Fractionation and partial characterization of macromolecular components from human ocular mucus

Crude human ocular mucus was extracted with 0·154 m-NaCl to separate soluble protein components from mucus. Small amounts of lipoglycoprotein of high molecular weight, as well as twelve plasma proteins, were detected in the soluble extract by gel filtration and immunodiffusion studies. After the NaCl extraction, the remaining mucus residue was further extracted with 6 m-urea-0·2 m-Tris-phosphoric acid buffer. From this portion of soluble extract, a relatively larger amount of lipoglycoprotein of high molecular weight, as well as a lower molecular weight fraction containing eight detectable plasma proteins, were both isolated by gel filtration. The glycoprotein moieties of the lipoglycoproteins of high molecular weight had similar chemical composition. Both contained approximately 40–43% protein and 57–60% carbohydrate, giving a carbohydrate-protein ratio of 1·30 to 1·48. Fucose, galactose, N-acetylhexosamine and N-acetylneuraminic acid comprised about 423–516 residues per 1000 amino acid residues, while serine and threonine constituted about 285–299. All analyses indicated mucin-like character in the lipoglycoproteins of high molecular weight.Plasma proteins constituted approximately three-fifths of the macromolecular components in ocular mucus. These proteins also appeared to be in complexes with lipids, but to a much lesser extent than the high molecular weight fractions. The relevance of present findings to the structure and composition of precorneal tear film is discussed.     

Studies on the isolation and composition of human ocular mucin

A method for the isolation and purification of human ocular mucin from the brief saline extract of human ocular mucus is reported. Initial purification of ocular mucin was achieved by sequential chromatography of the saline-soluble mucus extract from an individual donor's mucus pool on columns of Sephadex G-50 and Sepharose CL-4B. A portion of such mucin isolate was subjected to quantitative analysis of the O-seryl (threonyl)-N-acetylgalactosaminyl linkage, characteristic of mucins, by alkaline beta-elimination and tritiated borohydride reduction. Following Bio-Gel P-2 filtration, the mucin isolate whose cleaved oligosaccharides contained tritiated galactosaminitol greater than 0.5 microCi mg-1, a value that represents at least 64% of that observed for bovine and ovine submaxillary reference mucins, was considered to be mucin-rich. These isolates were subjected to further purification on Sephacryl S-500 and DEAE-Trisacryl M column chromatographies. The purified mucin had a minimum molecular weight of 120 kDa. It consisted of 25-30% protein and 54-55% carbohydrate. Its amino acid and carbohydrate compositions are characteristic of a mucin structure. The purity of the mucin was verified by SDS-gradient PAGE. Upon isoelectric focusing, polydispersity/microheterogeneity were exhibited in the pI range 5.0-6.6.     

SDS-gradient polyacrylamide gel electrophoresis of individual ocular mucus samples from patients with normal and diseased conjunctiva

Individual mucus samples were collected from normal individuals and from patients with ocular cicatricial pemphigoid (CP), Stevens-Johnson syndrome (SJS), and various types of conjunctival inflammation (rosacea, meibomianitis, atopy, keratoconjunctivitis sicca, etc.). The mucus samples were dissolved in sample buffer containing 8M urea, 2% SDS and 5% 2-mercaptoethanol and were electrophoresed on gradient 2-16% polyacrylamide gels. Four glycoproteins with molecular weights greater than 200,000 daltons were consistently observed in both individuals with normal conjunctiva and patients with CP, SJS, and other diseases exhibiting conjunctival inflammation. The amounts of each glycoprotein appeared to vary from one individual to another; however, the presence or absence of specific glycoproteins could not be correlated with the different ocular diseases. The techniques described for mucus analysis offer advantages over previously published techniques since improved resolution of the mucous glycoproteins can be achieved by electrophoresis on 2-16% gradient gels, and individual samples can be analyzed. Our results suggest that substantial amounts of ocular mucous glycoprotein are present in the eyes of patients with CP and SJS, diseases which have been previously described as mucin-deficient dry eye syndromes.     

A study of lectin-binding to the water-insoluble proteins of the lens

Binding of lectins to the water-insoluble fractions from human, monkey and mouse lenses was investigated. Lectins bound to glycoproteins blotted from SDS-polyacrylamide gels onto nitrocellulose paper. Use of multiple lectins showed that glycoproteins with apparent molecular weights of about 120 000 and 90 000 were present in all species. These proteins were found in the urea-insoluble fraction. Multiple lectins also bound to two bands around 67 000 and 63 000 molecular weight in both human and monkey lens fractions as well as a urea-soluble band at about 140 000. A 35 000 MW glycoprotein containing N-acetyl-galactosamine and fucose sugars was present in the capsule-epithelium of monkey lenses but was not detected in cortical or nuclear fractions. Glycoproteins from normal and cataractous mice were identified using this new methodology and compared with previously published work using sodium borohydride labelling of glycoproteins. Because of the numerous glycoproteins that can be detected and the ability to assign sugar residues to individual proteins, the lectin-binding method is a powerful tool to investigate lens glycoproteins.     

Immunological study of proteins and mucosubstance in saline soluble human ocular mucus

Proteins and mucosubstance of the saline extract of human ocular mucus were studied by immunological analysis. A minor study was made with human tears for comparison. Immunoelectrophoresis of proteins from these two sources consistently revealed similar characteristic gel patterns. Proteins were found as the major constituents of both samples. However, more mucosubstance was present in the saline extract of human ocular mucus than in tears. Seventeen proteins were identified in the mucus extract. Albumin, IgA, and lactoferrin appeared to be the three major proteins, while lysozyme, lactoferrin, tear prealbumin, and ocular mucoisolate were tear and ocular mucus specific. Although saline soluble mucoisolate is complex in structure, it seemed to resist electrical dissociation, producing only one major precipitation line along with a line of IgA during immunoelectrophoresis. The ocular mucoisolate accounted for about 12% of the saline extractable proteins of human ocular mucus.     

The glycoprotein (mucus) content of tears from normals and dry eye patients

Tears were sampled by two different methods from patients with ocular pemphigoid, Stevens-Johnson's Syndrome and Sjögren's Syndrome, as well as from normals. The tear glycoprotein (mucus) content was determined by measuring hexosamine after hydrolysis. Also hexuronic acid and protein levels were determined, as well as tear flow rate in some instances. The level of glycoprotein (mucus) in tears was markedly reduced in pemphigoid and Stevens-Johnson, diseases which are characterized by early break-up of the tear film and decreased content of mucus-producing conjunctival goblet cells. Mucus has earlier been shown to be necessary for the wetting of epithelium, but it is questionable whether mucus is reduced enough in pemphigoid and Stevens-Johnson to explain tear film instability in these disease entities.     

Ocular surface complex carbohydrates are modified with aging

Picric acid-paraformaldehyde-glutaraldehyde (PA-P-G) was used to stabilize chemically ocular surface-associated mucus in mice of various ages. Transmission electron microscopy was then used to examine those specimens stained with cationic ferritin (CF), dialysed iron and Alcian Blue. Collectively, all of these stains are markers for anionic sulfate or carboxyl groups. With each of them, positive labeling of the ocular surface was observed for all ages examined, even when mucus cannot be morphologically demonstrated. Except for dialysed iron, staining was weak in the youngest animals and heaviest in young adult and aged mice. The ocular surface was negative for high iron diamine (HID) in pups and older mice through 1 year of age. Scant positive staining for HID was seen at the ocular surface in 14-month-old mice indicating that mucus became slightly sulfated with aging. Treatment of eyes with neuraminidase prior to fixation reduced the number of CF binding sites in all ages of mice examined, confirming that many of the carboxyl groups at the ocular surface are associated with sialic acid residues. Comparison of percentage reduction in CF labeling following neuraminidase treatment of the eyes of 5- and 10-postnatal day mice with all other ages of mice suggested that fewer removable carboxyl groups at the ocular surface are associated with sialic acid residues in pups. The ocular surface of all eyes also stained positively at the TEM level when a periodic acid-thiocarbohydrazide-silver protein (PA-T-SP) staining sequence was used. Collectively, these data are of significance with respect to further characterization of the ocular surface, particularly with regard to development-associated changes and their possible role in defence of the eye surface.     

Human ocular mucus. Origins and preliminary characterisation.

A technique for collection of human ocular mucus was developed, and the solubility of the crude mucus clot in a variety of solvents, reducing agents and detergent solutions was assessed. Reduced and unreduced tears and mucus were separated by polyacrylamide gel electrophoresis on slab gels, and their mucous glycoprotein and other protein components identified by Coomassie Blue and periodic acid-Schiff staining. The principal mucin complex GP1 (mol. wt.>2×10⁶) and its subunit GP3M (mol. wt. about 200 000) were detected in unreduced mucus, while reduction of disulphide bonds gave rise to GP2 (mol. wt. about 1·3 × 10⁶). Human GP2 and GP3M each contained a high proportion of carbohydrate, but there was considerable variation among individual donors. Rerunning of the GP2 band isolated from gel gave GP2 and also a GP3M band. Immunofluorescent studies using rabbit antibody raised against human GP2 showed the site of origin of GP2 to be exclusively the conjunctival goblet cells and not lacrimal tissue. A single mucous glycoprotein GP3T, also about 200 000 mol. wt., was found in tears, but did not cross-react with anti-GP2 antibody.     

Studies on the solubilization of the water-insoluble fraction from human lens and cataract.

Studies were carried out comparing the ability of urea extraction and sonication to solubilize the water-insoluble (WI) protein fraction from human lens tissue. Sonication and urea extraction were able to solubilize greater than 80% of the insoluble protein whether whole lenses or lens nuclei were used. This was true for normal lens and +1 cataracts; however, only 60% solubilization was obtained with the WI fraction from more advanced cataracts. Equal aliquots of a WI fraction from both pooled normal and pooled cataract lens nuclei were solubilized with and without reducing agents. The addition of dithiothreitol (DTT) had no significant effect on solubilization of the normal lens WI fraction. DTT did increase the protein solubilized from the cataract WI fraction by 30% with urea extraction; however, no increase was seen with sonication. When sodium borohydride was used as the reducing agent, essentially the same results were obtained. The solubilized protein populations were identical by SDS-PAGE and amino acid analysis. The addition of reducing agents had no effect on the amino acid content of the solubilized proteins with the single exception of lysine. This amino acid was markedly decreased in the proteins extracted in the presence of 40 mM sodium borohydride, but not with DTT. These data suggest that the borohydride not only increased the amount of protein solubilized, but likely also stabilized glycated lysine residues during the acid hydrolysis. Therefore, sonication readily provides a soluble preparation of the WI proteins from normal and cataract lens nuclei without the need for denaturing agents, however, disulfide-linked and lysine modified crystallins were best solubilized with urea.     

Characterization of ocular mucus extracts by crossed immunoelectrophoretic techniques

Crossed immunoelectrophoresis (CIE) and crossed immunoelectrofocusing (CIEF) were used to characterize proteins and mucosubstance in the saline extract of human ocular mucus pooled from the normal eyes of donors. CIE resolved more components with greater specificity than previous techniques. Up to 25 components were identified. Lactoferrin, protein G, tear prealbumin, and ocular mucosubstance were found to be ocular-specific. CIE also allowed for the study of protein associations of: 1) albumin-alpha 1-antitrypsin; 2) albumin-tear prealbumin; and 3) IgA-secretory component and lactoferrin-mucosubstance. CIEF revealed that most of the proteins were in the pI range of 4.6-7.4. Up to 19 components were identified. Protein associations revealed by CIE were not evident by CIEF. These results provide a basis for future comparative analyses of tear and mucus from normal and diseased eyes, essential for a better understanding of tear and mucus function.     

High molecular weight mucin-like glycoproteins of the bovine interphotoreceptor matrix.

A very high molecular weight mucin-like glycoprotein was isolated by gel filtration of interphotoreceptor matrix (IPM) from fresh bovine eyes and purified to apparent homogeneity by cesium chloride/guanidine hydrochloride (GuHCl) equilibrium density gradient centrifugation. Although a molecular weight in excess of 10(7) Da is suggested by gel filtration, the presence of SDS or GuHCl did not alter its elution position, indicating that the large size was not simply due to aggregation. Treatment of this material with disulfide reagents, however, led to a decrease in molecular size. On a relative basis, substantially more of this glycoprotein is present in IPM prepared from retina than from retinal pigment epithelium. While the carbohydrate and amino acid composition are not those of a true 'mucin', the large size and many other properties are quite 'mucin-like'. The carbohydrate composition suggests the presence of both N- and O-glycosidically linked sugar chains. The presence of a mucin-type O-glycosidic linkage is indicated by its susceptibility to alkaline cleavage, with concomitant loss of serine and threonine and increase in 240 nm absorbance; production of a fluorescent product upon reaction with cyanoacetamide; lectin binding properties; and production of N-acetylgalactosaminitol upon alkaline borohydride elimination. This glycoprotein was digested by pronase and trypsin, confirming its protein nature, but was resistant to digestion with chondroitin ABC lyase, hyaluronidase and heparinase, as well as RNAase, indicating that these components were not present to any appreciable extent. ELISA for cartilage keratan sulfate was also negative. Centrifugation in CsCl/GuHCl gradients indicated a density much lower than that of a proteoglycan or nucleic acid as well. In vitro biosynthetic studies suggest that both retina and retinal pigment epithelium may be major sources of material in the IPM. The elution patterns of radioactivity were strikingly similar to the UV elution patterns of IPM. The medium from retinal incubations contained very high molecular weight material which was resistant to enzymes which hydrolyse glycosaminoglycans, suggesting that retina may be the source of this high molecular weight, mucin-like glycoprotein.     

Application of lectins for detection of goblet cell carbohydrates of the human conjunctiva

Paraffin-embedded and frozen biopsies from the human conjunctival epithelium were examined by fluorescence microscopy after labeling with eight fluorescein-conjugated lectins: wheat germ agglutinin; soybean agglutinin; peanut agglutinin; Ricinus communis agglutinin-1; Limulus polyphemus agglutinin; Ulex europaeus agglutinin-1; Dolichos biflorus agglutinin; concanavalin A. The lectins were used as specific molecular probes to detect carbohydrate composition of glycoproteins secreted from the conjunctival goblet cells. The labeling pattern of goblet cells and conjunctival epithelial surfaces with various lectins suggested that N-acetyl-glucosamine, galactose, N-acetyl-galactosamine and sialic acid are contained in goblet cells and contribute to the formation of tear mucus glycoprotein. Fucose and mannose, which are present in the tear mucus, were not detectable in goblet cells.     

Factors responsible for tear ferning

The biochemical agents involved in fern formation on drying tears were investigated by studying the different patterns of ferning of tears from normal and pathological eyes, mucus glycoprotein, saliva, serum, and various salt solutions. We conclude that the ferning phenomenon observed when a drop of tears is allowed to dry at room temperature on a clean microscope slide is to a large extent determined by the electrolyte concentration, especially the ratio of monovalent sodium and potassium ions to divalent calcium and magnesium ions. The presence of a biopolymer is essential, but this need not specifically be mucus as previously thought. The test may therefore be useful clinically in indicating the need for further tear analysis.     

Purification and characterization of rabbit ocular mucin

Ocular mucin, the major product of conjunctival goblet cells, constitutes the innermost layer of preocular tear film. Ocular mucin is known for its limited amount and inaccessibility. Using impression cytology, mucus strands collected from the inferior fornix of either rabbit or human eyes were found to contain inflammatory cellular debris. In order to circumvent these difficulties and to isolate native mucin molecule(s), we bathed rabbit eyes in fluid containing isotonic PBS and 5.5 X 10(-4) M acetylcholine for 4 or 12 hr. Bathing fluids containing rabbit ocular mucin (ROM), 1 ml per eye, were pooled and combined with 1M guanidine HCl and protease inhibitors containing EDTA, PMSF, and sodium azide to avoid any possible enzymatic degradation, and then separated under the same conditions by Sepharose CL-4B. In parallel, commercial porcine stomach mucin (PSM) was purified and used to compare with ROM. We also developed nitrocellulose-based dot semi-quantitative assays for nucleic acid, protein, and glycoprotein. PAS-positive fractions monitored by such a dot assay were collected at CL-4B void volume and then separated from nucleic acid contaminants by CsCl-gradient ultracentrifugation. A protein fraction, 65K, poorly-glycosylated, with high contents of Asx, Glx, and Gly was found strongly associated with both ROM and PSM, and was only separable by ultracentrifugation in 4M guanidine HCl and CsCl. Purification of the ROM was verified by SDS-polyacrylamide gel electrophoresis, amino acid analysis, and carbohydrate analysis. These results will allow future exploration of the molecular mechanism by which tear film is achieved.     

Mucus fishing syndrome: case report and new treatment option.

BACKGROUND: Mucus fishing syndrome (MFS) is a cascading cyclic condition characterized by continuous extraction of mucous strands from the eye. It is usually initiated by ocular irritation. In response to irritation, ocular surface cells produce excess mucus. A "snow balling" cycle begins when the patient extracts ("fishes") excess mucus from the ocular surface, thereby causing further irritation and a more-profound mucous discharge. To date, treatment includes eliminating the initiating element and educating the patient not to touch the eye when extracting the excess mucus, CASE REPORT: Presented is a case of mucus fishing syndrome initiated by dry eye. The patient's diagnosis, MFS, was identified by persistent mucous discharge, his admittance and demonstration of digitally extracting mucus from the ocular surface, and a characteristic rose bengal staining pattern. The conventional treatment initiated by using artificial tears for the dry eye condition and educating the patient not to touch the ocular surface did not provide relief from the excess mucous discharge. Therefore, a new approach to treatment was pursued. In order to break the cycle, a mucolytic agent and an antihistamine-mast cell stabilizer were prescribed, until the ocular surface healed. After treatment, the patient reported alleviation of symptoms and demonstrated improvement in ocular surface integrity by a profound reduction in rose bengal staining. CONCLUSION: Mucus fishing syndrome is challenging to resolve with conventional treatment because it requires a certain level of psychological tolerance and perseverance from the patient. By eliminating the present mucus and diminishing mucous production pharmacologically, the practitioner is able to remove the stimulus for digital extraction and thus accelerate ocular surface healing. We present a proposed new treatment option for patients who are refractory to conventional treatments.     

Analysis of glycoproteins expressed by isolates of herpes simplex virus causing different forms of keratitis in man

An in vitro analysis of glycoprotein produced by nine human ocular isolates of HSV-1 is reported. The source of the isolates was; three patients with recurrent dendritic keratitis, three with chronic stromal disease and three with primary keratoconjunctivitis. Virus strains were labelled with the radioactive precursors (35S) methionine and (14C) glucosamine. Radiolabelled viral glycoproteins were subsequently analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. Viral glycoproteins were further characterised by immuno-precipitation with polyclonal and monoclonal antibodies to HSV. The stromal isolates excrete larger amounts of 'soluble' precursor glycoprotein D than those in the other two disease categories. It is possible that the immune response to glycoprotein D is in part responsible for the severity of stromal disease.     

On the nature and the role of the subsurface vesicles in the outer epithelial cells of the conjunctiva.

The layer of the tear film in contact with the conjunctiva is mucus. This mucus comes from two sources, the conjunctival goblet cells and the subsurface vesicles. These vesicles are found just below the surface of the conjunctival cells. They contain long chain mucus glycoprotein molecules that are joined to the vesicle membrane. The vesicles fuse with the surface membrane of the conjunctival cells and expose their mucus glycoprotein chains to the overlying mucus. Chemical and physical bonds between the two types of mucus help to bind the mucus layer to the conjunctiva. The vesicle membrane becomes incorporated in the cell membrane and supplies the membranes for the microvilli that cover the exposed surface.     

Structure and Biological Roles of Mucin-type O-glycans at the Ocular Surface

Mucins are major components in mucus secretions and apical cell membranes on wet-surfaced epithelia. Structurally, they are characterized by the presence of tandem repeat domains containing heavily O-glycosylated serine and threonine residues. O-glycans contribute to maintaining the highly extended and rigid structure of mucins, conferring to them specific physical and biological properties essential for their protective functions. At the ocular surface epithelia, mucin-type O-glycan chains are short and predominantly sialylated, perhaps reflecting specific requirements of the ocular surface. Traditionally, secreted mucins and their O-glycans in the tear film have been involved in the clearance of debris and pathogens from the surface of the eye. New evidence, however, shows that O-glycans on the cell-surface glycocalyx have additional biological roles in the protection of corneal and conjunctival epithelia, such as preventing bacterial adhesion, promoting boundary lubrication, and maintaining the epithelial barrier function through their interaction with galectin-3. Abnormalities in mucin-type O-glycosylation have been identified in many disorders where the stability of the ocular surface is compromised. This review summarizes recent advances in understanding the structure, biosynthesis, and function of mucin-type O-glycans at the ocular surface and their alteration in ocular surface disease.     

Marihuana-derived material: biochemical studies of the ocular responses

Some biochemical factors of the iris-ciliary body of the rabbit have been examined for effects induced by water-soluble marihuana-derived material (MDM). Adenylate cyclase activity and sensitivity to beta-adrenergic agonists were unchanged, as measured 4 hours after MDM administration in vivo. Magnesium-dependent and anion-sensitive, but not sodium-potassium, ATPase activities were inhibited 6 hours after MDM administration in vivo, although they were unaffected by in vitro incubation. Topical administration of a potent substance P antagonist had no effect on the time course or magnitude of intravenous MDM-induced ocular effects in rabbit. Intravenously administered sugars antagonized the effects of MDM on intraocular pressure. A variety of drugs which display a range of biochemical effects varying from beta-adrenergic receptor agonism, to alteration of glycoprotein residues were employed. None of the agents employed, ranging from cAMP modifiers to protein synthesis blockers, had any effect on the MDM-induced response. It is apparent that the mechanism underlying the ocular hypotensive effect of MDM does not reside in mediation through adenylate cyclase, ATPase or substance P, but rather through a mechanism mediated by terminal sugar moieties on the molecule. The data suggest that modification of the surface membrane glycoprotein residues on the ciliary epithelium can induce marked alterations in aqueous humor flow rate.