From the New England Eye Center and Departments of
Ophthalmology,
Biochemistry, and § Pathology, Tufts
University School of Medicine, Boston, Massachusetts 02111 and the
¶ Department of Cell Biology, Albert Einstein College of Medicine,
New York, New York 10461
Received for publication, October 23, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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In this study we demonstrate that in
corneal epithelium there is cell-cell contact-regulated expression of a
145-kDa glycoprotein (GP) bearing the glycan determinant
Lewisx (Lex)
(Gal Corneal epithelium is a prototype-stratified squamous epithelium.
Its major function is to provide a barrier against fluid loss and
pathogen entrance. This function requires the cells of the epithelium
to remain tightly adherent to one another, as well as to the underlying
basal lamina. The structural integrity of corneal epithelium is
requisite for normal vision. Abnormality or loss of corneal epithelial
cell-cell adhesion may lead to the development of a number of ocular
surface disorders including corneal epithelial dysplasia and
dysmaturation (1-3), corneal epithelial hyperplasia, and corneal
epithelial in-growth, which is a rare but disastrous complication that
can occur following intraocular surgery (4-6). In many of these
conditions, enlarged intercellular spaces may be seen ultrastructurally
in the epithelium (1-6).
Numerous studies on skin epithelium have shown that the loss of
cell-cell adhesions in the epidermis produces life-threatening blistering skin diseases known as pemphigus foliaceus and pemphigus vulgaris (7-9). An understanding of the molecular mechanisms of
epithelial cell-cell adhesion is also crucial to the understanding of
complications relating to the failure of re-epithelialization of
wounds. In response to injury, cells at the leading edge undergo a
phenotypic conversion characterized by a dramatic reorganization of the
cytoskeleton, disruption of stable intercellular adhesion, and
redistribution of adhesion-related molecules. In fact, the breakage of
the stable intercellular contacts is prerequisite for initiating the
phase of re-epithelialization. Following re-epithelialization, reversion to the epithelial phenotype including the reformation of
stable intercellular contacts must occur if the function of the
epithelium is to be fully restored. The molecules that mediate cell-cell adhesion contacts also play crucial roles in important biological processes such as tissue morphogenesis and cell
differentiation (reviewed in Refs. 9-12).
In normal, intact epithelium, intercellular adhesion is brought about
by a variety of cell adhesion molecules, including cadherins, desmogleins, and desmocollins (7-12). Cell-cell adhesion might also be
mediated by the carbohydrate determinant Lewisx
(Lex,1
Gal Preparation of Rabbit Corneal Epithelial Cell
Cultures--
Rabbit corneal epithelial cells were grown in tissue
culture using eyes from Pel-Freez Biologicals (Rogers, AK) as described previously (21, 22). Sparse/exponentially growing cultures and
confluent/contact-inhibited cell cultures were analyzed. Sparse cultures were collected 3-4 days after starting the culture at which
time 30-50% of the culture dish was populated with rapidly dividing
cells that were migrating away from explants (Fig. 1). Confluent
cultures were collected 10-12 days after starting the culture when
90-95% of each dish was populated with tightly packed, polygonal
cells (Fig. 1).
Western Blot Analysis of Lex-Glycoproteins of
Confluent and Sparse Cultures of Corneal Epithelium--
mAb MMA,
which is specific for Lex
(Gal
To determine whether glycoproteins containing precursor,
non-fucosylated, lactosamine glycans were expressed in corneal
epithelium, Western blot analysis was performed using mAb 1B2 that
reacts with terminal (Gal Immunohistochemical Localization of Lex in Confluent
Cultures of Corneal Epithelium--
Explant cultures of corneal
epithelium were grown to confluence on coverslips. For immunostaining,
cells were fixed in 4% paraformaldehyde (15 min) and were then
sequentially treated with 3% H2O2 (10 min) to
block endogenous peroxidase and then 2.5% goat serum in PBS (20 min),
primary anti-Lex antibody (MMA, undiluted hybriodoma fluid;
anti-SSEA-1, 20 µg/ml (2 h)), and fluorescein isothiocyanate
anti-mouse goat IgM (1:400 to 1:800 (1 h)). Fluorescence was visualized
in a Leica confocal laser scanning microscope (Exton, PA). The images
were acquired using Tcsnt software from Leica.
Cell-Cell Adhesion Assay--
In this study, two different
anti-Lex antibodies, mAbs MMA (23) and anti-SSEA-1 (26),
were used. mAb CSLEX-1, which binds to sialyl-Lex (27),
served as control. One milligram each of mAb anti-SSEA-1 and CSLEX-1
were provided by Dr. Barry Potvin (Albert Einstein College of Medicine,
New York). mAb MMA (15 mg) was produced using the CELLMAX System
(Cellco, Spectrum Labs, Rancho Dominguez, CA) in media consisting of
DMEM and 5% fetal bovine serum. The antibody concentrations were
measured by radial immunodiffusion using the mouse IgM-NL radial
immunodiffusion kit (The Binding Site Inc., San Diego, CA). Prior to
use, all antibodies were dialyzed against EMEM/F-12 (1:1) and were
diluted as needed using dialyzed EMEM/F-12 containing 5% fetal bovine
serum (MFS). To determine the effect of anti-Lex mAbs on
cell-cell adhesion, primary cultures of rabbit corneal epithelium were
trypsinized and were plated in 24-well plates (3 × 105 cells/well in 0.6 ml) in SHEM media (22). After
allowing the cells to adhere for 3 h, they were rinsed with
EMEM/F-12 (1:1) and incubated overnight in the MFS media containing mAb
MMA or anti-SSEA-1 (0.8 mg/ml). At the end of the incubation period, the cells were washed with PBS, fixed with 1% glutaraldehyde, and
evaluated under a phase-contrast microscope for the presence of round
cells with intercellular spaces instead of tightly packed contact-inhibited polygonal cells. Control experiments involved incubation of the cells in MFS medium alone or in the medium containing mouse IgM (Calbiochem) or mAb CSLEX-1 (0.8 mg/ml). To determine whether
the effect of the antibody was dose-dependent, cultures were incubated with varying concentrations of mAb MMA (0.05-0.8 mg/ml).
In one experiment, to determine whether anti-Lex mAb was
able to disrupt a contact-inhibited monolayer, mAb MMA was added to tightly packed monolayer cultures of corneal epithelium. This assay was
carried out as described above except that cell cultures in 24-well
plates were incubated overnight instead of for 3 h in serum
containing SHEM media to obtain monolayers consisting of tightly packed
polygonal cells. These monolayers were rinsed with EMEM/F-12 (1:1) and
were then incubated with MFS medium in the presence and absence of mAb
MMA (0.05 to 0.8 mg/ml) and IgM (0.8 mg/ml) for 20-24 h. The cultures
were then evaluated under a phase-contrast microscope for cell rounding
and lifting of cell clumps from the culture dish.
RNA Isolation--
Poly(A)+ RNA from confluent and
sparse cultures of corneal epithelium and from Chinese hamster ovary
cell mutants LEC11 (28) and LEC30 (29) was isolated using FastTrack 2.0 kit mRNA Isolation System (Invitrogen, Carlsbad, CA). The RNA
preparation was treated with RQ, RNase-free DNase (2 units/µg RNA,
Promega, Madison, WI) for 15 min at 37 °C, and RNA was repurified by
phenol/chloroform extraction and ethanol precipitation. The yield of
poly(A)+ RNA was ~25 µg/108 cells.
Genomic DNA Preparation--
One gram of fresh liver (mouse or
rabbit) was cut into small pieces and was incubated with 12 ml of
digestion buffer consisting of 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 0.5% SDS, and
0.1 mg/ml proteinase K for 16 h at 55 °C. The digest was
extracted twice with saturated phenol and twice with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). DNA was precipitated by
the addition of 2 volumes of 7.5 M sodium acetate and 2 volumes of 100% ethanol. After rinsing the precipitate with 70%
ethanol, genomic DNA was dissolved in TE buffer and stored at
4 °C.
RT-PCR Amplification of Fut Genes Expressed by Rabbit Corneal
Epithelium--
For RT-PCR amplification, the Access RT-PCR System of Promega (Madison,
WI) was used. This is a single tube, two-enzyme system in which reverse
transcription and polymerase chain reaction are coupled in the same
tube. Reaction mixtures were prepared by combining 0.2 mM
dNTP mix, 1 µM each of gene-specific downstream and
upstream primers, 1 mM MgSO4, avian
myeloblastosis virus-reverse transcriptase (0.1 unit/µl), Tfl DNA
polymerase (5 units/µl), avian myeloblastosis virus/Tfl reaction
buffer, poly(A)+ RNA prepared from corneal epithelium
(0.7-1.0 µg), and nuclease-free water to 50 µl. Reactions were
incubated at 48 °C for 45 min to synthesize first strand cDNA,
denatured at 94 °C for 2 min, and subjected to 30 cycles of PCR
amplification. Annealing temperatures were 50 °C for
Fut4, 63 °C for Fut3/5/6, and 60 °C for
Fut9. PCR products were fractionated on 1% agarose gels,
DNA fragments were isolated from the gel using the Concert Rapid Gel
Extraction System (Life Technologies, Inc.), cloned into the pCR
2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen), sequenced,
and used as probes for Northern blot analysis.
Northern Blot Analysis--
Poly(A)+ RNA isolated
from confluent and sparse cultures (3 µg each) was fractionated on
1% agarose gels containing formaldehyde and transferred onto a Hybond
nylon membrane (Amershem Pharmacia Biotech). The blot was prehybridized
overnight in a solution consisting of 0.05 M PIPES, 0.1 M NaCl, 0.05 M NaPO4, 1 mM EDTA, 5% SDS, 60 µg/ml herring sperm DNA. The PCR
probes prepared as described in the previous section were labeled with
Immunohistochemical Localization of Lex
Glycoconjugates in Developing Rabbit Corneas--
New Zealand White
rabbits (Milbrook Farms, Amherst, MA) were used throughout this study.
All animal treatments in this study conformed to the Association for
Research in Vision and Ophthalmology Resolution on the Use of Animals
in Vision Research and the recommendations of the National Institutes
of Health Guide for the Care and Use of Laboratory Animals. Rabbit eyes
were removed at different stages of development including 21 and 27 days of gestation and from 1-, 7-, 10-, 12-, 14-day and 1-, 2-, and
3-month old offspring (n = 3 or more at each stage).
Depending on the age of the animal, either the eyes or the corneas with
2- to 3-mm scleral rims were embedded in optimal cutting temperature
medium immediately upon collection. Prior to embedding, convex shaped
buttons made of optimal cutting temperature medium were prepared using
a mold. These buttons were used as supports during embedding to protect the shape of the corneas. The embedded tissues were stored at
Unfixed longitudinal cryostat sections (5 µm thick) of whole
eyes or of corneas were incubated sequentially with 3%
H2O2 (37 °C, 10 min) and a solution of mouse
liver powder (100 µg/ml, 10 min) to block endogenous peroxidase
activity and nonspecific binding, respectively. The sections were
subsequently incubated with mAb MMA (undiluted hybridoma fluid, 1 h), biotinylated anti-mouse IgM (1:200, Vector Labs, Burlingame, CA), a
freshly prepared complex of avidin D, and biotin peroxidase and
diaminobenzidine/H2O2 reagent (21). For
negative controls, sections were treated with an irrelevant IgM mAb,
mAb 2D4 (ATCC), which is specific for asialo GM2, a
glycolipid not expressed in rabbit corneal
epithelium.2 To determine
whether the Lex antigen is widely expressed in epithelium
of adult tissues, a variety of rabbit epithelial tissues including
skin, conjunctiva, tongue, esophagus, and bladder were also stained
with the mAb MMA.
Cell-Cell Contact-regulated Expression of Lex GP in
Corneal Epithelium--
Confluent and sparse cultures of corneal
epithelium were analyzed to determine whether there is cell-cell
contact-regulated expression of glycoproteins containing
Lex determinants. Under the phase-contrast microscope,
confluent cultures contained polygonal cells with marked contact
inhibition, whereas sparse cultures contained mostly nonpolarized cells
(Fig. 1). Protein yield of sparse and
confluent cultures was ~1.7 and ~7 mg per 100-mm dish,
respectively. To assess whether there is a correlation between the
expression level of Lex-containing glycoproteins and
cell-cell adhesion in vitro, detergent extracts of three
different preparations of sparse (exponentially growing) and confluent
(contact-inhibited) cultures of corneal epithelium were analyzed for
reactivity with mAb MMA by Western blot analysis. When total extracts
were analyzed, no mAb MMA-reactive components were detected. Therefore,
fractions enriched in fucose-containing glycoproteins were prepared by
incubating total extracts with agarose beads conjugated with AAL, and
the proteins bound to the lectin were analyzed for reactivity with mAb
MMA. A neoglycoprotein, Lex-bovine serum albumin (Vector
Laboratories, Covington, LA) served as a positive control (Fig.
2, panel MMA, lane
BSA-Lex). One major mAb MMA-reactive glycoprotein
(145-kDa) was detected in the AAL-bound fraction of three independently
prepared confluent cultures (Fig. 2, panel MMA, lane C). In
contrast, in three independently prepared sparse cell culture extracts,
no mAb MMA-reactive glycoproteins were detected (Fig. 2, panel
MMA, lane S). There was a direct correlation between the
expression of the Lex-GP and the contact inhibition of cell
growth (Fig. 2, bottom panel). Rapidly dividing 4-day sparse
cultures did not express Lex-GP. The expression of
Lex-GP was first seen in 10-day-old early confluent cells
that exhibited significantly less [3H]thymidine
incorporation compared with sparse cultures. Even at this stage the
expression of the Lex-GP was relatively low. The expression
level of the Lex-GP was most robust in 15-day tightly
packed, fully confluent cultures that exhibited only background levels
of [3H]thymidine incorporation (Fig. 2, bottom
panel). The expression of Lex-GP was somewhat reduced
in 18-day post-confluent cultures that had begun to lift from the
culture dish in some areas.
To determine whether glycoproteins containing Lex precursor
lactosamine units are present in the sparse cell cultures of corneal epithelium, samples enriched in galactose-containing glycoproteins were
prepared by incubating total extracts with agarose beads conjugated
with RCA-I, and the proteins bound to the lectin were analyzed for
reactivity with mAb 1B2 by Western blot analysis. A major mAb
1B2-reactive component (~135-kDa) was detected in extracts of both
sparse and confluent cultures (Fig. 2, panel 1B2). These
data suggest that in corneal epithelium there may be cell-cell
contact-regulated Lex Antigen Is Expressed in Corneal Epithelial Cells at
Sites of Cell-Cell Adhesion--
To localize the Lex
antigen in corneal epithelium, confluent cultures grown on coverslips
were immunostained with two different Lex mAbs and were
examined by confocal laser scanning microscope. Whereas in some cases
there was random staining of the entire cell membrane, in most cases
the expression level of the antigen was significantly higher at sites
of cell-cell adhesion (Fig. 3A). Both anti-Lex
mAbs, anti-SSEA-1 (Fig. 3, A(i) and
A(ii)) and MMA (Fig. 3,
A(iii) and A(iv)), gave
similar results.
Anti-Lex Antibodies but Not Anti-sialyl-Lex
Perturb the Formation of Corneal Epithelial Cell-Cell Adhesion
Contacts--
To determine whether the anti-Lex antibodies
inhibit the formation of cell-cell adhesion contacts, corneal
epithelial cells were seeded in 24-well culture plates, allowed to
adhere to the culture dish, and were then incubated in culture medium
in the presence and absence of various antibodies and purified IgM.
Cultures incubated in medium alone (Fig. 3B(i))
and medium containing anti-sialyl-Lex mAb CSLEX-1 (Fig.
3B(ii)) or IgM (not shown) exhibited tightly packed polygonal cells with marked contact inhibition. In contrast, cultures incubated in the presence of anti-Lex mAbs, MMA
(Fig. 3B(iii)), and anti-SSEA-1 (Fig.
3B(iv)) contained a large number of round cells.
The cell rounding was more pronounced in cultures incubated in the
presence of MMA compared with those incubated with anti-SSEA-1.
Overall, cultures incubated with MMA and anti-SSEA-1 contained
~80-90 and ~50-60% cells with round morphology, respectively.
The inhibitory effect of mAb MMA on the formation of cell-cell adhesion
was dose- dependent (Fig. 3C(i)). For each
antibody, duplicate wells were used. Experiments with mAb MMA and IgM
were performed at least three times with reproducible results. Due to
limited availability of the antibodies, mAbs anti-SSEA-1 and CSLEX-1
were used only in one experiment.
To determine whether the anti-Lex is able to disrupt the
cell-cell adhesion contacts, monolayer cultures of corneal epithelium consisting of tightly packed polygonal cells in 24-well plates were
treated overnight with mAb MMA. In this experiment, mAb MMA was found
to induce rounding and lifting of cells from the culture dish in a
dose-dependent manner (Fig.
3C(ii)).
Corneal Epithelial Cells Express Transcripts for the Fut4 and
Fut3/5/6 Genes--
To assess which Fut genes are expressed
in rabbit corneal epithelium, RT-PCR experiments were performed on four
different preparations of poly(A)+ RNA from confluent
cultures. PCR products of the predicted size were cloned into a
pCR2.1-TOPO vector and sequenced in both strands. All four RNA
preparations of confluent cultures of corneal epithelium produced the
expected size Fut4 (213 bp) and Fut3/5/6 (258 bp) products (Fig. 4A, lanes RCE).
In both cases, when reaction mixtures lacked reverse transcriptase, no
components were amplified (not shown). Poly(A)+ RNA
isolated from Chinese hamster ovary cell mutants LEC30 and LEC11 served
as positive controls for Fut4 (29) and Fut3/5/6 (28), respectively (Fig. 4A). ClustalW multiple sequence
alignment analysis (Fig. 4B) showed that the deduced amino
acid sequence of the rabbit Fut4 PCR fragment was closely
related to that of human, mouse, and hamster Fuc-TIV (human, 90%
similarity and 88% identity; mouse and hamster, 87% similarity and
84% identity). The deduced amino acid sequence of rabbit
Fut3/5/6 PCR fragment was most similar to that of human
Fuc-TIII, Fuc-TV, and Fuc-TVI (74% similarity and 71% identity to
Fuc-TIII, -V, and -VI each); the degree of similarity was somewhat less
with the equivalent region of bovine Fuc-T and hamster Fuc-TVI (bovine,
68% similarity and 66% identity; hamster, 69% similarity and 65%
identity). Numerous efforts were made utilizing a variety of annealing
temperatures and cycling parameters to detect Fut9 gene
transcripts in confluent cultures of rabbit corneal epithelium by
RT-PCR. In no case was a Fut9-related PCR product detected.
In contrast, when PCR was performed using rabbit genomic DNA, the
expected size (500 bp) Fut9 PCR product (31) was produced
(not shown). These data lead us to conclude that rabbit corneal
epithelium expresses the Fut4 and Fut3/5/6 genes
but not the Fut9 gene. In the present study, no attempts
were made to determine whether Fuc-TVII is expressed in corneal
epithelium, because this enzyme fucosylates only sialylated precursors.
We have previously shown that sialyl-Lex is not present in
the basal and middle cell layers of rabbit corneal epithelium (21).
Corneal Epithelial Cell-Cell Adhesion Contact-regulated Expression
of Cell Differentiation Stage-specific Expression of Lex
Determinant in Adult Rabbit Corneas--
In the avascular corneal
epithelium, stem cells are confined to a vascularized outer rim of the
cornea known as the limbus, the transition zone between cornea and
conjunctival epithelium (Fig. 6,
regions marked L). These stem cells undergo differentiation as their daughter cells migrate centripetally from the limbus toward
the center of the cornea (33-35). As a result there is
differentiation-related hierarchy of cells with primitive stem
cells in the limbus at the edge of the cornea and an increasing degree
of maturation of cells toward the center of the cornea. This sequential
arrangement of stem, early differentiate, and terminally differentiated
cells makes corneal epithelium an excellent model in which to study the
molecular mechanisms of epithelial cell differentiation.
The characteristic anti-Lex staining pattern of adult
corneal, limbal, and flanking conjunctival epithelium is shown in Fig. 6. Because limbal epithelium overlies a "loose" connective tissue, it was easily differentiated from the corneal epithelium that overlies
compact, plywood-like stroma (36). Conjunctival epithelium was
identified based on the presence of the mucin-secreting goblet cells.
The anti-Lex mAb MMA did not react with limbal epithelium
(Fig. 6, A and B(i), regions
marked L) but reacted intensely with peripheral corneal epithelium
(Fig. 6, A and B(i),
arrowhead, and 6B(iii)). The
Lex immunoreactivity of corneal epithelium decreased toward
the center of the cornea with an increasing degree of maturation of
cells (Fig. 6, A and B(vii)). The
immunoreactive distribution of Lex determinant appeared to
be largely membranous at the site of cell-cell and cell-matrix
interactions (Fig. 6, B(iii) and
B(v)). In the present study, a total of 17 rabbit
corneas (from adult, 2-3-month-old animals) were analyzed.
Lex immunoreactivity was not detected in limbal epithelium
in any of the 17 corneas analyzed. The characteristic Lex
distribution pattern shown in Fig. 6 indicating progressive decrease in
the Lex immunoreactivity toward the center of the cornea
was seen in 14 of the 17 corneas analyzed. In the remaining three
corneas, the entire epithelium stained weakly with no significant
difference in the staining intensity between the central and peripheral
regions. Lex immunoreactivity was also detected in
conjunctival epithelium of all corneas analyzed, but there was a
characteristic difference between the Lex distribution
pattern of conjunctival and corneal epithelium. Although, in corneal
epithelium, Lex immunoreactivity was predominantly detected
in basal and middle cell layers (Fig. 6, B(i),
arrowhead, B(iii) and
B(v)), in conjunctival epithelium the
Lex expression was seen largely in superficial cell layers
(Fig. 6B(i), black arrow). No
Lex immunoreactivity was detected in basal or suprabasal
cell layers of conjunctival epithelium in any of the 17 specimens
analyzed. For comparison purposes, three rabbit corneas were also
stained with another anti-Lex mAb, mAb 7A (provided by Dr.
F. B. Jungalwala; originally prepared by Dr. G. Schwarting) (37).
Similar results were obtained with both mAbs 7A and MMA except that the
staining intensity with mAb 7A was often less than that observed with
mAb MMA. No staining was detected when tissue sections were treated
with a control mAb (mAb 2D4, not shown).
To determine whether the Lex antigen is widely expressed in
the stratified epithelia of adult tissues at the site of cell-cell adhesion, we surveyed a variety of epithelial tissues of three adult
rabbits for Lex expression by immunohistochemical analysis.
In epithelia of six different tissues analyzed, Lex
immunoreactivity in the basal and the middle cell layers was seen only
in the cornea. In other stratified epithelia, Lex
immunoreactivity was either not detected (epidermis) or was detected only in the superficial cell layers (conjunctiva, tongue, esophagus, and bladder).
Expression of Lex Glycoconjugates in the Cornea Is
Developmentally Regulated--
To further determine whether there is a
correlation between Lex expression and the differentiation
stage of corneal epithelial cells, we analyzed the Lex
expression pattern of developing corneas. Frozen sections of 21- and
27-day-old rabbit embryos and of offspring of various age groups (1 day
to 12 weeks) were stained with mAb MMA. Table I shows a summary of the Lex
expression pattern of developing corneas. No Lex
immunoreactivity was detected in the corneas of rabbit embryos or of 1- to 10-day-old animals. At postnatal day 12, around the time of eyelid
opening, Lex immunoreactivity was transiently detected in
central corneal epithelium in three of the seven corneas analyzed (Fig.
7A and Table I). In these
three corneas, no immunoreactivity was detected in peripheral (Fig.
7C) corneal epithelium. In fact, the Lex
distribution pattern detected in these corneas, i.e. robust
expression in central corneal epithelium with the antibody staining
diminishing progressively toward the periphery of the cornea (Fig. 7),
was exactly the opposite of that seen in the adult corneas (Fig. 6). No
Lex immunoreactivity was detected in epithelia of 14- or
15-day-old corneas (Table I). Weak Lex immunoreactivity of
peripheral corneal epithelium was first detected in 1-month-old
corneas. With increasing age of the animal, the Lex
immunoreactivity in peripheral corneal epithelium progressively increased. Between the 2nd and 3rd month of age, the Lex
distribution pattern approached that of adult corneas with peripheral epithelium staining intensely with the anti-Lex mAb and
epithelium in the central region either not staining or staining only
weakly (Fig. 6 and Table I).
In the present study, we demonstrate cell-cell contact-regulated
expression of a 145-kDa glycoprotein bearing the Lex
determinant (Lex-GP) in corneal epithelium. Whereas
confluent cultures of corneal epithelium were found to robustly express
Lex-GP, sparse cultures did not express detectable levels.
[3H]Thymidine incorporation and analysis of
Lex-GP at various cell densities revealed a direct
correlation between the contact inhibition of cell growth and
expression of Lex-GP. Rapidly dividing cells exhibiting
high levels of [3H]thymidine incorporation did not
express Lex-GP, whereas contact-inhibited cultures that
incorporated only background levels of [3H]thymidine
robustly expressed Lex-GP. By using immunofluorescence
staining in conjunction with confocal microscopy, we further
demonstrated that in confluent cultures of corneal epithelium,
Lex antigen is located in high density at sites of
cell-cell adhesion. In in vitro cell-cell adhesion assays,
antibodies to Lex but not to sialyl-Lex
inhibited the formation of cell-cell adhesion contacts. Although cultures incubated with the anti-sialyl-Lex and IgM
successfully formed monolayer cultures consisting of tightly packed
polygonal cells, those incubated with anti-Lex antibodies
failed to form the monolayer and contained largely round cells.
Moreover, when added to confluent cultures of corneal epithelium,
anti-Lex disrupted the monolayer, causing polygonal cells
to round up and lose their close apposition with the neighboring cells.
Cell-cell contact-regulated expression of Lex-GP in corneal
epithelium could be the result of either the de novo
synthesis of carrier protein or of the oligosaccharide chains carrying
the Lex epitope. Lex epitope is synthesized by
Together, our findings suggest a role for the Lex-GP in
events that mediate corneal epithelial cell-cell adhesion. Previous studies have suggested that Lex side chains of plasma
membrane glycoconjugates play an essential role in cell-cell
interactions during embryogenesis in the mouse (13-17). Moreover, it
has been suggested that the Lex-recognizing molecule in
adjacent cells is Lex itself, such that homotypic
Lex-Lex interactions occurring between opposing
cell surfaces mediate cell recognition during early development (15,
16). It has been further shown that homotypic
Lex-Lex interactions are catalyzed by
Ca2+ and other divalent cations (15, 16, 38, 39).
Alternatively, a previously unidentified Lex-binding lectin
may be present in corneal epithelium. Thus, it remains to be determined
whether the Lex-GP identified in this study represents a
novel selectin ligand or whether a
Lex-Lex-mediated cell-cell recognition system
operates in the cornea.
In the present study, we also investigated the putative role of
Lex in corneal epithelial cell differentiation. There are
numerous examples of studies showing that homotypic interactions
between identical cells as well as heterotypic interactions between two different cell types induce the process of differentiation (12, 40,
41). We show here that the expression of Lex
glycoconjugates in corneal epithelium correlates with the stage of
differentiation of the cells. In the immunohistochemical study of adult
rabbit corneas using anti-Lex mAbs, no immunoreactivity was
detected in limbal epithelium, which contains relatively
undifferentiated cells. Intense immunoreactivity was detected in
peripheral corneal epithelium, which contains early differentiated
cells, and little or no immunoreactivity was seen in the terminally
differentiated, central corneal epithelium. Such a region-specific
expression leads us to speculate that the absence of Lex in
the limbal region may provide a mechanism by which stem cells of the
cornea are maintained in the undifferentiated state and that the
induction of Lex may signal cells to enter the pathway of
terminal differentiation.
If Lex indeed plays a role in corneal epithelial cell-cell
adhesion, it is reasonable to ask whether cell-cell interactions are
perturbed in limbal epithelium that lacks Lex. Matic
et al. (42) have reported that two gap junction connexin proteins, CX43 and CX50, which are abundantly expressed in corneal epithelium, are absent in the limbal epithelium. Also, functional gap
junctions are absent in limbal epithelium. It is thus tempting to
speculate that the lack or paucity of connexins and functional gap
junctions in the limbal epithelium may be related to the absence of
Lex in this region. It is known that selectin-carbohydrate
interactions during the rolling of leukocytes along the endothelium
activate the leukocytes and up-regulate its integrin receptors; this
causes leukocytes to adhere to ICAM-1 and transmigrate through the
capillary wall (32). It is thus conceivable that the
Lex-mediated cell-cell adhesion may facilitate the signals
required for the induction of connexins in the corneal epithelium.
In the present study, we also examined Lex expression
pattern of developing corneas. It is known that in prenatal and early postnatal developing corneas, undifferentiated stem or stem-like cells
are present across the entire corneal epithelium (43-47). We reasoned
that if the lack of Lex expression in limbal epithelium is
related to the undifferentiated state of the cells, the corneal
epithelium of fetal and young rabbits, like that of limbal epithelium
of adult animals, would not react with anti-Lex mAbs.
Indeed, in our study, no Lex immunoreactivity was detected
in the epithelia of fetal and young rabbit corneas. The appearance of
the Lex antigen in peripheral corneal epithelium was first
seen in the corneas of 1-month-old animals, and even at this stage, the
Lex expression level was relatively low. Only when rabbits
reached about 10 weeks of age was the Lex staining
intensity of peripheral corneal epithelium, similar to that seen in
adult corneas, detected. Consistent with the report of Zieske and
Wasson (47), our findings also suggest that the process of maturation
of epithelial cells begins in the central cornea around the time of
eyelid opening. In three of seven 12-day-old corneas, around the time
of eyelid opening, Lex immunoreactivity was detected in
central corneal epithelium but not in peripheral corneal epithelium.
This heterogeneity among rabbits of the same age group suggests
transient Lex expression at this stage. If the duration of
Lex expression were no more than several hours, it would be
expected that at the time the corneas were collected, whereas some
animals may not have yet begun to express, others may have already
ceased to express the antigen.
In brief, the present study provides evidence that a 145-kDa
Lex-bearing glycoprotein plays a role through the
Lex determinant in corneal epithelial cell-cell adhesion.
We propose that Lex-mediated cell-cell interactions most
likely contribute to the mechanisms that mediate corneal epithelial
cell differentiation and that they may play a role in the induction of
connexins and formation of gap junctions in corneal epithelium.
(1,4)[Fuc
(1,3)]GlcNAc). This glycoprotein
(Lex-GP) was expressed in confluent/contact-inhibited
cultures but not in sparse cultures of corneal epithelium. In contrast,
a 135-kDa glycoprotein bearing precursor, unfucosylated,
lactosamine-containing glycans (Gal
1-4GlcNAc
1-R) was expressed
in sparse cultures. Immunofluorescence staining and confocal microscopy
of confluent cultures revealed that in corneal epithelium,
Lex antigen is located in high density at sites of
cell-cell adhesion. In in vitro cell-cell adhesion assays,
anti-Lex, but not anti-sialyl-Lex monoclonal
antibodies, inhibited the formation of corneal epithelial cell-cell
adhesion. Also, when added to confluent cultures, antibodies to
Lex disrupted the monolayer and caused tightly packed
polygonal cells to round up. Analysis of the expression of
Fut genes that encode
-1,3-fucosyltransferases,
the enzymes that generate the Lex determinant, revealed
that confluent/contact-inhibited cultures of rabbit corneal epithelium
contain markedly elevated levels of Fut4 and
Fut3/5/6 gene transcripts compared with sparse cultures. These data suggest that the Fut4 and Fut3/5/6
genes are targets of cell-cell contact-regulated signals and that
Fut gene products direct cell-cell contact-associated
expression of Lex on the Lex-GP in corneal
epithelium. Immunohistochemical analysis revealed that the expression
of Lex antigen in the epithelium of adult and developing
corneas is related to the stage of differentiation of the cells.
Although early differentiated cells robustly expressed Lex,
relatively undifferentiated cells did not, and the expression level was
relatively low in terminally differentiated cells. Overall, these data
provide evidence that a Lex-bearing glycoprotein plays a
role through the Lex determinant in corneal epithelial
cell-cell adhesion, and these data suggest that
Lex-mediated cell-cell interactions contribute to
mechanisms that mediate corneal epithelial cell differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1,4)[Fuc
(1,3)]GlcNAc). Several studies have suggested that Lex side chains of plasma membrane glycoconjugates play an
essential role in cell-cell interactions during embryogenesis in the
mouse (13-17). In the developing mouse embryo, the Lex
antigen appears after the third cleavage (8-cell stage) at the time of
onset of compaction and disappears around the 32-cell stage after
completion of the compaction process. Multivalent lysyllysine
conjugates of Lex (lacto-N-fucopentaose III)
cause individual blastomeres of fully compacted 16-cell embryos to
round up and lose their close apposition of membranes (14). Eggens
et al. (15) and Kojima et al. (16) demonstrated
that lacto-N-fucopentaose III also inhibits the
intercellular adhesion of teratocarcinoma cells. In these cells and in
developing mouse embryos, Lex-Lex interactions
occurring between opposing homotypic cell surfaces have been postulated
to mediate cell-cell adhesion (15, 16). Several other studies have also
implicated Lex as a possible adhesion molecule during
embryogenesis (18-20). In the present study we show that in rabbit
corneal epithelium: (i) there is cell-cell contact-regulated expression
of a Lex-bearing 145-kDa glycoprotein (Lex-GP)
and of
-1,3-fucosyltransferase genes that mediate the synthesis of
the Lex side chains; (ii) Lex antigen is
located in high density at sites of cell-cell adhesion; (iii)
antibodies to Lex inhibit the formation of cell-cell
adhesion contacts; and (iv) there is cell differentiation
stage-specific and developmentally regulated expression of the
Lex antigen.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-4[Fuc
1-3]GlcNAc
1-R) (23), and mAb 1B2, which binds
to terminal N-acetyllactosamine disaccharide
(Gal
1-4GlcNAc
1-R), the unfucosylated precursor of
Lex (24), were used. Hybridomas secreting both mAbs were
purchased from American Type Culture Collection (Manassas, VA). To
isolate fractions enriched in Lex-containing glycoproteins
(Lex-GPs), 1.0 ml of cold radioimmunoprecipitation assay
(RIPA) buffer (50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 0.1% Nonidet P-40, and 0.5% deoxycholic acid)
was added to 10-cm dishes of washed confluent and sparse primary cell
cultures. After 30 min on ice, cell extracts were clarified by
centrifugation (30 min, 100,000 × g, 4 °C), and the
protein concentration was estimated using the BCA protein assay reagent
(Pierce). Clarified extracts (~7.0 mg of protein in 1.0 ml) were
incubated with 50 µl of agarose beads conjugated with Aleuria
aurantia lectin (AAL, a fucose-specific lectin; Vector
Laboratories, Burlingame, CA) for 1 h at 4 °C. After the
incubation period, the lectin-agarose beads were washed five times with
RIPA buffer, and 26 µl of electrophoresis sample buffer (50 mM Tris-HCl containing 2% SDS, 10% glycerol, and 100 mM dithiothreitol, pH 6.8) was added. The samples were
boiled for 4 min and centrifuged, and the supernatants were
electrophoresed on 10% polyacrylamide gels in the presence of SDS.
Proteins from the gels were blotted onto nitrocellulose paper. To
identify Lex-GPs of corneal epithelium, gel blots were
treated with 2% bovine serum albumin in PBS (2 h, room temperature) to
block nonspecific binding and were then incubated with mAb MMA
(undiluted hybridoma fluid, overnight, 4 °C). Gel blots were
developed with peroxidase-labeled anti-mouse IgM using a
chemiluminescent detection system (25) (Kirkegaard & Perry
Laboratories, Inc., Gaithersburg, MD). To determine whether there is a
correlation between the contact inhibition of cell growth and the
expression of the Lex-GP, cell cultures at various
densities (day 4, sparse; day 10, early confluent; day 15, confluent;
and day 18, post-confluent) were incubated in SHEM medium containing
[3H]thymidine (2 µCi/ml, 6.7 Ci/mmol) for 16-18 h. At
the end of the incubation period, cells were extensively rinsed with
PBS and harvested for estimating [3H]thymidine
incorporation and Western blot analysis.
1-4GlcNAc
1-R) side chains. Briefly,
detergent extracts of cell cultures of rabbit corneal epithelium were
incubated with agarose beads conjugated with a
-galactose-specific
lectin, Ricinus communis agglutinin-I (RCA-I). The
RCA-I-bound glycoproteins were electrophoresed, and gel blots were
analyzed for reactivity with mAb 1B2 as described above.
-1,3-Fucosyltransferases genes known to direct the
synthesis of the Lex epitope include
Fut3-Fut6 and Fut9. The human
FUT3, FUT5, and FUT6 genes
share 90% sequence identity and cannot be differentiated from one
another by RT-PCR or Northern blot analysis with coding region probes
or primers. On the other hand, Fut4 and Fut9 are considerably different from one another as well as from
Fut3/5/6 (30-32). To determine which Fut genes
were expressed in corneal epithelium, oligonucleotide primers
corresponding to the conserved regions of Fut4,
Fut3/5/6, and Fut9 were designed using Oligo 6.0 software (Molecular Biology Insights, Cascade, CO) unless stated
otherwise. Nucleotide sequences of primers used are shown as follows:
Fut4, forward, 5'-TAYCTRGCNTTTGAGAACTC, and reverse, 5'-GGAAGTAGCGACGATAGAC; Fut3/5/6, forward,
5'-GCCCTACGGCTGGCTSGAGC, and reverse, 5'-GTGATGTAGTCGGGGTGCARGGAGTTCTC;
and Fut9 (from Kudo et al. (31)), forward,
5'-CAGCTGGGATCTGACTAACTTACC, and reverse,
5'-CCACATGAATGAATGAATCAGCTGG.
-[32P]dCTP using the Prime-It RmT Random Primer
Labeling Kit (Stratagene, La Jolla, CA). Unincorporated nucleotides
were removed from the labeled probes using the Push Column Beta Shield
Device and NucTrap probe purification columns (Stratagene). The
specific activity of the labeled probes was ~109 cpm/µg
DNA. The probe was denatured at 100 °C for 5 min and chilled on ice
briefly and was then added to prehybridization buffer to the final
concentration of 106 cpm/ml. Hybridization was performed
overnight at 60 °C. Blots were washed with 2× SSC buffer containing
0.1% SDS for 30 min at 60 °C and were then subjected to
autoradiography. Approximate densities of various components in the
autoradiographs were estimated using the Bio-Image Whole Band Analysis
System (Millipore, Ann Arbor, MI).
80 °C until used for sectioning.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Phase contrast micrographs of
sparse/exponentially growing (top) and
confluent/contact-inhibited (bottom) cultures of
corneal epithelium. Bar, 450 µm.
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Fig. 2.
Western blot analysis showing that
contact-inhibited but not sparse/exponentially growing cultures of
corneal epithelium express a glycoprotein bearing the Lex
determinant chains, whereas sparse cultures express a glycoprotein
lacking Lex but containing Lex precursor side
chains. To detect glycoproteins containing Lex
determinant, protein extracts of confluent (C) and sparse
(S) cultures were incubated with AAL-agarose; AAL-bound
glycoproteins were electrophoresed on SDS-polyacrylamide gels, and the
protein blots were processed for immunostaining with an
anti-Lex mAb, MMA. To detect glycoproteins bearing terminal
lactosamine units, protein extracts were incubated with RCA-I-agarose,
and RCA-I-bound glycoproteins were electrophoresed and immunostained
with mAb 1B2 which binds to unfucosylated lactosamine precursor of
Lex. Note that confluent cultures (panel MMA, lane
C) contained a major mAb MMA-reactive glycoprotein (145-kDa) that
was not detected in the sparse cultures (panel MMA, lane S).
In contrast, sparse cultures contained a major mAb 1B2-reactive
component (135-kDa) (panel 1B2, lane S). Samples derived
from equal amount of original cell protein were electrophoresed in both
S and C lanes. Right panel shows the
Coomassie Blue staining pattern of the total protein extracts of
confluent and sparse cultures, 16 µg of protein each. Bovine serum
albumin-Lex (BSA-Lex, 0.5 µg) served
as a positive control for mAb MMA. The bottom panel shows
direct correlation between the contact inhibition of cell growth as
measured by [3H]thymidine incorporation and the
expression of the Lex-GP. S, sparse;
C1, early confluent; C2, tightly packed fully
confluent; C3, postconfluent cultures beginning to lift from
culture wells in some areas; ND, not determined.
-1,3- fucosylation of the Lex
precursor on the ~135-kDa glycoprotein.
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Fig. 3.
A, Lex antigen is
expressed at sites of corneal epithelial cell-cell adhesion. Confluent
cultures of corneal epithelium grown on coverslips were stained with
the anti-Lex mAb anti-SSEA-1 (i and
ii) or MMA (iii and iv). The primary
antibody was detected using an anti-mouse secondary antibody coupled to
fluorescein isothiocyanate. Confocal microscopy images demonstrating
localization of the antigen at sites of cell-cell adhesion are shown.
Two different fields are shown for each antibody staining. Cells
stained with mAb MMA are shown at higher magnification compared with
those stained with anti-SSEA-1 (bar, 18 µm).
B, antibodies to Lex inhibit the formation of
cell-cell adhesion contacts. Primary cultures of rabbit corneal
epithelial cells were plated in 24-well culture plates (5 × 105 cells/ml, 0.6 ml/well). After allowing the cells to
adhere to the wells (3 h), the cells were incubated overnight with
medium alone (i) or medium containing 0.8 mg/ml of mAb
CSLEX-1 (ii), mAb MMA (iii), or mAb anti-SSEA-1
(iv). At the end of the incubation periods, cells were fixed
with glutaraldehyde and evaluated under a phase-contrast microscope.
Note that cultures incubated in medium alone (i) or in
medium containing mAb CSLEX-1 (ii) and purified IgM (not
shown) successfully formed tightly packed contact-inhibited cultures.
In contrast, cultures incubated in the presence of anti-Lex
antibodies, MMA (iii) and anti-SSEA-1 (iv),
remained round presumably due to the inhibition of the formation of
cell-cell adhesion contacts. Prior to glutaraldehyde fixation, the cell
borders of cultures shown in panels (i) and (ii)
were markedly distinct and were similar to those shown for cultures in
Fig. 1, bottom panel. C, the effect of mAb MMA on
the formation of cell-cell adhesion contacts and disruption of the
monolayers is dose-dependent. (i) cells were
allowed to adhere to culture wells for 3 h and were then incubated
overnight with the mAb MMA; (ii) tightly packed monolayer
cultures were incubated overnight with mAb MMA. Cell rounding: ,
<5%; +
, 5-10%; +, >10 to <25%; ++, >25 to <50%, +++,
>50%.
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Fig. 4.
Rabbit corneal epithelial cells express
Fut4 and Fut3/5/6. A,
Poly(A)+ RNA (0.7-1.0 µg) from confluent cultures of
rabbit corneal epithelial cells (RCE) was subjected to
RT-PCR as described under "Experimental Procedures." The expected
fragments of 213 and 258 bp were amplified using Fut4 and
Fut3/5/6 gene-specific primers, respectively.
Poly(A)+ RNA isolated from Chinese hamster ovary cell
mutants LEC30 and LEC11 served as positive controls for Fut4
and Fut3/5/6, respectively. In each case, no components were
amplified when reaction mixtures lacked reverse transcriptase (not
shown). B, (i), ClustalW analysis of deduced
amino sequence of the rabbit Fut4 PCR fragment compared with
human, mouse, and hamster Fuc-TIV. First amino acid represents residues
294, 323, and 290 of human, mouse, and hamster Fuc-TIV, respectively.
(ii), rabbit Fut3/5/6 fragment compared with
human, bovine, and hamster Fuc-TIII/V/VI. First amino acid represents
residues 178, 177, and 191 of human Fuc-TIII, Fuc-TV, and Fuc-TVI,
respectively. In bovine and hamster, first amino acid represents
residues 182 and 179, respectively. Solid boxes indicate
identity; shaded boxes indicate similarity.
-1,3-Fucosyltransferase Genes--
Northern blot analysis using
poly(A)+ RNA from two different preparations each of
confluent and sparse cultures was performed to determine whether in
corneal epithelium there is cell-cell contact-regulated expression of
the Fut4 and/or Fut3/5/6 genes. In confluent as
well as sparse cultures of corneal epithelium, a 3.3-kilobase pair
Fut4 gene transcript and a 2.1-kilobase pair Fut3/5/6 gene transcript were detected (Fig.
5). In both preparations, confluent/contact-inhibited cultures were found to contain markedly elevated levels of Fut4 and Fut3/5/6 mRNA
transcripts compared with sparse cultures that lack stable cell-cell
contacts. Overall, compared with sparse cultures, confluent cultures
contained ~6 times more Fut4 and ~3 times more
Fut3/5/6 gene transcripts, respectively.
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Fig. 5.
Cell-cell contact-regulated expression of
Fut4 and Fut3/5/6 in corneal
epithelium. Poly(A)+ RNA (3 µg) from confluent
(C) and sparse (S) cultures of corneal epithelium
was electrophoresed on 1% agarose-formaldehyde gel, blotted onto
Hybond nylon membrane and probed with a 213-bp 32P-labeled
rabbit corneal epithelial Fut4 cDNA fragment. A
duplicate blot was probed with a 258-bp Fut3/5/6 probe.
Blots were subsequently stripped and hybridized to a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. Note
that confluent cultures contain higher levels of both Fut4
and Fut3/5/6 mRNA transcripts compared with the sparse
cultures. C:S ratio based on the density of each band is
shown below each panel. kb, kilobase pairs.
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Fig. 6.
Region-specific expression of Lex
glycoconjugates in an adult rabbit cornea. Unfixed frozen sections
of corneas were immunostained with mAb MMA as described under
"Experimental Procedures." A, a composite photograph
showing the staining pattern of the epithelium across the cornea. The
region between the red arrows was constructed into a
composite using the first 25% (2 cm) width of each of the 17 consecutive prints. Note that the anti-Lex mAb did not
react with limbal epithelium (L), the transitional zone
between the cornea (the region between the limbus) and conjunctiva (the
region on either side of the limbus) but reacted intensely with
peripheral corneal epithelium. The immunoreactivity of corneal
epithelium decreased toward the center of the cornea with the
increasing degree of maturation of cells. Also, the superficial cell
layers of conjunctival epithelium stained intensely with the antibody.
B, photographs showing representative regions from limbal
(i and ii), peripheral (iii and
iv), midperipheral (v and vi), and
central (vii and viii) epithelium of the cornea
shown in A. Tissue sections stained with mAb MMA (i,
iii, v, and vii) and Mayer's hematoxylin (ii,
iv, vi, and viii) are shown. Limbal epithelium
(L) is shown with flanking conjunctival (black
arrow) and peripheral (arrowhead) corneal epithelium.
Note the characteristic difference in the Lex distribution
pattern of corneal and conjunctival epithelium, whereas predominantly
basal and middle cell layers of corneal epithelium (iii and
v) were stained with mAb MMA, largely superficial cell
layers of conjunctival epithelium (i, black
arrow) reacted positively with the antibody. Bar, 50 µm.
Lex expression pattern in developing rabbit corneal epithelium
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Fig. 7.
Transient expression of Lex
antigen in central corneal epithelium of a 12-day-old rabbit.
Unfixed frozen sections of whole eyes were immunostained with mAb MMA
as described under "Experimental Procedures." Representative
regions from central (A), midperipheral (B), and
peripheral (C and D) corneal epithelium are
shown. A-C, sections stained with mAb MMA; D,
section stained with Mayer's hematoxylin. Note that unlike corneal
epithelium of the adult animals (Fig. 6), which showed intense
Lex immunoreactivity in the peripheral region with
decreasing staining intensity toward the center of the cornea, that of
a 12-day-old cornea showed intense immunoreactivity in the central
corneal epithelium with decreasing intensity toward the periphery of
the cornea. Bar, 50 µm. Basal cell layer in C
is indicated by arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,3-fucosylation of oligosaccharide chains bearing terminal
(Gal
1-4GlcNAc
1-R) disaccharides. Western blot experiments
revealed that a 135-kDa glycoprotein bearing the Lex
precursor disaccharide (Gal
1-4GlcNAc
1-R) is robustly expressed in sparse cultures of corneal epithelium. Thus cell-cell
contact-regulated expression of the Lex-GP in corneal
epithelium appears to be due to the de novo synthesis of the
Lex epitope due to cell-cell contact-regulated
-1,3-fucosylation of Lex precursor-terminated side
chains on the 135-kDa glycoprotein. The
-1,3-fucosyltransferases
known to generate Lex include Fut4,
Fut9, and a family of highly homologous Futs
referred to herein as Fut3/5/6 (30-32). Sequencing of
RT-PCR products showed that transcripts for both Fut4 and
Fut3/5/6 but not Fut9 are expressed in cultures
of corneal epithelium. Northern blot analysis showed that in corneal
epithelium there is cell-cell adhesion contact-regulated expression of
both Fut4 and Fut3/5/6.
Confluent/contact-inhibited cultures of corneal epithelium were found
to contain markedly elevated levels of both Fut4 and
Fut3/5/6 gene transcripts compared with sparse cultures that
lack stable cell-cell contacts. These data led us to conclude that the
Fut4 and Fut3/5/6 genes are targets of cell-cell
contact-regulated signals and that these Fut gene products
direct cell-cell contact-associated expression of Lex on
the Lex-GP in corneal epithelium.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Barry Potvin for anti-SSEA-1 and CSLEX-1 mAbs, JiZeng Qiao for help with confocal microscopy, and Dr. Robert M. Lavker and Santosh K. Patnaik for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants EY07088 and EY09349, a sabbatical award from Research to Prevent Blindness (to N. P.), and National Institutes of Health Grant R01 36434 (to P. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Ophthalmology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6776; Fax: 617-636-0348; E-mail: Noorjahan.Panjwani@tufts.edu.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M009672200
2 N. Panjwani and Z. Zhao, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: Lex, Lewisx; GP, glycoprotein; PBS, phosphate-buffered saline; mAb, monoclonal antibody; AAL, A. aurantia lectin; RCA-I, R. communis agglutinin-I; EMEM, Eagle's minimum essential medium; MFS, EMEM/F12 (1:1) containing 5% fetal bovine serum; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; bp, base pair; PIPES, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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