of both lacrimal tissue (Jensen et al., 1969) and lacrimal gland fluid (Kreuger et al., 1976) and staining of lacrimal tissues with dyes indicative of acid glycoproteins (Allen et al., 1972). While these data indicate the presence or secretion of highly glycosylated molecules, direct proof of mucin gene expression by the lacrimal gland is lacking.

2. MUCINS, AN EMERGING DEFINITION Mucins are high-molecular-weight glycoproteins that have a high proportion of their mass (at least 50-80%) as carbohydrate (Strous and Dekker, 1992). The carbohydrate is linked by O-glycosidic bonds to serine or threonine residues which are present at high levels in the protein backbone (Strous and Dekker, 1992). Because of their large size and heavy glycosylation, further characterization of mucins has been slow, and thus, the characteristics of high carbohydrate content and O-linkage of carbohydrates formed the basis of the definition of mucins well into the molecular biology era. Indeed difficulty in isolation and

characterization has been compounded by heterogeneity in glycosylation. Thus, antibodies to carbohydrate epitopes on mucins often do not provide specific markers for specific mucin gene products. Only recently has cDNA cloning and sequencing provided information regarding the primary structure of mucins (for reviews, see Gum, 1992; Rose, 1992; Gendler and Spicer, 1995). To date, nine different human mucin genes have been cloned. Most mucin genes have been cloned using expression cDNA libraries, which have been screened with antibodies to deglycosylated mucin proteins (Gum et al., 1989; Gendler et al., 1990; Gum et al., 1990; Lan et al., 1990; Porchet et al., 1991; Toribara et al., 1993; Shankar et al., 1994). Several mucin genes have been cloned in rat and mouse; mucins have been cloned from porcine stomach and submaxillary gland, and several amphibian integumentary mucins have been described (as reviewed by Gendler and Spicer, 1995). By human genome mapping conventions, the human mucins are designated by 'MUC' followed by a number that indicates the order in which cloning of the mucin was reported (Gendler and Spicer, 1995). Mouse homologues to the human mucin genes are designated by 'Muc' plus the correlative number (Gendler and Spicer, 1995). A summary of human mucin genes cloned to date and some of their characteristics are given in Table 1. Of the human mucin genes, only MUC1, 2, and 7 cDNAs have been completely sequenced. From this data and partial cloning

Table 1. Human Mucin Genes

Designation MUC1 MUC2 MUC3 MUC4 MUC5AC

MUC5B MUC6 MUC7

Type if sequence verified

c D N A clone source

Chromosomal mapping

A m i n o acids in tandem repeat

Membrane-spanning

Mammary/ pancreatic t u m o r Intestine Intestine Trachea Trachea

lq21q24

20

llp15 7 3 llp15

23 17 16 8

Trachea Stomach Salivary gland

l lp15 l lp15 4

29 169 23

Trachea

12

13/41

Gel-form:tng/secretory

Gel-forming/secretory

Soluble, monomer/secretory

MUC8 N D = N o t Determined.

References Gendler et al., 1987; Lan et al., 1990 G u m et al., 1989 G u m et al., 1990 Porchet et al., 1991 Meerzaman et al., 1994; G u y o n n e t Duperat et al., 1995 Dufosse et al., 1993 Toribara et al., 1993 Bobek et al., 1993 Shankar et al., 1994

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data on MUC3, 4, 5AC, 5B, 6, and 8, generalizations about the primary structure of mucins have been made. As a result, an additional characteristic has been added to the general definition of mucins as being highly glycosylated glycoproteins whose carbohydrates are O-linked. This characteristic is the presence of tandem repeats of series of amino acids. Each mucin has unique tandem repeats, with the only common feature between mucins being the rich content of serine and/or threonine which provide sites for O-glycosidic linkage of carbohydrates. The number of amino acids per tandem repeat of the nine human mucin genes cloned to date varies from 8 to 169, but the number of tandem repeats per mucin molecule can also vary in individuals as a result of genetic polymorphism. The full and partial cloning of mucin cDNA has added other information about mucin primary structure that has led to categorization of the mucins into transmembrane and secretory types and further subdivision of secretory mucins into gel-forming and soluble types (Gendler and Spicer, 1995).

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MUC1 mucin, the only transmembrane mucin known to date, is integral to the apical membrane of cells of many epithelia (Gendler et al., 1990). Initially isolated and characterized biochemically and through use of antibodies, various names were given to the cell surface glycoprotein. These include episialin, epithelial membrane antigen, and polymorphic epithelial mucin, to name a few (for review see Gendler and Spicer, 1995). Originally cloned from both breast carcinoma and pancreatic tumor, it is the most studied and best characterized of the mucins, probably because expression of the mucin has been found to be upregulated by carcinomas (Dahiya et al., 1993; Ho et aI., 1993). Full-length cDNA and genomic clones for both human (MUC1) and mouse (Mucl) have been published (for a complete review of MUC1 gene and cDNA structure, see Gendler and Spicer, 1995). A diagram demonstrating its transmembrane structure and its cytoplasmic and

Mucins of the Ocular Surface extracellular domain is shown in Fig. 1. The extracellular domain consists of a variable number of 20 amino acid tandem repeats. This highly glycosylated region forms a rigid structure that extends some 200-500 nm above the glycocalyx of the apical membrane of the cell (Hilkens et al., 1992). Homology of the extracellular domain between human MUC1 and a mouse homologue is only 34% in the tandem repeat region, whereas, the transmembrane and cytoplasmic domains are well conserved (Spicer et al., 1991). The cytoplasmic domain consists of 69 amino acids and has been reported to be as,;ociated with actin filaments (Parry et al., 1990). Detection of MUC1 extracellular domain in blood, particularly that of cancer patients, indicates that the domain is cleaved from the cell (Burchell et al., 1984). It is not known whether this simply reflects degradation or whether free MUC1 extracellular domain serves a function. The core protein of MUC1 appears to be identical between ~fissues, but there appears to be a tremendous variation in glycosylation from one tissue type to another, resulting in huge variations in molecular weights. Tissue-specific glycosylation of MUC1 may alter the cell surface function of MUCI at various sites to facilitate tissue-specific surface requirements. MUCI is aberrantly glycosylated in a number of tumor cells (Devine and McKenzie, 1992). Post-transcriptional modifications of MUC1 (i.e. its glycosylation) can be hormonally regulated (Parry et al., 1992). In addition to the regulation of glycosylation, other possible regulations of MUC1 transcription have been proposed. Several regulatory regions of the 5' region of the MUC1 gene and their binding proteins have been identified (Abe and Kufe, 1993; Kovarik et al., 1993; Shirotani et al., 1994). Hormonal regulation of MUC1 expression has been observed in human and mouse uterus, even though the specific promoter elements have not been identified (Parry et al., 1992; Braga and Gendler, 1993; Hey et al., 1994). A variety of functions have been suggested for MUC1 (Gendler and Spicer, 1995). A function for which there is supportive experimental evidence is MUCI's role in preventing cell adhesion. When high levels of the., transmembrane mucin were induced by transfection in a mammary epithelial

85

cell line and a melanoma cell line, cell aggregation in vitro was suppressed (Ligtenberg et al., 1992). In the endometrium of mice that were in the secretory phase of the estrus cycle, progesterone downregulated MUC1 expression, thereby allowing low expression of MUC1 on the endometrial surface at the time of embryo implantation (Braga and Gendler, 1993). This cell-disadhesive function may be the result of MUCI's large extracellular domain in combination with the negativelycharged sialic acid residues on the molecule.

2.2. Secretory Mucins

The full length of MUC2 and MUC7 cDNAs have been sequenced, and the cloning of these two mucins provides the detailed information available on secretory mucins to date (Gendler and Spicer, 1995). Initially, MUC2 through MUC8 were categorized as secretory mucins due to their expression by simple secretory epithelia. They are still so categorized even though only MUC2 and MUC7 have definitively been shown to lack a transmembrane domain. Generally, except for MUC7, these mucin cDNAs are much larger than MUC1, but they all share the characteristic tandem repeat in the protein backbone. MUC2, MUC5AC and their rodent homologues have cysteine-rich domains that are homologous to each other and to the yon Willebrand factor (Gum et al., 1994; Guyonnet Duperat et al., 1995). These homologous regions may allow disulfide bonding between individual mucin molecules which enables them to form gels. Unlike the gel-forming mucins, MUC7 lacks the cysteine-rich domain, and appears to be a soluble monomer. Thus, secretory mucins are subdivided into two types, gel-forming and non-gel-forming mucins (Gendler and Spicer, 1995).

2.2.1. Gel-forming mucins The complete sequence of MUC2 cDNA, 15 563 bp, was obtained from a small intestine cDNA library by Gum et al. (1994). This enormous effort revealed the primary structure of a gel-forming mucin. MUC2 contains two tandem repeat regions

86

I. K. Gipson and T. Inatomi

and four cysteine-rich D domains. Interestingly, these D domains are homologous to prepro-von Willebrand factor, which suggests that they are responsible for the packing and oligomerization of each molecule. It is noteworthy that cysteine-rich domains are also conserved in rat Muc2, MUC5AC, mouse Muc5, an amphibian mucin, and porcine and bovine submaxillary mucin (Gendler and Spicer, 1995). Thus, the cysteine-rich domain appears common to the gel-forming mucins. Several clones of MUC5 cDNA have been reported from trachea and stomach cDNA libraries (Dufosse et al., 1993; Meerzaman et al., 1994; Guyonnet Duperat et al., 1995; Ho et al., 1995). Initially termed MUC5A, B, or C, it has been demonstrated that MUC5A and C are part of the same mucin gene (Guyonnet Duperat et al., 1995). This mucin gene is referred to as MUC5AC. MUC5B appears to be a distinct gene. Further sequencing is necessary to clarify the detailed gene structure of MUC5AC and MUC5B.

library, and only the sequence of 1.83 Kb which contains the 48-bp tandem repeat unit that is repeated 39 times, is available (Porchet et al., 1991). A variety of simple epithelia express MUC4 mRNA, but the details of structure and function of this mucin await further sequence data. MUC6 cDNA, cloned from a stomach cDNA library, has tandem repeats of 169 amino acids, the longest repeat unit reported to date (Toribara et al., 1993). Although scant sequence is available, it is noteworthy that the MUC6 gene localizes on chromosome 1lp15.4-15.5, where MUC2 and MUC5 genes are also placed. MUC8 cDNA has recently been cloned from trachea. It contains imperfect 41-bp nucleotide tandem repeats that encode two types of consensus repeat peptides. This is a unique type of peptide organization for mucin tandem repeats described to date (Shankar et al., 1994).

2.2.2. Non-gel-forming mucin

Recent research from our laboratory indicates that at least three mucin genes are expressed by human ocular surface epithelium (Inatomi et al., 1995, 1996). They include the transmembrane mucin MUC1 (Inatomi et al., 1995), and the secretory mucins MUC4 and MUC5AC (Inatomi et al., 1996). Message for MUC2, MUC3, 5B MUC6, or MUC7 has not been detected by northern blot analysis. Several abstracts report MUC2 presence in conjunctival epithelia and tear film, respectively (Jumblatt et al., 1995; Bolis et al., 1995). We have not been able to detect MUC2 by northern blot analysis or in situ hybridization (Inatomi et al., 1996). The reason for the disparity in our results is unclear, but to date, full reports on MUC2 in ocular surface epithelium are not available. Data is not available for MUC8 expression by ocular surface epithelia.

The full-length sequence of MUC7 cDNA has been reported (Bobek et al., 1993). In contrast to other mucin genes, MUC7 cDNA (2350 bp) is relatively short. MUC7 mRNA codes the so-called MG2 protein found in human salivary gland. MG2 exists as a single polypeptide chain without the intermolecular disulfide bonds that are a distinct property of gel-forming mucins (Bobek et al., 1993). To date, MUC7 mRNA expression is reported only in submandibular and sublingual glands (Bobek et al., 1993).

2.2.3. Less well-characterized mucins At this writing, there is insufficient published sequence of MUC3, 4, 6, or 8 to truly categorize them as secretory mucins, although Gendler and Spicer (1995) do list them as such. MUC3 cDNA was cloned from a small intestine cDNA library, and the tissues it is expressed in are small intestine, colon, and colonic tumors (Gum et al., 1990). MUC4 cDNA was cloned from a trachea cDNA

3. MUCIN GENES EXPRESSED BY THE OCULAR SURFACE EPITHELIUM

3.1. M U C l - - t h e Transmembrane Muein of Corneal and Conjunctival Epithelia

The stratified squamous epithelia of both cornea and conjunctiva express the trans-

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Fig. 2. (A) Northern blot analysis using MUC1 cDNA probe in cultured human corneal epithelium (CHCE), mammary gland (MG), two breast carcinoma cell lines (BT-20 and MCF-7), and human umbilical vein endothelial cells (HUVEC). Note that CHCE, IVIG,BT-20, and MCF-7 have a common 4.4 kbp mRNA, that MG and MCF-7 have a second 7.1 kbp mRNA, and that CHCE has a second mRNA larger than 7.4 kbp. These variations of transcripts may be due to the polymorphism present in MUC1 genes as described by Gendler et al. (1990). (B) Immunoblot of CHCE extract using HMFG-1 monoclonal antibody to MUC1 core protein. Strips were pretreated with neuraminidase to remove sialic acid (N) or without treatnaent (C). HMFG-1 binding in the > 200 kD range was greatly increased after neuraminidase treatment.

membrane mucin MUC1 (Inatomi et al., 1995). Northern blot analysis of R N A from cultured h u m a n corneal epithelium indicates message of appropriate size (Fig. 2A). Two different sizes of transcripts within the appropriate range were detected. Swallow et al. (1987) have reported that transcripts of different size are the result of codominant expression of the polymorphic MUC1 gene. R T - P C R of in vivo human conjunctival epithelial[ samples obtained by filter paper stripping also showed MUC1 m R N A . Immunoblot analysis using an antibody to the core protein of MUC1, designated H M F G - 1 , demonstrates presence of MUC1 protein in cultured human corneal epithelium (Fig. 2B). Neuraminidase pretreatment of the protein blots enhanced the antibody binding. In situ hybridization using riboprobes to the tandem repeat sequence of MUC1 shows message in all cell layers of corneal epithelium (Fig. 3A). Unlike the m R N A distribution, antibodies to the protein core of MUC1 ( H M F G - 1 and 139H2) localize the protein to apical membranes of the superficial cells of both cornea and conjunctiva and

to basal regions of the conjunctival epithelium (Fig. 3C,D). The reason for the difference in localization of protein compared to m R N A is unclear, perhaps the access of antibody to MUC1 protein is masked in some regions of the epithelium. Expression of MUC1 by goblet cells could not be discerned. The exact function of MUC1 is not known, but since it is a transmembrane mucin, it may facilitate spread of secreted mucins over the wet-surfaced epithelium. At the ocular surface specifically, it may facilitate spread of the tear film mucus gel over the eye. Negatively charged sialic acid residues on M U C I may prevent adhesion of the secreted mucus of the tear film, thereby facilitating movement of the mucus gel over the epithelium. The movement of the secreted mucus and its own inherent disadhesive function, plus that of MUC1 extracellular domain may together prevent adherence of foreign debris and pathogens. Mucus (Fleiszig et al., 1994) and intestinal mucins (Chen et al., 1993) are known to inhibit P s e u d o m o n a s aeruginosa and rotavirus adherence, respectively.

88

I . K . Gipson and T. Inatomi

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Fig. 3. MUC1 mRNA distribution in cornea, using in situ hybridization with 35S-labeled antisense (A) and sense (B) riboprobes to the MUCI tandem repeat region. Expression of MUC1 mRNA was observed in all layers of corneal epithelium. MUCI protein distribution in the ocular surface, using HMFG-I antibody (C,D). In sections of a cornea, HMFG-I bound to the apical membrane. Some binding was observed in the cytoplasm of apical and subapical cells (C). In sections of a conjunctiva, HMFG-1 binding was observed on the apical membrane and the basal regions, but not in goblet cells (D). Bars: A,B = 100 t~m; C,D = 10 #m.

3.2. MUC4---a Less Well-characterized Mucin of the Conjunctival Epithelium

Recent data from our laboratory demonstrate m R N A for M U C 4 in human conjunctival epithelium (Inatomi et al., 1996). Northern blot analysis, using an oligonucleotide probe to the tandem repeat region of MUC4, demonstrates M U C 4 m R N A in forniceal conjunctival R N A isolates (Fig. 4A). A polydispersed pattern of binding of the M U C 4 probe is evident. Such a pattern is typical in northern blot analyses of other mucins, except MUC1 (Gum, 1992). In situ hybridization, using both 35S and digoxygeninlabeled probes show that the cells of the stratified squamous epithelia express M U C 4 m R N A (Fig. 4B,C). As assayed by northern blot analysis, no M U C 4 m R N A could be found in cultured human corneal epithelium, nor could M U C 4 m R N A be detected in human corneal epithelium by in situ hybridization. There is insufficient sequence information to designate M U C 4 as a secreted mucin or to lend insight into its function. Since there is so little information on the mucin, real understanding of

M U C 4 function at the ocular surface awaits more complete c D N A and genomic D N A sequencing.

3.3. MUC5---a Conjunctival Goblet Cell Mucin

Goblet cells of human conjunctiva express M U C 5 A C m R N A (Inatomi et al., 1996). Northern blot analysis using a M U C 5 c D N A probe to the tandem repeat region indicates message in conjunctival R N A (Fig. 5A). The typical polydispersed pattern of the secreted mucin m R N A (Toribara et al., 1993) is obvious in conjunctival as well as stomach and tracheal RNA. In situ hybridization using riboprobes to the tandem repeat of M U C 5 A C demonstrates heavy labeling of goblet cells (Fig. 5B,C). Most, but not all, goblet cells were labeled. This may indicate state of differentiation of the goblet cell or heterogeneity of mucin genes expressed by goblet cells. No message over background was detected in stratified epithelia of either cornea or conjunctiva and northern blot analysis of R N A from cultured human corneal epithelium showed no M U C 5 A C m R N A . These data suggest that M U C 5 A C is

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28S& Fig. 4. (A) Northern blot analysis using 48-0,3bp oligonucleotide probe to MUC4 tandem repeat region. MUC4 mRNA expression was detected in conjunctiva as well as trachea, which is a positive control. Both signals show the polydispersed pattern. 28S ribosome RNA stained with ethidium bromide shows the integrity of RNA (A, lower, inset). (B,C) In situ hybridization using digoxigenin-labeled antisense (B) and sense (C) oligonucleotide probes demonstrated MUC4 mRNA distribution in conjunctiva. Intense signal was observed in stratified epithelium of the conjunctiva. Bar = 20 ~m.

secreted by the conjunctival goblet cells and is a gel-forming mucin of the ocular surface tear film. Whether MUC5AC is the only secreted gel-forming mucin on the ocular surface, awaits complete characterization of MUC4 and determination of whether the ocular surface epithelium expresses unique or uncloned mucins.

3.4. Are There Additional and/or Unique Mucins Expressed by the Ocular Surface Epithelium?

From the screening of the ocular surface epithelium for expression of the cloned mucins, MUC1-MUC7, we have shown that three of these are expressed. The goblet cells of the conjunctival epithelium express MUC5AC; the conjunctival epithelium expresses MUC 4, and the stratified epithelia of both conjunctiva and cornea express MUC1. Assay for expression of MUC8 by the ocular surface epithelium has not been reported.

Major questions remain. Are additional mucins expressed by the ocular surface epithelia? Are there ocular-surface-specific mucins?

3.4.1. Corneal epithelial glycocalyx mucin of rat Several years ago we began to characterize a high molecular weight, highly glycosylated glycoprotein that is present in the glycocalyx region of the apical cell membrane of the apical cells of the stratified epithelia of both cornea and conjunctiva of the rat (Gipson et al., 1992) (Fig. 6). Immunoelectron microscopy using a monoclonal antibody, R339, to the glycoprotein demonstrated that the glycoprotein is present not only along the glycocalyx of the apical membrane of apical cells, where it was particularly prominent at the tips of the microplicae, but it was also present within small cytoplasmic vesicles in the cytoplasm of 2-3 cell layers of flat subapical cells of the corneal and

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A Fig. 5. (A) Northern blot analysis using cDNA probe to MUC5AC tandem repeat region. An intense polydispersed binding was detected in conjunctiva as well as the positive controls of trachea and stomach. 28S ribosome RNA stained with ethidium bromide shows the integrity of the RNA (A, lower, inset). (B,C) In situ hybridization using 35S-labeled antisense (B) and sense (C) riboprobes demonstrated MUC5AC mRNA distribution in the conjunctiva. MUC5 mRNA was limited to the conjunctival goblet cells. Bar = 50 pm.

conjunctival epithelia (Fig. 6). Studies of the developmental appearance of the glycoprotein indicate that eyelid opening induces expression of the molecule in the 12-day-old rat pup. Artificial eyelid opening at earlier time points also induces expression as indicated by binding of monoclonal antibody R339 (Watanabe et al., 1993). Two other stratified epithelia of the rat produce the glycoprotein recognized by R339. The stratified epithelia of rat trachea and vagina express the glycoprotein in exactly the same subcellular localization pattern as that of the cornea (Gipson et al., 1995). Isolation of the glycoprotein from both corneal and vaginal epithelia of the rat was accomplished by lectin affinity chromatography, the lectin being Dolichos biflorus agglutinin (Fig. 7). The isolates, free of contaminating protein, were further characterized by determining carbohydrate-to-protein ratio and carbohydrate and amino acid composition (Gipson et al., 1995).

These data showed that the molecule has the classic characteristics of a mucin. The mass of the glycoprotein is approximately 60% carbohydrate; it has a high concentration of carbohydrates characteristic of O-linked sugars. Molar percentages of the six monosaccharides detected were: N-acetylgalactosamine--37%, N-acetylglucosamine--23%, mannose--14%, galactose-14%, xylose--11%, and fucose--2%. Amino acid analysis demonstrated high percentages of serine and threonine (Gipson et al., 1995).

3.4.2. Ocular surface glycocalyx glycoprotein of humans We asked the question--is there a comparable glycoprotein in the human ocular surface epithelium? By Jacalin affinity chromatography, a glycoprotein was isolated from cultured human corneal epithelium, which has many of the same characteristics as the rat ocular surface mucin

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Fig. 6. Immunolocalization of monoclonal antibody R339 to rat corneal epithelium. The antibody recognizes a carbohydrate epitope on a mucin-i[ike glycoprotein. In fluorescence micrograph inset at top, labeling to three to four layers of flattened

cells of the corneal epithelium is shown. By immunoelectron microscopy (A) the binding can be seen along the apical tear-facing membrane of the apical cell. In subapical cells, boundaries of which are indicated by arrows, binding is present within the cell's cytoplasm. At higher magnification, binding of the silver-enhanced, 1 nM immunogold can be seen to be prevalent on microplicae of the apical cells (B). In (B) the arrows indicate membrane of subapical cell. (C) At higher magnification one can discern binding on vesicle membranes within the cytoplasm of subapical cells. Bars: A inset = 20/zm; A = 1 #m; B,C = 0.2 #m. (Fig. 7). We developed a m o n o c l o n a l antibody, designated H185, to the glycoprotein (Watanabe et al., 1995). The binding o f H185 to cornea and conjunctival epithelia matches precisely the tissue and subcellular binding patterns o f R339 seen in the rat (Fig. 8). H185 binds to the glycocalyx o f the apical m e m b r a n e o f apical cells o f the cornea and conjunctiva and to small vesicles in the cytoplasm o f subapical cells. Binding is particularly prominent on the tips of the

microplicae. The isolated glycoprotein has a high molecular weight and is highly glycosylated. Furthermore, binding o f H185 to blots o f the protein can be prevented by O-glycanase treatment (Fig. 7). Are these glycocalyx-located, mucin-like molecules f r o m rat and h u m a n ocular surface epithelia cloned mucins or are they unique a n d / o r uncloned mucins? Since the mucins recognized by antibodies R339 and H185 are p r o d u c e d by the corneal

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Fig. 7. (A) Binding of monoclonal antibody R339 to total proteins of rat corneal epithelium, lane 1; vaginal epithelium, lane 2; and tracheal epithelium, lane 3, and binding of H185 to total protein of human corneal epithelium, lane 1; cultured human corneal epithelium, lane 2. Note similarities in binding patterns. (B) lmmunoblot analysis of mucin isolates recognized by R339 (lanes 1-3 on left) and H185 (1 lane on right). The mucin-like glycoprotein recognized by R339 was isolated by dolichos bifluorus agglutinin lectin affinity chromatography. Lane 1, isolate from vaginal epithelia; lane 2, isolate from corneal epithelium; lane 3, silver-stained gel of DBA isolate showing lack of contaminating proteins. The R339 isolate does not stain by silver or Coomassie blue, which is characteristic of highly glycosylated glycoproteins. Lane 1 on right shows the H185 mucin which was isolated by Jacalin lectin affinity chromatography. (C) O-glycanase treatment of proteins of human corneal epithelium destroys H185 binding. Lane 1 is untreated control, lane 2 is protein pretreated with neuraminidase only, lanes 3 and 4 are neuraminidase- then O-glycanase-treated proteins, and lane 5 is protein treated with neuraminidase and heat-inactivated O-glycanase. Note complete lack of binding of H185 to lanes 3 and 4. (D) Immunoblots showing comparison of binding of H185 and MUC1 protein core antibodies to soluble and membrane fractions of proteins from cultured human corneal epithelium. Note that H185 binds to both soluble (S) and membrane (M) fractions, whereas HMFG1 binds to a different molecular weight region and to membrane fractions only.

e p i t h e l i u m a n d since the o n l y c l o n e d m u c i n f o u n d to be expressed b y the c o r n e a l e p i t h e l i u m to d a t e is M U C 1 , it w o u l d a p p e a r t h a t M U C 1 is the o n l y c a n d i d a t e for c o m p a r i s o n . D e f i n i t i v e p r o o f o f w h e t h e r the m u c i n r e c o g n i z e d b y H 1 8 5 is M U C 1 a w a i t s c l o n i n g o f the H 1 8 5 a n t i g e n . A t t e m p t s to

screen a n e x p r e s s i o n l i b r a r y with a p o l y c l o n a l a n t i b o d y to a d e g l y c o s y l a t e d p r e p a r a t i o n o f the h u m a n c o r n e a l m u c i n h a v e b e e n u n s u c c e s s f u l , to date. It has p r o v e n difficult to o b t a i n e n o u g h o f the m u c i n - l i k e m o l e c u l e f r o m c o r n e a l epithelial c u l t u r e s to be able to m a k e repetitive a t t e m p t s

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Fig. 8. Binding of monoclonal antibody H185 to the human corneal epithelium. A light micrograph of human corneal epithelium (A) is shown for comparison to immunolocalization of H185 to three to four layers of flattened apical cells in B (arrows indicate base of epithelium). Immunoelectron microscopiclocalization of H 185 to apical cells is shown in C and D. Curved arrow in C indicates boundary between apical and subapical cells. Note prevalence of binding to microplicae at tear-epithelial cell interface (open arrow in C) and binding to cytoplasmic vesiclesof subapical cells (solid arrows in C and D). Bars: A,B = 10/~m; C,D = 0.2 #m.

at deglycosylation of the isolate and antibody production to the core protein. We have made comparisons of binding of antibodies H185 and H M F G - 1 (antibody to the core protein of M U C 1 ) in adjacent lanes on the same blot of soluble and membrane fractions from human corneal epithelial cell cultures. M e m b r a n e fractions were isolated from third passage cells, using techniques described by Azzarolo et al. (1995). H185 binds to a glycoprotein that is of higher molecular weight than the MUC1 glycoprotein detected by H M F G - 1 (Fig. 7). Additionally, H185 binds to both soluble and membrane fractions, whereas H M F G - 1 binds to membrane fractions only. However, caution in interpretation of these data is warranted. Comparison of binding of antibodies that bind to carbohydrate epitopes (H185) to that of binding of antibodies to the protein core of

mucins ( H M F G - 1 ) may not be appropriate. A carbohydrate-binding antibody may bind a population of mucin molecules that are more heavily glycosylated and are thus of apparent higher molecular weight. Secondly, binding of an antibody to the protein core of a heavily glycosylated mucin molecule may be prevented by the carbohydrate side chains. As stated above, definitive comparison between our corneal epithelial mucinlike isolate and MUC1 awaits molecular characterization of the corneal isolate.

3.5. Role of Specific Mucins in the Tear Film

Use of molecular biology techniques have facilitated the determination that at least three mucin genes are expressed by the ocular surface

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L--. MUC 5 MUC 1

Fig. 9. Diagram depicting our hypothesis of the roles of the transmembrane mucin MUC1, and the gel-forming mucin MUC5AC in the tear film on the surface of the cornea. We hypothesize that MUC1 extends from the microplicae into the glycocalyx region and that the negative charges, indicated by minus signs at the tips of the MUC1 extracellular domain, facilitate surface wetting and loose association of the gel layer. The loose association of the MUC5AC gel, with its associated non-mucin proteins and fluid, may be required to allow movement and facilitate the cleansing function of the mucus layer.

epithelium. This new data demonstrates that the corneal epithelium expresses MUC1 and that conjunctival epithelium expresses MUC1, 4, and 5AC. It is not known what the functions of the specific mucins are, nor whether their functions vary. There is, however, sufficient structural information regarding the transmembrane mucin MUC1 and the gel-forming mucins (MUC2 and MUC5) to develop an hypothesis regarding the functions of M U C 1 and M U C S A C at the epithelial tear film interface (Fig. 9). We hypothesize that MUC1, which extends from the apical membrane of the apical cells of the stratified epithelia of both cornea and conjunctiva, is an important base or substrate that facilitates the spread of the secreted, gel-forming mucin (MUC5AC) and the aqueous components of the tear film. The negatively charged character of the carbohydrates on MUC1 may repel other negatively charged cells and/or mucins. This 'disadhesive' character may facilitate free movement of the gelled mucus layer over the surface of the eye when blinking occurs. Free movement of the viscous mucus may facilitate removal of cellular and other debris from the surface of the eye.

3.6. Mucins and Ocular Surface Disease

Although several clinical tests have been proposed and developed to evaluate tear film stability, tear break-up time (BUT) may be the most helpful assay, and it offers a valuable parameter for diagnostic criteria for dry eye syndrome (Lemp, 1995). B U T has been shown to be dependent on reduction of tear surface tension by mucins (Dohlman et al., 1976; Holly, 1980; Tiffany et al., 1989). A significant reduction of BUT is commonly observed in pathological conditions that are associated with conjunctival goblet cell loss, such as Stevens-Johnson syndrome, ocular cicatricial pemphigoid and xerophthalmia (Lemp, 1973; Kinoshita et al., 1983; Nelson and Wright, 1984). In these patients, the reduction of BUT m a y reflect deficiency of the secretory mucins such as M U C 5 A C , derived from conjunctival goblet cells. The etiology of the deficiency of the secretory mucins in these diseases may be lack of differentiation of conjunctival stem cells to goblet cells, lack of expression of mucin genes, or alteration of glycosylation of mucins. Having determined which of the cloned mucins are

Mucins of the Ocular Surface expressed in goblet ce.lls, it will be possible to assay conjunctival tissue fi'om mucus-deficient patients to ascertain if appropriate mucin gene expression is occurring. Does decreased BUT occur even if a normal goblet cell population exists? Recently, our studies have demonstrated that the stratified epithelia of the cornea and conjunctiva express the transmembrane mucin, MUC1, and that it is present on the apical membrane of the apical cells of the epithelia. MUC 1 may play an important role in the interaction of the secreted mucins and the epithelial surface at the base of the tear film. Thus, absence of MUC1 or alteration of its glycosylation may also be a major cause for reduction of BUT. Furthermore, it is this kind of transmembrane mucin deficiency that may be detected by Rose bengal staining. Clinically, abnormal Rose bengal staining is commonly observed in a variety of pathological conditions from dry eye to severe keratinization. It is possible that the absence of or alteration of MUC1 may correlate with penetrance of Rose bengal into cells in areas where tear film is absent and 'dry spots' are present. In preliminary studies, we used monoclonal H185 which recognizes a carbohydrate epitope on a human ocular surface mucin-like molecule (described above), to determine the mucins' distribution in apical cells of seven normal conjunctival specimens and 15 conjunctival specimens from patients with chronic dry eye syndrome (Tisdale et al., 1994). Apical cells from four quadrants of bulbar conjunctiva were obtained by placing strips of filter paper on the conjunctival surface. H185 binding was assayed by immunofluorescence microscopy. Diagnosis of dry eye patients included positive Rose bengal staining and symptoms of sandy gritty sensation, burning, stinging, irritation, and dryness. In normals, H185 binding to apical cells had a cobblestone appearance. Cells bound the antibody in light, medium, and dark patterns. In 14 out of 15 dry eye patients, we found that H185 binding was diminished or lacking on the apical cells of the conjunctival epithelium. Curiously, staining of goblet cells in dry eye patients was enhanced. These preliminary data suggest alteration of mucin's distribution on the apical surface of conjunctival stratified epithelial cells of patients with chronic dry eye. It remains to be determined whether the

95

alteration of mucin distribution or character detected with H185 binding correlates to changes in synthesis of mucins or whether altered H185 binding results from aberrant or altered glycosylation of the mucins brought about by altered glycosyl transferase activity. It may be useful to assay MUC1 and MUC5AC gene expression by in situ hybridization on filter-paper-derived conjunctival epithelial samples to begin to sort these questions out. Assay of mucus at the ocular surface has depended primarily on assays that detect carbohydrate content of lacrimal fluid or conjunctival biopsies. Three techniques have been used; they include PAS or Alcian blue staining of conjunctival biopsies to determine presence or absence of goblet cells with their secretory product, PAS staining of cotton strips that had been placed in the inferior cul-de-sac, and hexosamine assay of tear fluids (Doughman, 1973; Dohlman et al., 1976). In the latter study, hexosamine content was significantly lowered but not absent in tears of patients with Stevens-Johnson syndrome and ocular pemphigoid. Since hexosamine content was not completely absent, Dohlman et al. (1976) were concerned that the diseases were not truly mucus deficiencies. Studies of mucin gene expression may help to clarify this issue and may be useful in the future for diagnosis of variations of dry eye syndrome.

4. SUMMARY AND FUTURE DIRECTIONS Molecular characterization of mucins has begun to yield information regarding their primary structure and tissue-specific expression. Because of their huge size and heavy glycosylation, progress in their characterization has been slow. Much remains to be determined regarding their primary structure and the regulation of their expression, particularly at the tissue level. Much remains to be learned of their glycosylation and the regulation of the process, again at the tissue-specific level. Little is known of the tissue-specific expression of the glycosyltransferases that are involved in the O-linked glycosylation of the mucin gene products. Future directions in mucin characterization in general will undoubtedly lead in these directions.

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W o r k cited in this review demonstrates that at least three mucin genes are expressed by the ocular surface epithelium in a region-/cell-specific pattern. M a n y questions and future experiments are suggested by these data. Several are noted below. Are additional and/or unique mucins expressed by the ocular surface epithelium and its accessory glands? In the G I tract at least six different mucin genes are expressed in a region-specific pattern. It is likely that additional ocular surface mucin genes will be identified. Do the lacrimal and accessory lacrimal glands express mucin genes? These questions need to be answered in order to have a more complete understanding of mucins in the tear film. Recent work demonstrates that goblet cells of the conjunctiva are derived from the same stem cells as those of the stratified epithelium of the conjunctiva. Since M U C 5 A C appears to be a goblet-cell-specific mucin, M U C 5 A C may be a useful marker in studies to determine factors involved in goblet cell differentiation. Previously it has been impossible to identify early stages of goblet cell differentiation. If M U C 5 A C message appears early in differentiation, it will be a useful tool to discern the position and morphology of early goblet cells and to test factors which might effect goblet cell differentiation in vitro. These studies are especially important in understanding ocular surface diseases in which goblet cell populations become depleted, i.e. Stevens-Johnson syndrome, vitamin A deficiency, ocular cicatricial pemphigoid and alkali injury. Using antibodies to carbohydrate epitopes of ocular surface mucins, we have discerned an alteration in mucin distribution on surfaces of stratified epithelium of patients with dry eye syndrome. The question arises, is this alteration a result of altered expression of mucin genes, or altered glycosylation of the mucin gene products? Identification of the mucin genes expressed by the ocular surface will allow studies to answer the first aspect of this question. I f mucin gene expression is not altered, then investigation of glycosyltransferase expression and activity in ocular surface epithelium may be warranted. Major questions remain regarding the functions of the different mucins at the ocular surface.

One can envision differing functions of transmembrane and gel-forming mucins, but the roles of each mucin have not been experimentally determined. One approach toward discerning function of mucins is development of transgenic mice null for specific mucins. Indeed, a recent report describes development of a MUC1 null mouse. The only alteration in phenotype that was discerned was delay of m a m m a r y tumor progression (Spicer et al., 1995). Perhaps function of MUC1 is reiterated in other uncharacterized transmembrane mucins. Development of other mucin null mice awaits complete cloning of mouse mucin genes. Understanding tissue-specific regulation of mucin gene expression will be of particular relevance to ophthalmology. Identification of ocular-surface-specific promoters for mucin gene expression may provide a system for insertion of genes for ocular surface therapy or for delivery of other therapeutic proteins. In summary, the understanding of the molecular nature of ocular surface mucins is in its early stages. Future studies will yield information relevant to treatment of ocular surface disease, mucin function, and pathogen adherence. wish to acknowledge the support of National Institutes of Health/National Eye Institute (Grant No. R37 EY03306) and Massachusetts Lions Eye Research. Acknowledgements~e

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