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 membrane 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 molecular 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 conjunctiva and cornea is nonkeratinizing, stratified, and
squamous and is made up of three to seven cell layers.
Theflattenedouter squames of the epithelium are covered at their apical membrane by the tear film, which
is generally considered to be subdivided—from air interface to epithelial apical membrane—into oil,
aqueous, and mucus layers. The mucus layer is secreted onto the ocular surface epithelium by goblet
cells in the conjunctival region. In guinea pigs, the
mucus layer varies in thickness from 1.0 nm over cornea to 2-7 jim over conjunctiva.1 As has been elegantly demonstrated in the electron micrographs of
the rapid-freeze, freeze-substitution-prepared ocular
surface epithelium of the guinea pig, the mucus layer
is intimately associated with the glycocalyx of the apical cell.' The glycocalyx is a carbohydrate-rich, extrinsic cell surface coat that forms a layer along the apical
membrane to which the mucus layer binds, presumably loosely. Electron microscopy of ocular surface
tissue stained with tannic acid demonstrates that the
glycocalyx is a fine, filamentous layer. Each filament
inserts into the cell membrane and has angular bends
and branches distally.2 These filaments are particularly prominent at the tips of the microplicae.
Very little is known about the biochemical nature
of the glycocalyx, and even less is known of its interaction with or role in the spread of mucus over the apical cells. That the glycocalyx contains many highly
charged polyanions is demonstrated by the intense
binding of ruthenium red tofixedtissue.23 Other studies demonstrate binding of Alcian blue, dialyzed iron,
cationized ferritin, periodic acid-Schiff reagent, and
several lectins to the ocular surface.4"8 These studies
indicate that the ocular surface is rich in carbohydrate
moieties, but they do not give specific molecular information nor do they differentiate totally between
glycocalyx and mucus layers.
We have produced a monoclonal antibody that
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 glycoprotein recognized by this antibody.
From the *Eye Research Institute, and the f Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Supported by grant R37-EY-03306 from the National Eye Institute, National Institutes of Health, Bethesda, Maryland.
Submitted for publication: April 29, 1991; accepted July 22,
1991.
Reprint requests: llene K. Gipson, Eye Research Institute, 20
Staniford Street, Boston, MA 02114.
Materials and Methods
All investigations involving animals reported in
this study conform to the ARVO Resolution on the
Use of Animals in Research. Adult male Sprague-
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OCULAR SURFACE GLYCOCALYX GLYCOPROTEIN / Gipson er ol
Dawley rats, New Zealand white rabbits, and Hartley
guinea pigs were used. Animals were sacrificed with
an overdose of sodium pentobarbital.
Monoclonal Antibody Production
Preparation of immunogen and immunization:
Apical cells of the corneas of adult Sprague-Dawley
rats (175-225 g) were obtained by gentle brushing of
corneas that had been excised, pinned on paraffin
posts, and incubated overnight in low-Ca2+ minimum
essential medium (MEM) (Gibco, Grand Island,
NY).9 The cells were centrifuged at 1,000 X g for 15
min. The cell pellet was resuspended in MEM with
10% dimethyl sulfoxide (Sigma, St. Louis, MO), frozen, and stored in liquid nitrogen until enough cells
were obtained for the immunizations. Prior to immunization, the cells were thawed in a 37°C water bath
and washed two or three times in MEM and once in
phosphate-buffered saline (PBS). The cells were resuspended in equal amounts of PBS and RIBI adjuvant
(RIBI; Immunochem Research, Hamilton, MT). One
times 107 apical cells prepared in this way were injected intraperitoneally into 6-week-old female
BALB/cByJ mice (Jackson Laboratories, Bar Harbor,
ME). A booster injection of 7.5 X 105 cells in PBS/
RIBI adjuvant was given one month later.
Cell fusion and hybridoma cloning: Four days after
the boost, cell fusion was carried out according to a
modification of the procedure of Kohler and Milstein.10 Briefly, spleen cells from an immunized
mouse were mixed with P3/NSl/l-Ag4-l(NS-l)
(ATCC, Rockville, MD) myeloma cells in a ratio of
5:1 in serum-free medium. Cells were centrifuged at
200 X g for 10 min at room temperature. The supernatant was gently removed and the tube transferred to
a 37°C water bath where 1 ml of 50% polyethylene
glycol (PEG) (Boehringer Mannheim Biochem, Indianapolis, IN) in 75raMHEPES (Gibco) was added.
After 1 min, the PEG was diluted out by adding 1, 2,
and 4 ml of serum-free Iscove's Modified Dulbecco's
Medium (IMDM; Gibco) after 1,2, and 4 min, respectively. The dilution was completed by adding 8 ml of
IMDM with 10% fetal calf serum (FCS). The cells
were centrifuged at 200 X g for 10 min and resuspended in 50 ml of IMDM plus FCS and HAT
(Sigma). One hundred microliters per well of this suspension was plated in flat-bottom, 96-well plates containing a feeder layer of BALB/cByJ mouse peritoneal
macrophages. After 7 days, 100 /A of HAT was added
to each well. After 2 weeks, the cultures were fed with
HT-containing medium and screened by ELISA for
IgG production using the Bio-Rad (Richmond, CA)
Clone Selector Mouse Monoclonal Antibody Screening Kit. Positive cultures were screened for hybridomas of interest on cryostat sections of rat corneas by
219
immunofluorescence (IF) microscopy. Hybridomas
with apical cell binding by IF were cloned by limiting
dilution (0.5 cell/well) two consecutive times. Antibodies from tissue culture medium were concentrated
by ammonium sulfate precipitation.
Immunofluorescence Localization
Six-micrometer cryostat sections of rat cornea, eyelid, skin, esophagus, lacrimal gland, oral mucosa,
liver, pancreas, ileum, lung, and colon were placed on
gelatin-coated slides and dried overnight at 37 °C. Sections were similarly prepared from guinea pig and
rabbit corneas and human corneas obtained from National Disease Research Interchange. Sections were
rehydrated in PBS, pH 7.2, and blocked in PBS with
1% bovine serum albumin (BSA) for 10 min. Primary
antibody (hybridoma tissue culture media or monoclonal antibody) was applied for 1 hr at room temperature in a moist chamber. Sections were rinsed with
PBS followed by 10 min in PBS with 1% BSA. Fluorescein isothiocyanate (FITC)-goat anti-mouse IgG (Calbiochem, La Jolla, CA) was similarly applied for 1 h at
room temperature. After a PBS wash, coverslips were
mounted with a medium consisting of PBS, glycerol,
and para-phenylenediamine." Negative control tissue sections (primary antibody omitted) were routinely included in each antibody-binding study. The
sections were viewed and photographed on a Zeiss
photomicroscope III (AZI, Avon, MA) equipped for
epi-illumination.
Neuraminidase treatment: Cryostat sections of rat
cornea were treated with 1.25 U/ml neuraminidase
isolated from Clostridium perfringens (Sigma, St.
Louis, MO) in PBS, pH 5.5, for 20, 40, or 60 min at
37°C.4 As a control, adjacent serial sections were simultaneously incubated in PBS, pH 5.5. Following
the neuraminidase or control buffer incubation, the
sections were rinsed in PBS, pH 7.2, and the normal
immunofluorescence labeling procedure was followed. The neuraminidase used for this treatment
was checked for contaminating protease activity using
the Bio-Rad Protease Detection Kit. Dispase II
(Boehringer-Mannheim, Indianapolis, IN), a neutral
bacterial protease, was used as the standard for the
assay. The neuraminidase was found to be free of protease activity at 62.5 U/ml (50 times the concentration used in the treatment).
Periodate treatment: The effect of strong, moderate, and mild oxidation on antibody binding to cryostat sections was examined using sodium periodate
(NaIO4), according to the method of Basbaum et al.12
Cryostat sections of rat cornea were incubated at
room temperature in the dark, overnight, in 100 mM
NaIO4 (strong oxidation; Sigma), at 4°C for 1 hr in 50
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1992
raM NaIO4 (moderate oxidation), or at 4°C for 10
min in 10 mM NaIO4 (mild oxidation). The NaIO4
solutions were made up in 50 mM sodium acetate, pH
4.5. As controls, adjacent sections were similarly incubated in the NaIO4 solution plus 0.1 M glucose or
0.015 (volume/volume) ethylene glycol. Following
the incubations in NaIO4, the sections were incubated
in 10 mM sodium borohydride (Sigma) in PBS, pH
7.2, for 30 min at room temperature. The sections
were rinsed well in five changes of PBS, and the usual
immunofluorescence labeling procedure was followed.
N-glycanase treatment: Cryostat sections of rat cornea were incubated in 60 U/ml N-glycanase peptide
—N-glycosidase F (Genzyme, Boston, MA) in 0.55 M
sodium phosphate, pH 8.6, overnight, at 44°C. As a
control, adjacent sections were incubated in 60 U/ml
N-glycanase in 30 mM citrate buffer, which inhibits
glycanase activity. Following the overnight incubation, the sections were rinsed well in PBS, pH 7.2, and
the usual immunofluorescence localization protocol
was followed.
Immunoelectron Microscopy (IEM)
Following fixation in 4% paraformaldehyde and
0.2% glutaraldehyde in 0.1 M PO4 buffer, pH 7.4, for
1 h at 4°C, corneas were rinsed in 0.1 M PO4 buffer
and cut into quarters. They were dehydrated in a
graded ethanol series, then were embedded in medium-grade LR White resin (London Resin Co.; Ernest F. Fullam, Latham, NY) in gelatin capsules followed by heat-curing at 50°C. Sections 1 nm thick
were stained with toluidine blue for orientation.
Thick sections also were mounted on gelatin-coated
slides for immunofluorescence localization (see protocol above) to verify antibody binding to the LR
White fixed and embedded tissue. Thin sections were
mounted on 3-mm, 200-mesh nickel grids (Ernest F.
Fullam). Rat apical monoclonal antibodies were localized using post-embedding immuno-gold labeling following the protocol described by the manufacturers of
the gold-conjugated secondary antibody (Janssen,
Ted Pella, Redding, CA). Janssen Auroprobe One affinity-purified, goat anti-mouse IgG antibody linked
to 1-nm colloidal-gold particles (Ted Pella) was used
as the secondary antibody. Gold signal was visualized
by silver amplification with IntenSE M according to
the Janssen protocol (Ted Pella).
Electrophoresis and Immunoblotting
Limbal to limbal corneal epithelium was scraped
from rat eyes and solubilized in 500 /xl of 7.5 mM
Tris, pH 8.9, 12.5% glycerol, 0.05% SDS, 5 mM urea
solution run through 18 G, 20 G, 21 G, 25 G, and 27
G needles, and then homogenized with two or three
Vol. 03
30-sec pulses of the polytron (Brinkmann Instruments, Westbury, NY). The solution was diluted to a
protein concentration of 1.6 /ig/Vl (via Bio-Rad protein assay) with 2X reaction mix (60 mM Tris, 0.25%
glycerol, 0.5% SDS, 45 mM dithiothreitol, 4 mM
urea). SDS-PAGE was performed using the buffer system described by Miles Laboratories,13 which is an
adaptation of Jovin's discontinuous (multiphasic)
buffer system14 using the mini-protean II dual slab gel
apparatus from Bio-Rad. Gels 0.75 mm thick, 6% (6%
T, 2.75% C), were run at constant voltage of 200 V for
1.25 hr using reagents from Bio-Rad. Prestained molecular weight markers included myosin (205 kD) and
b-galactosidase (118 kD). After the SDS-PAGE, gels
were stained by a modification of an Alcian bluesilver staining method designed to stain highly glycosylated glycoproteins that will not stain by Coomassie
Blue or silver.15 Gels were fixed and stained with Alcian blue15 followed by silver staining using the protocol of Wray et al.16
Proteins in gels were transferred to nitrocellulose
paper as described by Towbin et al.17 Blotted antigens
then were detected using the Vectastain elite mouse
IgG kit (Vector Labs, Burlingame, CA) and the manufacturer's protocol with several exceptions. Tween-20
(Sigma) was eliminated from the blocking buffer because it was determined that it interfered with antibody binding. Ten percent horse serum (Gibco) in
Tris-buffered saline, pH 7.5, was used as a blocking
agent during initial blocking of unbound nitrocellulose binding sites, as well as during primary and secondary antibody incubations. The NaCl concentration in the ABC reagents was increased to 0.5 M to
decrease nonspecific staining.
Periodate oxidation: To determine if periodate oxidation removed the antibody binding to immunoblots, blotted nitrocellulose strips were soaked in 50
mM sodium acetate, pH 4.5, for 5 min and transferred to 0.1, 1,5, 10, or 20 mM NaIO4 (Sigma), pH
4.5, for 1 hr in the dark at 23°C, according to the
technique of Woodward et al.18 Antigens then were
detected using the Vectastain kit as already described.
Results
Two fusions (one did not yield hybridomas of interest) yielded 12 cell lines. Supernatant from these lines
yielded several types of antibodies. Eight of the 12
supernatants bound apical cells. We selected one hybridoma specific for apical cells for further cloning.
Several clones from the hybridoma were selected for
characterization. Each clone from the same hybridoma had similar characteristics, and we believe they
all recognize the same epitope. The data reported here
are from monoclonal antibody 13G5.339. In the re-
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OCULAR SURFACE GLYCOCALYX GLYCOPROTEIN / Gipson er al
suits described below, we refer to the antibody as rat
ocular surface glycocalyx antibody, or ROSG antibody.
By immunofluorescence microscopy, we found
that binding of antibody was to all the flattened cell
layers or squames along the entire ocular surface epithelium (Fig. 1A). Binding ended abruptly where the
epithelium becomes keratinized at the lid margin
(Fig. 1C). Goblet cells within the conjunctival epithelium also bound the antibody (Fig. IB). Lacrimal
gland did not bind the antibody, but an occasional
duct cell did (data not shown). Binding was not found
in the other species tested (rabbit, guinea pig, human)
nor in other rat tissues tested, including skin, esophagus, oral mucosa, liver, pancreas, ileum, lung, and
colon (data not shown).
Immunoelectron microscopic localization studies
showed that in the outer squames adjacent to the tear
film, binding was in the outer apical membrane (Fig.
2A). At higher magnification, binding was particularly prominent at the tips of the microplicae (Fig.
2B). The antigen was present within small vesicles in
the cytoplasm of the squames below the apical cell
221
(Figs. 2A, B). In the cytoplasmic vesicles, the binding
appeared to be along the internal face of the vesicle
membrane (Fig. 2C). These vesicles that bind the antibody occur only in flattened squames of the ocular
surface epithelium. There was variability in the
amount of gold label present on apical cells (compare
Figs. 2A and D). In a loosened cell that appears to be
desquamating (Fig. 2D), there is no binding to its apical surface. The cell beneath the loosened cell shows
binding along its apical membrane.
In rats, goblet cells occur in groups or clusters of
cells that in some respects have the appearance of
acini. Immunoelectron microscopy of the goblet cell
cluster showed that only some of the goblet cells of the
cluster bind the antibody. In Figure 3, three adjacent
cells of a cluster of goblet cells show different binding
patterns. A cell in the center of the cluster shows no
binding; an adjacent cell shows binding in the Golgi
region outside the mucin granules, and adjacent to
this cell near the outer edge of the cluster, binding is
present on the mucin granules. Study of the localization along mucin granules showed a prevalence of
binding to the membrane region of the granule (Fig.
Fig. 1. Immunofluorescence micrographs
demonstrating localization of the ROSG antibody in the corneal epithelium (A) and conjunctival epithelium (B). Binding in the cornea is present on the several layers of apical
flattened squames. In the conjunctiva, in addition to apical squame binding, intense binding to goblet cells is present. (C) The abrupt
end to binding is seen where the nonkeratinized ocular surface epithelium joins keratinized epidermis at the lid margin. The hair follicle of the eyelash is seen at the upper left
between arrows (A-C, X300).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1992
Vol. 03
>;
I
i
•••.
Fig. 2. Immunoelectron microscopic localization of the ROSG antibody in corneal epithelium. (A) At low magnification, the silver-enhanced 1 -nm immunogold can be seen along the apical tear-facing membrane of the apical cell. In subapical cells, the label is present within the
cytoplasm of the cells. Arrows indicate cell boundaries of subapical cells (X7200). (B) Higher magnification electron micrograph demonstrating prevalence of antibody binding on microplicae of apical cells. The membrane of the abutting subapical cell is indicated by the arrows
(X21,300). (C) Higher magnification electron micrograph showing labeled cytoplasmic vesicles of subapical cells; the label is particularly
prevalent on the vesicle membrane (arrows) (X21,300). (D) Variation in amount of binding to apical cells was noted. In this electron
micrograph of a loosely adherent cell, there is no apical membrane binding. The cell beneath has binding in its apical membrane (x21,300).
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OCULAR SURFACE GLYCOCALYX GLYCOPROTEIN / Gipson er ol
223
B
Fig. 3. (A) Immunoelectron microscopic localization of the ROSG antibody on a cluster of goblet cells. The cell in the center of the cluster
(1) is not labeled, even though it has mucin granules. In the adjacent cell (2), the antigen is localized to the Golgi apparatus (arrows) between
mucin granules. The cell at the periphery of the cluster (3) has intensely labeled mucin granules (X9000). The inset shows these mucin granules
at higher magnification. The label appears to be more prevalent along the peripheral membrane region of the granule (X36.000). (B) Electron
micrograph of epithelium processed as a secondary antibody control in which incubation with primary antibody was omitted (X9000).
3A, inset). Some binding is in the "lumen" region
above the goblet cell cluster. Because secondary antibody controls (Fig. 3B) appear free of gold, the luminal binding may be from secretory products in the
lumen.
In developing the post-embedding technique for
immunoelectron microscopic localization of the antigen, we found that although we preserved antigenicity
after fixation and embedding in LR White resin (as
judged by immunofluorescence localization on the
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / January 1992
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1-^m sections), we could not label with secondary antibodies conjugated to 5-nm gold particles. Only secondary antibodies conjugated to 1-nm immuno-gold
followed by silver enhancement allowed localization
at the electron microscope level.
Electrophoretic Mobility and Immunoblots
By immunoblot analysis, the ROSG antibody
reacted with a prominent band that has a molecular
weight greater than 205,000 (Fig. 4). Coomassie or
silver staining of the gel in that region demonstrated
no band of similar molecular weight. Periodic acidSchiff reagent did stain a band in the same region
(data not shown) as did Alcian blue followed by silver
staining (Fig. 4). These data plus the observation that
the reactive band was more diffuse at lower acrylamide concentrations (6% compared to 10-12%) suggest
that the antigen is a highly glycosylated glycoprotein.
Because highly glycosylated glycoproteins run anomalously on SDS-PAGE, an accurate molecular weight
determination was not possible.
Periodate Incubation
To determine whether the epitope of the antigen
recognized by the ocular-surface monoclonal antibody was to the carbohydrate portion of the glycoprotein, periodate oxidation of immunoblots and frozen
sections were done. The effect of increasing concentrations of periodate on binding of ROSG monoclonal antibody to immunoblots is shown in Fig. 5.
Treatment of immunoblot with 5 and 10 mM periodate for 1 hr completely removed antibody binding.
At 1 mM periodate, binding was partially removed,
and at 0.1 mM, binding was similar to that of the
control incubation. Treatment of cryostat sections
with increasing periodate concentrations and incuba-
O.I
77 —
Fig. 4. Left lane, 6% SDS-PAGE of cornea! epithelium stained
with alcian blue followed by silver. Right lane, immunoblot showing antibody binding to a band of similar molecular weight. Molecular masses (in kilodaltons) determined from standard proteins
(myosin, 205; /3-galactosidase, 118; bovine serum albumin, 77) are
noted.
5.0
10*0
mM Periodate
Fig. 5. Blots of 6% SDS-PAGE treated with increasing concentrations of sodium periodate to remove carbohydrate. The arrow indicates remaining reactive bands in the control and low periodate
concentration lanes. At higher concentrations, binding is completely lost.
tion time also showed a dose-, and treatment time-,
dependent removal of antibody binding (Fig. 6).
Under mild periodate oxidation conditions (10 mM,
1 hr incubation), partial binding remained. Under
moderate (50 mM, 1 hr) or strong (100 mM, overnight) oxidation conditions, all binding was lost.
Enzyme Treatments
Having evidence that the epitope recognized by
ROSG monoclonal antibody is carbohydrate, two
glycosidases were used on cryostat sections to determine whether antibody binding was lost after incubation in their presence. Neuraminidase treatment at
1.25 U/ml for increasing periods of incubation up to 1
hr did not affect antibody binding (data not shown).
Incubation of sections with N-glycanase to remove
asparagine-linked glycoproteins abolished antibody
binding as compared to controls (Fig. 7).
205 —
118.
1.0
Discussion
Our immunohistochemical and immunoelectron
microscopy findings suggest that the antigen recognized by the ROSG antibody is a component of the
glycocalyx of the ocular surface epithelium. That it is
a glycocalyx component rather than a goblet cell mucin product is supported by these lines of evidence: (1)
the antigen is localized in the cytoplasm in subapical
squames; (2) fixation does not remove the antigen
from the surface of the eye. Nichols et al1 have shown
that the mucus layer is not preserved by conventional
fixation for electron microscopy; and (3) incubation
of cryostat sections with N-glycanase destroys anti-
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OCULAR SURFACE GLYCOCALYX GLYCOPROTEIN / Gipson er ol
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Fig. 6. Immunofluorescence micrographs of cryostat sections treated with 100 mM (A; region between arrows is epithelium) and 10 mM (B)
sodium periodate. Control incubations had ethylene glycol (C) or glucose (D) added to incubation solutions. (A-D, X75O).
body binding. These data suggest that the epitope recognized by the ROSG antibody is on an N-linked sugar chain. In mucins, sugar chains are attached to the
central core protein primarily through O-glycosidic
linkage.19
An argument could be made that the binding of the
antibody to goblet cells refutes the claim that the antigen recognizes an N-linked glycocalyx component.
Maybe the sugar epitope is common to cell surface
glycoprotein and a goblet cell mucin. The pattern of
immunoelectron microscopic localization of the antigen in the goblet cell and the Western blot data suggest that the goblet cell binding is to a nonmucin component of the mucin granule. Binding of the antibody
in clusters of goblet cells varies among cells of the
cluster, with Golgi binding and no mucin packet binding in some cells and mucin packet membrane binding in others. This suggests that the antigen is added to
the mucin granule late in the maturation of the goblet
cell, after mucin production and packaging into granules. The predilection to bind to the granule membrane may be similar to that on the apical membrane
of the apical cells of the stratified epithelium. The mucin granule membrane and the apical membrane of
the epithelium are assumed to require a loose association of mucus to them. In the case of the mucin gran-
ule, loose association would be required to allow mucin discharge from the granule and its membrane.
Loose association of the mucus coat to the apical
membrane is presumed necessary to allow movement
of the mucus to act as a debris removal system for the
ocular surface much as it acts in the trachea.20 Perhaps antigen recognized by our antibody is a glycocalyx component that facilitates such loose association.
If mucins and glycocalyx glycoproteins bound the antibody, binding to several protein bands could be anticipated. By Western blot analysis, only one reactive
band was present.
In the electron microscopic localizations of the antigen in the apical-most cell glycocalyx, we noted a
difference among cells regarding the amount of antigen detected. The cells that were less adherent to underlying cells and that had the appearance of cells
about to desquamate bound less antibody. Perhaps as
cells age, glycocalyx components are lost from the cell
surface by movement of the mucus layer along the
microplicae during blinking or by active shedding
from the cell surface. As the cell ages and loses its
mucin-interacting glycoproteins of the glycocalyx,
perhaps the mucus layer sticks more tightly to the cell,
inducing or facilitating desquamation. Wells and
Hazlett have reported an increase in mucus on "dark"
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INVESTIGATIVE OPHTHALMOLOGY & VI5UAL SCIENCE / January 1992
Fig. 7. Immunofluorescence micrographs of section treated with
N-glycanase (A) or N-glycanase plus citrate (B) as control. Antibody binding is lost in (A), where epithelium is barely visible between arrows (A, B, X370).
cells of the ocular surface.4 By scanning electron microscopy, "light, medium, and dark" cells have been
noted,421 and these authors postulate that dark cells
are the "oldest" cells on the ocular surface. If there is
loss of glycocalyx components from cells into the tear
film as the cells age, their presence in tear fluid presumably would be detectable. In preliminary studies,
we have detected antibody binding to rat tear samples
obtained with a capillary pipette and dried onto glass
slides. In another tissue, surface glycoconjugates from
ciliated cells have been demonstrated to be components of the tracheal "mucus" secretions.22
The immunoelectron microscopy data on localization in the stratified ocular surface epithelium show
that most of the antigen in the apical-most cell is in
the glycocalyx with little cytoplasmic binding. Because the antigen appears within small, membranebound vesicles within subapical squames, a signal to
move the vesicles to the membrane must be generated
as the cell takes up its apical-most position adjacent
to the tear film. That signal may be tight junction formation. In simple columnar epithelium, the tight
junction is known to be the structure along the cell
membrane that segregates the apical membrane components from basolateral components.23 The establishment of apical-basal polarity through cell-substrate and cell-cell contact leads to tight junction for-
Vol. 33
mation. Formation of the tight junction is known to
be responsible for the targeting of secretory products
or membrane components to the appropriate pole of
the cell.23 Numerous examples of such targeting have
been provided for products of simple epithelium.23 To
our knowledge, no such movement has been demonstrated for stratified squamous epithelia.
Apically targeted membrane components of simple
epithelia, including MDCK cells, thyroid epithelia,
and colonic epithelia, are stored within a novel vacuolar apical compartment termed VAC.24 Initiation of
cell-cell contact induces rapid formation of tight junctions formation in cultured MDCK cells. Upon cellcell contact, the VAC is exocytosed toward the region
of cell-cell contact, where it contributes significantly
to the formation of the apical surface.24 Culture conditions that prevent cell-cell contact, ie, culture in low
Ca+2, prevent VAC exocytosis; with increased Ca"1"2
concentrations, cell-cell contact is initiated and VAC
exocytosis ensues.25
Perhaps the subapical squames of the ocular surface
epithelium store apical membrane or glycocalyx components in a VAC, and perhaps movement of the
VAC to the apical membrane occurs as the tight junction forms between apical squames. Vinculin, a tight
junction component, has been localized to the region
of contact between apical cells of the corneal epithelium of the rat.26 Verification of tight junction induction of movement of the glycocalyx glycoprotein recognized by the ROSG antibody to the ocular surface
awaits double-labeling experiments that will allow
correlation of junction formation with movement of
vacuoles to the apical surface.
Movement of vesicles to the surface of apical conjunctival epithelial cells has been proposed.52627
Greiner et al5 suggested that vesicle delivery provided
a second source of mucin to the ocular surface. The
stains these investigators used to follow the vesicles
bind highly glycosylated molecules of either N- or Olinkage. Whether the products carried within the vesicles are cell surface glycocalyx components or mucins
remains to be determined. Possibly the antigen detected by our ROSG antibody is the highly glycosylated molecule detected by these investigators.
The antigen detected by immunoblot analysis in
our study has a molecular weight greater than 205 kD,
just entering a 6% gel. In an SDS-PAGE analysis of
individual ocular mucus samples from normal and
diseased human conjunctivas, high-molecular-weight
glycoproteins were found in 2-16% gradient gels.28
Although the two gel systems in these studies are different, comparing the data is tempting. The most prevalent band within the human gels, GP2, appears at
approximately the same region of the gel as the reactive band in our immunoblots.
The Western blot data, the Alcian blue-silver stain
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No. 1
OCULAR SURFACE GLYCOCALYX GLYCOPROTEIN / Gipson er ol
of the SDS-PAGE gels, and the periodate oxidation
data in this report suggest that the antigen recognized
by the ROSG antibody is a highly glycosylated glycoprotein greater than 205 kD in molecular weight. In
addition, the epitope recognized by the antibody is to
a sugar portion of the molecule. The immunohistochemical data suggest that the sugar epitope is not
sialic acid and that it is an N-linked sugar. These later
studies are, however, not definitive. Attempts to confirm the immunohistochemical data by Western blot
procedures were unsuccessful. Because such protocols
are more successful with purified glycoproteins, definitive classification of the glycoprotein into cell surface or mucin categories awaits purification of the antigen.
In summary, we have developed a monoclonal antibody that recognizes a sugar epitope on a high-molecular-weight, highly glycosylated glycoprotein found
in the ocular surface glycocalyx of the rat. Further
characterization of the glycoprotein may yield information relevant to the biological properties of the ocular surface, such as mucin spread and resistance to
pathogen adherence.
Key words: ocular surface, glycocalyx, tear film spread,
monoclonal antibody, goblet cells
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