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Characteristics of a Glycoprotein in the Ocular Surface Glycocalyx

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Characteristics of a Glycoprotein in the Ocular Surface Glycocalyx Investigative Ophthalmology & Visual Science, Vol. 33, No. 1, January 1992 Copyright © Association for Research in Vision and Ophthalmology Characteristics of a Glycoprotein in the Ocular Surface Glycocalyx llene K. Gipson,*t Michelle Yankauckas,* Sandra J. Spurr-Michaud,* Ann S. Tisdale,* and William Rineharr* A monoclonal antibody has been produced that binds to the apical squames (flattened cells) of the rat ocular surface epithelium and to the goblet cells of the conjunctiva. Immunoelectron microscopic localization of the antigen indicates that in apical cells it is present along the apical-microplical mem- brane in the region of the glycocalyx. In subapical squames, the antigen is in cytoplasmic vesicles. In some goblet cells, the antigen is in the Golgi network, and in others, it is located primarily in the membrane of the mucous granules. SDS-PAGE and immunoblot analysis demonstrate that the molecu- lar weight of the antigen is greater than 205 kD, and the electrophoretic band stains with Alcian blue followed by silver stain. Periodate oxidation of immunoblots and cryostat sections removes antibody binding. Neuraminidase treatment of cryostat sections does not remove antibody binding, whereas N-glycanase does. Taken together, these data indicate that the antigen recognized by the monoclonal antibody is a carbohydrate epitope on a high-molecular-weight, highly glycosylated glycoprotein in the glycocalyx of the ocular surface epithelium and goblet cell mucin granule membrane. The antigen appears to be stored within cytoplasmic vesicles and reaches the glycocalyx when cells differentiate to the apical-most position where the glycocalyx interfaces with the mucin layer of the tear film. Invest Ophthalmol Vis Sci 33:218-227,1992 The ocular surface epithelium that covers the con- tissue stained with tannic acid demonstrates that the junctiva and cornea is nonkeratinizing, stratified, and glycocalyx is a fine, filamentous layer. Each filament squamous and is made up of three to seven cell layers. inserts into the cell membrane and has angular bends The flattened outer squames of the epithelium are cov- and branches distally.2 These filaments are particu- ered at their apical membrane by the tear film, which larly prominent at the tips of the microplicae. is generally considered to be subdivided—from air in- Very little is known about the biochemical nature terface to epithelial apical membrane—into oil, of the glycocalyx, and even less is known of its interac- aqueous, and mucus layers. The mucus layer is se- tion with or role in the spread of mucus over the api- creted onto the ocular surface epithelium by goblet cal cells. That the glycocalyx contains many highly cells in the conjunctival region. In guinea pigs, the charged polyanions is demonstrated by the intense mucus layer varies in thickness from 1.0 nm over cor- binding of ruthenium red to fixed tissue.23 Other stud- nea to 2-7 jim over conjunctiva.1 As has been ele- ies demonstrate binding of Alcian blue, dialyzed iron, gantly demonstrated in the electron micrographs of cationized ferritin, periodic acid-Schiff reagent, and the rapid-freeze, freeze-substitution-prepared ocular several lectins to the ocular surface.4"8 These studies surface epithelium of the guinea pig, the mucus layer indicate that the ocular surface is rich in carbohydrate is intimately associated with the glycocalyx of the api- moieties, but they do not give specific molecular in- cal cell.' The glycocalyx is a carbohydrate-rich, extrin- formation nor do they differentiate totally between sic cell surface coat that forms a layer along the apical glycocalyx and mucus layers. membrane to which the mucus layer binds, presum- We have produced a monoclonal antibody that ably loosely. Electron microscopy of ocular surface binds to apical cells of the ocular surface epithelium of the rat and that appears to recognize a component of the glycocalyx. We have begun to characterize the gly- From the *Eye Research Institute, and the f Department of Oph- coprotein recognized by this antibody. thalmology, Harvard Medical School, Boston, Massachusetts. Supported by grant R37-EY-03306 from the National Eye Insti- tute, National Institutes of Health, Bethesda, Maryland. Materials and Methods Submitted for publication: April 29, 1991; accepted July 22, 1991. All investigations involving animals reported in Reprint requests: llene K. Gipson, Eye Research Institute, 20 this study conform to the ARVO Resolution on the Staniford Street, Boston, MA 02114. Use of Animals in Research. Adult male Sprague- 218 Downloaded from iovs.arvojournals.org on 09/27/2021 No. 1 OCULAR SURFACE GLYCOCALYX GLYCOPROTEIN / Gipson er ol 219 Dawley rats, New Zealand white rabbits, and Hartley immunofluorescence (IF) microscopy. Hybridomas guinea pigs were used. Animals were sacrificed with with apical cell binding by IF were cloned by limiting an overdose of sodium pentobarbital. dilution (0.5 cell/well) two consecutive times. Anti- bodies from tissue culture medium were concentrated Monoclonal Antibody Production by ammonium sulfate precipitation. Preparation of immunogen and immunization: Apical cells of the corneas of adult Sprague-Dawley Immunofluorescence Localization rats (175-225 g) were obtained by gentle brushing of corneas that had been excised, pinned on paraffin Six-micrometer cryostat sections of rat cornea, eye- posts, and incubated overnight in low-Ca2+ minimum lid, skin, esophagus, lacrimal gland, oral mucosa, essential medium (MEM) (Gibco, Grand Island, liver, pancreas, ileum, lung, and colon were placed on NY).9 The cells were centrifuged at 1,000 X g for 15 gelatin-coated slides and dried overnight at 37 °C. Sec- min. The cell pellet was resuspended in MEM with tions were similarly prepared from guinea pig and 10% dimethyl sulfoxide (Sigma, St. Louis, MO), fro- rabbit corneas and human corneas obtained from Na- zen, and stored in liquid nitrogen until enough cells tional Disease Research Interchange. Sections were were obtained for the immunizations. Prior to immu- rehydrated in PBS, pH 7.2, and blocked in PBS with nization, the cells were thawed in a 37°C water bath 1% bovine serum albumin (BSA) for 10 min. Primary and washed two or three times in MEM and once in antibody (hybridoma tissue culture media or mono- phosphate-buffered saline (PBS). The cells were resus- clonal antibody) was applied for 1 hr at room tempera- pended in equal amounts of PBS and RIBI adjuvant ture in a moist chamber. Sections were rinsed with (RIBI; Immunochem Research, Hamilton, MT). One PBS followed by 10 min in PBS with 1% BSA. Fluores- times 107 apical cells prepared in this way were in- cein isothiocyanate (FITC)-goat anti-mouse IgG (Cal- jected intraperitoneally into 6-week-old female biochem, La Jolla, CA) was similarly applied for 1 h at BALB/cByJ mice (Jackson Laboratories, Bar Harbor, room temperature. After a PBS wash, coverslips were ME). A booster injection of 7.5 X 105 cells in PBS/ mounted with a medium consisting of PBS, glycerol, RIBI adjuvant was given one month later. and para-phenylenediamine." Negative control tis- Cell fusion and hybridoma cloning: Four days after sue sections (primary antibody omitted) were rou- the boost, cell fusion was carried out according to a tinely included in each antibody-binding study. The modification of the procedure of Kohler and Mil- sections were viewed and photographed on a Zeiss stein.10 Briefly, spleen cells from an immunized photomicroscope III (AZI, Avon, MA) equipped for mouse were mixed with P3/NSl/l-Ag4-l(NS-l) epi-illumination. (ATCC, Rockville, MD) myeloma cells in a ratio of Neuraminidase treatment: Cryostat sections of rat 5:1 in serum-free medium. Cells were centrifuged at cornea were treated with 1.25 U/ml neuraminidase 200 X g for 10 min at room temperature. The super- isolated from Clostridium perfringens (Sigma, St. natant was gently removed and the tube transferred to Louis, MO) in PBS, pH 5.5, for 20, 40, or 60 min at a 37°C water bath where 1 ml of 50% polyethylene 37°C.4 As a control, adjacent serial sections were si- glycol (PEG) (Boehringer Mannheim Biochem, In- multaneously incubated in PBS, pH 5.5. Following dianapolis, IN) in 75 raM HEPES (Gibco) was added. the neuraminidase or control buffer incubation, the After 1 min, the PEG was diluted out by adding 1, 2, sections were rinsed in PBS, pH 7.2, and the normal and 4 ml of serum-free Iscove's Modified Dulbecco's immunofluorescence labeling procedure was fol- Medium (IMDM; Gibco) after 1,2, and 4 min, respec- lowed. The neuraminidase used for this treatment tively. The dilution was completed by adding 8 ml of was checked for contaminating protease activity using IMDM with 10% fetal calf serum (FCS). The cells the Bio-Rad Protease Detection Kit. Dispase II were centrifuged at 200 X g for 10 min and resus- (Boehringer-Mannheim, Indianapolis, IN), a neutral pended in 50 ml of IMDM plus FCS and HAT bacterial protease, was used as the standard for the (Sigma). One hundred microliters per well of this sus- assay. The neuraminidase was found to be free of pro- pension was plated in flat-bottom, 96-well plates con- tease activity at 62.5 U/ml (50 times the concentra- taining a feeder layer of BALB/cByJ mouse peritoneal tion used in the treatment). macrophages. After 7 days, 100 /A of HAT was added Periodate treatment: The effect of strong, moder- to each well. After 2 weeks, the cultures were fed with ate, and mild oxidation on antibody binding to cryo- HT-containing medium and screened by ELISA for stat sections was examined using sodium periodate 12 IgG production using the Bio-Rad (Richmond, CA) (NaIO4), according to the method of Basbaum et al. Clone Selector Mouse Monoclonal Antibody Screen- Cryostat sections of rat cornea were incubated at ing Kit.
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