Download Expression of mucin peptide and blood group ABH

Expression of Mucin Peptide and Blood Group ABH- and
Lewis-Related Carbohydrate Antigens
in Normal Human Conjunctiva
Catherine Garcher * Jacques Bara,\ Alain Bron,* and Rafael Oriol%
Purpose. The immunohistochemical characterization of mucin peptide antigens and identification of blood group ABH- and Lewis-related carbohydrate antigens expressed in epithelium of
normal human conjunctiva.
Methods. Immunoperoxidase characterization of conjunctival glycoproteins was performed
using antibodies against peptide and saccharide moieties of gastrointestinal mucins on conjunctival biopsy specimens from 89 healthy individuals of known ABO and Lewis red cell
phenotypes.
Results. Ocular mucins had epitopes in common with the peptidic core of gastric mucin (Ml
epitopes), but also contained sialylated saccharide chains like those of intestinal mucins. AntiMi gastric mucins, anti-type 1 precursor, and anti-T antibodies strongly stained the cytoplasm
of goblet cells. Anti-Lea, anti-NeuAc-Lea, and anti-Leb antibodies stained both epithelial and
goblet cells of Lewis-positive individuals only. Anti-H type 1, anti-H type 2, anti-A, and anti-B
stained epithelial cells of ABH secretors. In addition, A-like epitopes independent of secretor
phenotype were found in epithelial and goblet cells of all donors.
Conclusions. The conjunctival mucins cross-react with antibodies specific for digestive mucins
and for blood group-related carbohydrate epitopes. The distributions of Lewis and secretor
phenotypes in conjunctiva are: 19% Lewis-positive nonsecretors, 71% Lewis-positive secretors, and 10% Lewis-negative secretors. Lea and Leb antigens are present on epithelial cells of
Lewis-positive nonsecretors and Lewis-positive secretors, respectively, as expected for antigens under control of Lewis and secretor genes. The secretor a-2-fucosyltransferase is not
expressed on goblet cells, as described for the goblet cells of the distal rectal colonic mucosae.
ABH antigens synthesized by the products of Al, A2, B, and Se genes and modulated by the
presence of the product of the Lewis gene are found in epithelial cells. In addition, A-related
epitopes independent of the A1-A2 subtype, secretor, and Lewis phenotypes also are present
in both epithelial and goblet cells. Invest Ophthalmol Vis Sci. 1994;35:1184-1191.
JVlucins are the major components of the mucus gel
overlaying the ocular surface, and help to maintain
tear film stability. Earlier attempts to study conjunctival mucins by histochemistry were limited to the identification of sialylated, sulfated, or neutral mucins.1
Mucins have a flexible peptidic core coated with
oligosaccharide chains, showing alternating poorly
From the "Department of Ophthalmology, University of Burgundy, Dijon,
VNSERM U-55, Hopital Saint Antoine, Paris, and {iNSERM U-178, Villejuif
France. J. Bara and R. Oriol are full time researchers from the Centre National
de la Recherche Scientifique (CNRS), France.
Submitted for publication July 13, 1993; revised September 29, 1993; accepted
October 5, 1993.
Proprietary interest category: N.
Reprint requests: Catherine Garcher, M. D., Service d'Ophtalmologie, 3 rue du
Faubourg Raines, Dijon 21000, France.
1184
(naked) and highly glycosylated (T domain) regions.2
They may be considered a mosaic of epitopes,3 some of
which are associated with the peptide core of the
naked regions (Ml or M3SI epitopes),4"5 and others
with the terminal blood group-related carbohydrate
epitopes (ABH and Lewis).6 These histo-blood group
antigens are made by the sequential addition of monosaccharides onto precursor glycolipid or glycoprotein
carrier molecules.7 Six main types of disaccharide precursor chains have been proposed,8 and all but type 5
already have been found in human tissues:
Type 1: 0Gall->3/3GlcNAcl^R
Type 2: |SGall^4|3GlcNAcl^R
Type 3: /3Gall^3«GalNAcl^R
Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3
Copyright © Association for Research in Vision and Ophthalmology
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Mucin Antigens of Normal Human Conjunctiva
1185
Type 4: /3Gall-»3j3GalNAcl^R
Type 5: ,3Gall->3,3Gall^R
Type 6: /3Gall-^4/3Glcl-»R
The different carbohydrate epitopes are made by
the addition of terminal monosaccharide units to these
precursor chains by specific glycosyltransferases encoded by the genes of the ABO, H, secretor, and Lewis
systems.
Two main tissue compartments express these antigens in humans, the mesodermal and epithelial tissues.
Mesodermal tissues such as erythrocytes and vascular
endothelium have ABH antigens under control of the
ABO and H genes, and the epithelial tissues producing
exocrine secretions have ABH and Lewis antigens
under control of ABO, Se, and Le genes. 6 Most of the
Lewis antigens synthesized by epithelial tissues are
found in exocrine secretions, but a small proportion is
shed into the bloodstream as glycosphingolipids that
are passively adsorbed at the surface of red and white
cells. Therefore, red cells have two types of histoblood group antigens: adsorbed type-1 ABH and
Lewis antigens under control of ABO, Le, and Se
genes; and intrinsic type 2 ABH antigens under control of ABO and H genes. 9
The structures found in exocrine secretions are
mainly based on type 1 (/3Gall^3GlcNAcl^R) and
type 2 ( i 8Gall-^4 J 8GlcNAc^R) precursors. Table 1
summarizes the different chemical structures, en-
zymes, and genes involved in the synthesis of type 1
carbohydrate epitopes.
Addition of fucose in al—>2 onto the terminal
jSGal of the type 1 precursor by the secretor enzyme
(encoded by the Se gene) makes the H type 1 epitope.
Addition of fucose in al—>4 onto the subterminal
/JGlcNAc by the Lewis enzyme (encoded by the Le
gene) makes the Le a epitope. Addition of the same
al—»4 fucose onto the H structure makes the Le b epitope. The combined effects of the products of the Se
and Le genes give the four phenotypes illustrated on
Table 1.
Two additional modifications can be obtained by
addition of A or B terminal saccharides in secretors or
addition of neuraminic acid (NeuAc) on to the precursor carbohydrate chains. The A or B enzymes use the
monofucosylated H structures made by the products
of H or Se genes as acceptors, and therefore secretors
also can make A or B epitopes. A specific sialyltransferase can add NeuAc in a2—*3 linkage onto the terminal jSGal of the precursor chains, and the subsequent
addition of the al—»4 fucose results in the NeuAc-Lea
epitope in Lewis-positive individuals.
All these carbohydrate structures can be important for a better understanding of the quality of the
mucin gel on the surface of the ocular globe, and
might be useful in studying glycoprotein changes associated with diseased conjunctiva. This study investigates the epitopes of normal conjunctival antigens in
l. Genes, Enzymes, and Antigens Based on Type 1 Structures Found in Exocrine
Secretions and Red Cells of the Four Main Combinations of Secretor and Lewis Phenotypes
TABLE
Chemical Structure
jSGal
|1,3
aFucl -*> 4j8GlcNAc
aFucl -+ 2j3Gal
|1,3
aFucl -*• 4/3GlcNAc
R
Antigen Name
0Gal
|1,3
/SGlcNAc
R
aFucl
11,3
iSGlcNAc
I
R
R
(precursor)
Lec
2j8Gal
(H type 1)
Led
Lea
Le b
+
+
—
;
Nonsecretor
Le(a+b-)
Secretor
Le(a-b+)
Nonsecretor
Le(a-b-)
Secretor
Le(a-b-)
Secretor
Lewis
se/se
Le/-
Se/Le/-
se/se
le/le
Se/le/le
Frequency
20%
70%
1%
9%
Enzymes Found
Secretor
Lewis
Phenotype
Secretion
Red cell
Genotype
The secretors can have, in addition, on top of the /3Gal of H or Leb structures, the terminal aGalNAc (if they are of group A) or the terminal aGal (if they are of group B). The addition of a terminal aNeuAc on top of the /3Gal gives the NeuAc-Le" or CA-19.9 antigen on
the precursor chains of Lewis positive individuals only.
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Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3
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patients undergoing cataract surgery, who were typed
on erythrocytes for ABO and Lewis antigens.
MATERIALS AND METHODS
Monoclonal Antibodies Against Peptidic Core
of Mucin
Monoclonal antibodies (MAbs) 1-13M1, 2-11 Ml,
2-12M1, 9-13M1, 58M1, 19M1, and 45M1 (IgGl)
were prepared as described.10 These MAbs recognize
conformational Ml epitopes associated with the peptide core of mucins. A combination of these seven different anti-Mi MAbs staining all mucous cells of human surface gastric epithelium, regardless of the ABH
or Lewis phenotype, was used. MAb 168M3SI (IgGl)
recognized the NeuAc-Tn epitope and cross-reacted
with the peptide core of the small intestine mucin.5
Antibodies Against Saccharide Moieties of
Mucin
Anti-T MAb (AH9-17, IgM) was a gift of Dr L. Lamontagne (Chembiomed, Alberta Research Council, Edmonton, Alberta, Canada); anti-Lea (7LE, IgGl), antiLeb (2-25LE, IgGl) cross-reacting slightly with Lea,
and anti-H type 2 (19-OLE, IgM) were from our laboratory11; anti-NeuAc-Lea was the NS 19-9 MAb
(IgGl).12 Affinity-purified rabbit polyclonal antibodies,13 anti-H type 1 (Led) cross-reacting with Leb, and
anti-type 1 precursor (Lec) were obtained from Dr. S.
Henry (Blood Transfusion Service, Auckland Hospital, New Zealand).
The anti-B MAb (26W3) reacting with fucosylated
blood group B epitopes and a broad anti-A MAb
(34W1) reacting with all types of linear and fucosylated blood group A epitopes, were obtained from the
First International Workshop on Monoclonal Antibodies Against Human Red Cells and Related Antigens
(Paris, France).14 Anti-A MAb (016) reacting with all A
types, anti-A MAb (013) reacting preferentially with A
type 2 structures, and anti-AB MAb (050) reacting
only with A type 2 and B type 2 structures, were obtained from the Second International Workshop on
Monoclonal Antibodies Against Human Red Cells and
Related antigens (Lund, Sweden).15 The detailed specificity of these antibodies was determined with synthetic blood group-related oligosaccharides.16
The main biochemical structures recognized by
these antibodies are shown in Table 2. All reacted
strongly with mucous cells of gastroduodenal mucosa
according to the Lewis and secretor phenotype of the
tissue donor: MAbs against Lea and T antigens react
on the gastric mucosa of all the nonsecretor individuals, and anti-NeuAc-Lea MAbs react on the digestive
mucosae of all the Lewis-positive individuals.
Tissue Samples
The guidelines of the Declaration of Helsinki were followed. Conjunctival biopsy specimens were collected
with fully informed, written consent, and authorization of the local ethical committee in 89 patients undergoing cataract surgery (age range, 18 to 96 years).
Biopsy specimens measuring 4 mm2 were taken from
the inferior fornix of the conjunctiva under topical
anesthesia (oxybuprocaine chlorhydrate) with nontoothed forceps.
Preparation of Tissues
Conjunctival biopsy specimens were fixed immediately
in 95% ethanol overnight, processed by routine histology, and then embedded in paraffin wax. Serial sections (3 /tm thick) were cut using an Autocut (Reichert-Jung, Heidelberg, Germany).
Immunoperoxidase
Sections were deparaffinized, incubated for 3 minutes
in phosphate-buffered saline (PBS) containing 0.1%
Tween-20, and then incubated for 30 minutes with the
TABLE 2.
Main Histo-Blood-Group Related Structures Recognized
by the Anti-Carbohydrate Antibodies
Epitope Names
Oligosacchari.de Structures
Antibodies
Type 1 precursor
Lea
Leb
H type 1
H type 2
NeuAc-Lea
NeuAc-Tn
T or type 3 precursor
A histo-blood-group
B histo-blood-group
/3Gall - • 30GlcNAc
Lec
7LE
2-25LE
Led
19-OLE
NS19-9
168M3SI*
AH9-17
013, 016, 050, 34W1
050, 26W3
/3Gall -*• 3(aFucl -»• 4)/?GlcNAc
aFucl -*• 2jSGall -»> 3(aFucl -+ 4)/3GlcNAc
aFucl -•• 2/?Gall -•• 3/3GlcNAc
aFucl ->• 2/3Gall -*• 4j8GlcNAc
«NeuAc2 -* 3/3Gall - • 3(aFucl -»• 4)j8GlcNAc
«NeuAc2 -+ 6aGalNAcl -* Ser/Thr
jSGall - * 3aGalNAc
aGalNAcl ->• 3(aFucl ->• 2)/3Gal
aGall -* 3(aFucl -» 2)0Gal
Gal = galactose; GlcNAc = ./V-acetylglucosamine; GalNAc = iV-acetylgalactosamine; Fuc = fucose; NeuAc = N-acetylneuraminic acid.
* The 168M3SI antibody cross-reacts with the peptide core of small intestine mucin.
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Mucin Antigens of Normal Human Conjunctiva
undiluted hybridoma supernatants or the affinity-purified antibodies at 20 Mg/ml- They were washed in
Tween-20-PBS and then incubated for 30 minutes
with anti-IgG antibodies (diluted 1/50) linked to peroxidase (Diagnostics Pasteur, Marnes-la-Coquette,
France). Finally, the sections were washed with Tween20-PBS and incubated for 4 minutes with amino-ethylcarbazole (Carlo Erba, Milano, Italy) and hydrogen
peroxide.17 Before microscopic examination, cell nuclei were stained with 1% haematein for 2 minutes.
The specificity of immunoreactivity was controlled
by inhibition of staining after incubation of hybridoma
supernatants with gastric mucin preparations or red
blood cells containing the epitopes corresponding to
each antibody.
ABO and Lewis Phenotypes
Blood samples were obtained from the 89 patients to
determine ABO and Lewis erythrocyte phenotypes by
routine hemagglutination tests performed at the
blood bank of the Dijon hospital. Al and A2 subtypes
of blood group A donors were determined by agglutination with lectins: Dolichos biflorus (reacting better
with Al than A2) and Ulex europaeus I (reacting better
with A2 than Al).
RESULTS
Mucin Peptide Epitopes
Conjunctiva is a stratified epithelium containing epithelial cells and mucus-secreting goblet cells. The cytoplasm of goblet cells of all donors was strongly
stained with anti-Mi gastric mucin MAbs (Fig. 1). On
the contrary, the MAb against intestinal mucin
(168M3SI) did not stain either mucous cells or epithelial cells in any donor.
Carbohydrate Non-ABO Epitopes
T (type 3 precursor) and type 1 precursor epitopes
were present in the mucous secretion of conjunctival
goblet cells.
The H type 1 and H type 2 epitopes were found in
cytoplasm and membranes of suprabasal epithelial
cells (Fig. 2). The basal cells and adjacent layers of the
conjunctiva did not express these epitopes.
Lewis-related epitopes were detected on epithelial
and goblet cells of Lewis-positive donors only. The
NeuAc-Lea epitope was present on both epithelial and
goblet cells, regardless of their secretor phenotype
(Fig. 3), illustrating that this antigens is under the control of the Le gene and independent of the Se gene.
No Lea or Leb antigens were found on epithelial
cells of Lewis-negative individuals. In contrast, Lea was
strongly expressed on Lewis-positive nonsecretors and
1187
Leb on Lewis-positive secretors, suggesting that these
two antigens are under control of both Le and Se
genes. The weak reaction of anti-Leb on nonsecretor
individuals is probably due to the Leb- Lea cross-reactivity of this antibody.
The anti-H type 2 and anti-H type 1 antibodies
were positive on epithelial cells of secretors and were
negative on epithelial cells of nonsecretors, suggesting
that they are under the control of the Se gene (Table 3).
A quite different pattern was observed on mucins
of goblet cells; the Lea and NeuAc-Lea antigens were
present in both Lewis-positive phenotypes regardless
of the secretor character, confirming that in these cells
Lea and NeuAc-Lea are not modified by the product of
the Se gene. A weak reaction of anti-Leb with Lewispositive donors is probably secondary to the Leb-Lea
cross-reactivity of this antibody. No expression of H
type 2 or H type 1 could be detected in goblet cells
(Table 3).
Carbohydrate ABO Epitopes
A series of tissue donors were selected because they
represented the main Al, A2, B, O, Lewis, and secretor phenotypes, and they were all tested immunohistologically with well characterized anti-A, anti-B, and
anti-AB reagents (Table 4).
The positive or negative results on the mucins of
goblet cells were independent of the Lewis and secretor phenotypes of the tissue donors, whereas the same
secretor and Lewis phenotypes did modulate the expression of ABO antigens on epithelial cells.
Two different patterns of anti-A reactivity were
observed: (1) A epitopes were revealed by the broad
anti-A MAb (34W1) on goblet cells and epithelial cells
of all the blood group A individuals, regardless of their
A1-A2, secretor, and Lewis phenotypes; and (2) A epitopes were revealed by the restricted anti-A (013) and
anti-AB (050) MAbs, which are present only on epithelial cells, and whose expression is modulated by the
A1-A2, secretor, and Lewis phenotypes.
The 016 anti-A MAb reacted weakly with some
goblet cells and strongly with epithelial cells, suggesting that its reactivity pattern is intermediate between
the two patterns described above.
The positive reactions of three anti-A MAbs (013,
016, and 050) on epithelial cells of A individuals were
modulated by three parameters: secretor phenotype,
A1-A2 subtypes, and Lewis phenotype (Table 4). Epithelial cells of nonsecretors were negative with these
three anti-A MAbs, except for the conjunctivas of Al
nonsecretors, which gave a weak positive reaction with
one anti-A MAb (016). In contrast, epithelial cells of
secretors were in general positive with the three MAbs;
however, this reactivity was modulated by the Al or A2
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Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3
FIGURE l. Anti-Mi (mixture of seven different MAbs)
strongly stained conjunctival goblet cells and did not stain
epithelial cells in any tissue donor, regardless of their ABO,
Lewis, and secretor phenotypes (X450). Similar staining was
obtained with anti-T MAb (AH9-17) and anti-type 1 precursor antibodies (Lec).
FIGURE 3. Anti-NeuAc-Lea (NS 19-9) strongly stained both
goblet cells and epithelial cells of Lewis-positive secretor individuals (X450). The same staining was obtained with antiLe a (7LE) on Lewis-positive nonsecretors and with anti-Le b
(2-25LE) on Lewis-positive secretors. No staining was obtained with anti-NeuAc-Lea, anti-Le3, or anti-Le b on Lewisnegative donors.
subtype and the Lewis phenotype. Al individuals gave
stronger reactions than A2 donors, and among A2 individuals, Lewis-negative donors gave stronger reactions than Lewis-positive donors.
A similar pattern was observed with the anti-B
(26 W3) and the anti-AB (050) MAbs on conjunctivas
of blood group B individuals. Almost no reactivity was
detected on goblet cells. Epithelial cells of nonsecretors were mainly negative, and only one of the two
nonsecretor individuals tested reacted with the anti-B
MAb. In contrast, all secretor individuals gave strongly
positive results with both anti-B and anti-AB MAbs
(Table 4).
Comparison of the Lewis Phenotypes Using
Hemagglutination and Immunohistology
All Lewis-positive nonsecretor tissue donors had
Le(a+b—) phenotypes on red cells, and all Lewis-negative tissue donors were of the red cell Le(a—b—) phenotype (Table 1). Most of the Lewis-positive secretors
were of the Le(a—b+) red cell phenotype, with the
exception of nine individuals who were typed as
Le(a—b—) by hemagglutination and as Lewis-positive
secretors by immunohistology. These nine individuals,
giving discordant red cell and tissue Lewis results,
were all of the Al blood subgroup. This is a frequent
problem in red cell serology with some anti-Leb reagents, because the Al red cells have fewer Leb epitopes than the other blood groups.18 Therefore, immunohistologic Lewis typing of Al individuals is more
reliable than the hemagglutination test.
DISCUSSION
FIGURE 2. Anti-H type 2 MAb (19-OLE) stained conjunctival
suprabasal epithelial cells and did not stain goblet cells of
Lewis-positive secretor individuals (X450). No staining was
obtained on nonsecretor donors.
The peptide mucin epitopes of the normal gastrointestinal tract have organ specificity.3'19 The Ml epitopes
are restricted to the surface gastric epithelium,1019
whereas the M3SI epitopes are positive only on small
intestine goblet cells.5 Ml epitopes, however, also have
been found in epithelium embryologically derived
from the anterior gut (gastric and tracheal epithelium)
and mullerian ducts (endocervical mucosa and mucous cells near the prostatic utriculus).19'20 These Ml
epitopes are strongly expressed in conjunctival goblet
cells, which also are positive with anti-T, as is gastric
mucosa. In addition, conjunctival goblet cells also dis-
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Mucin Antigens of Normal Human Conjunctiva
TABLE 3.
Summary of the Immunoperoxidase Reactions Obtained with Non-A, Non-B
Anti-Carbohydrate Antigens on the 89 Biopsies of Human Conjunctiva
According to Their Lewis and Secretor Phenotypes
Phenotype of Tissue Donor
Le
Lewis
a
b
d
Positive
Positive
Negative
Secretor No.*
%
Le"
17
63
9
19
71
10
+++
No
Yes
Yes
Goblet Cells
Epithelial Cells
N-W
Le"
Ht2
Tip
Htl
T
Le°
N-W
W
Ht2
Tip
Htl
T
r xxx x ~ ~i + xxx
:
XXX +X+ ++ +++
— —
Le" = anti-Le a (7LE); N-Le a = anti-NeuAc-Le a (NS19-9); Le b = anti-Le b (2-25LE); Ht2 = anti-H-type-2 (19-OLE); H t l = anti-H-type-1
(Led); T i p = anti-type-1-precursor (Lec); T = anti-type-3-precursor (AH9-17); Le = abbreviation combining Lewis and secretor phenotypes. Positive reactions from + to + + + represent increasing number of stained cells and intensity of staining.
* No. of individuals tested.
played NeuAc-Lea epitopes that normally are found in
intestinal mucosa.21
The conjunctival epithelial cells produce Lea and
NeuAc-Lea in Lewis-positive nonsecretors; NeuAcLea, Leb, H type 1, H type 2, A, and B in Lewis-positive
secretors; and H type 1, H type 2, A, and B in Lewisnegative secretors, suggesting that most of the carbohydrate antigens produced by these cells are under the
genetic control of the products of Le and Se genes. The
presence, however, of some A-like epitopes, detected
by the 34W1 MAb, in all individuals regardless of their
Lewis and secretor phenotypes, suggests that they also
produce some blood group-related antigens not controlled by Se and Le genes.
The two major allele variants of the A gene, Al
and A2, have been sequenced.22 The A2 allele has a
single base deletion in the coding region, which results
in a severe loss of transferase activity of the encoded
A2 enzyme. Therefore, it is not surprising that A2 tissue donors express less A antigen than Al individuals.
The goblet cells express large amounts of Lea and
NeuAc-Lea in Lewis-positive individuals, regardless of
their secretor phenotype, suggesting that these cells
can produce fucosylated epitopes under control of the
Le gene product, but that they do not express the product of the Se gene, as has already been found in the
goblet cells of distal rectal colonic mucosa. In good
agreement with this hypothesis, all the A-like epitopes
detected on goblet cells were independent of the secretor phenotype of the tissue donor, suggesting that
another gene product, different from the secretor enzyme, may be responsible for the synthesis of the precursors of these A-like epitopes detected by MAbs 016
and 34W1 on goblet cells.
TABLE 4.
Summary of the Immunoperoxidase Reactions Obtained on Epithelial and Goblet
Cells With Anti-A (013, 016, 34W1), Anti-B (26W3), and Anti-AB (050) Monoclonal
Antibodies on Some Tissue Donors Selected Because They Were of Known Al and A2
Subtypes and/or Represented the Main ABO, Lewis, and Secretor Phenotypes
Phenotype of Tissue Donor
Epithelial Cells
ABO
Le
Lewis
Secretor
No.
A2
Al
a
a
b
b
d
d
a
b
d
a
b
d
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Positive
Negative
Positive
Positive
Negative
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
2
5
1
4
1
3
2
3
2
1
1
1
A2
Al
A2
Al
B
B
B
O
O
O
013
016
Goblet Cells
34W1
26W3
050
013
016
34W1
Le = abbreviation combining Lewis and secretor phenotypes. Positive reactions from + to + + + represent increasing number of stained
cells and intensity of staining.
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Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3
Another difference in the carbohydrates expressed by epithelial and goblet cells is the presence of
large amounts of precursor chain epitopes in goblet
cells, whereas there is none on epithelial cells, suggesting that these last cells are more efficient in the fucosylation of precursor chains, and that they have transformed all the precursor chains into the corresponding Lea, Leb, and ABH antigens.
Using immunoperoxidase, we found 19% Lewispositive nonsecretors (Le/—, se/se), 71% Lewis-positive
secretors (Le/—, Se/—), and 10% Lewis-negative secretors (le/le, Se/—). These results are very close to the
expected incidences of the Lewis and secretor phenotypes in the French population. Because the carbohydrates expressed by each of these phenotypes in normal individuals are different, it would be interesting to
determine the frequency of the ABO, Lewis, and secretor phenotypes in the different conjuncdval pathologies to see if the different carbohydrates expressed
by each phenotype play a role in susceptibility to certain diseases.
Changes in components of conjunctival mucus
probably are responsible for the poor quality of tears
associated with distinctive clinical features seen in dry
eyes, neuroparalytic keratitis, or pterygium.* >23'24 Characterization of mucins with MAbs has been used essentially to characterize conjunctival features of cells collected on limbus or on cell cultures.2526 Histochemical
techniques using Alcian blue or periodic acid—Schiff
reagents already have suggested that the shift in acid
glycoprotein induces increased viscosity.27"29 With the
lectin-colloidal gold technique, it was found that dryeye patients presented a decrease in neuraminic acid,
JV-acetylgalactosamine, JV-acetylglucosamine, and galactose-7V- acetylgalactosamine.30 Previous work suggested that the NeuAc terminal should be able to influence the viscous properties of mucus.31 Furthermore,
other authors assumed that the degree of sulfation of
the mucin is closely correlated with mucous disturbance, and consequently with conjunctival metaplasia
as well.32
We have found that according to ABO, A1-A2,
Lewis, and secretor phenotypes, conjunctival mucins
are differently glycosylated. The above-mentioned results strongly suggest that the genetic controls of the
expression of carbohydrate epitopes by goblet cells
and epithelial cells are not identical. The effect of the
Le gene product predominates in goblet cells, whereas
both the products of Se and Le genes seem to play
equivalent roles in epithelial cells. The result of this
genetic difference is a difference in the expression of
the final carbohydrate epitopes in these two types of
cells. Mucins are found mainly in goblet cells. Epithelial cells, however, could be the source of a second type
of mucin, and differences between the secretions of
goblet and epithelial cells already have been described
in contact lens wearers.3334
Key Words
mucin, conjunctiva, ABO, Lewis, blood groups
References
1. Adams AD. The morphology of the human conjunctival mucus. Arch Ophthalmol. 1979;97:730-734.
2. Carlstedt I, Lindgren H, Sheehan JK. The macromolecular structure of the human cervical-mucus glycoproteins. BiochemJ. 1983;213:427-435.
3. BaraJ. Organ-specific mucin antigens and gastrointestinal carcinogenesis. Italian Journal of Gastroenterology.
1991;23:29-35.
4. BaraJ, Gautier R, Le Pendu, Oriol R. Immunochemical characterization of mucin: polypeptide (Ml) and
polysaccharide (A and Leb) antigens. Biochem J.
1988;254:185-193.
5. Kordari P, Zamora PO, Kjeldsen T, Gautier R, BaraJ.
Comparative study of 4 Mabs against sialyl-Tn-related
antigens in normal, fetal and cancerous colonic mucosae. Tumor Biol. 1990; 11:111. Abstract.
6. Oriol R, Le Pendu J, Mollicone R. Genetics of ABO,
H, X, and related antigens. Vox Sang. 1986;51:161171.
7. Clausen H, Hakomori SI. ABH and related histoblood group antigens: immunochemical differences in
carrier isotypes and their distribution. Vox Sang.
1989;56:l-20.
8. Oriol R. Genetic control of the fucosylation of ABH
precursor chains: evidence for new epistatic interactions in different cells and tissues. J Immunogenet.
1990; 17:235-245.
9. Oriol R, Le Pendu J, Sparkes RS, et al. Insights into
the expression of ABH and Lewis antigens through
human bone marrow transplantation. Am J Hum
Genet. 1981;33:551-560.
10. BaraJ, Gautier R, Mouradian P, Decaens C, Daher N.
Oncofetal mucin Ml epitope family: characterization
and expression during colonic carcinogenesis. Int J
Cancer. 1991; 47:304-310.
11. BaraJ, Daher N, Mollicone R, Oriol R. Immunohistological patterns of 20 monoclonal antibodies against
non-A, non-B glycoconjugates in normal human pyloric and duodenal mucosae. Blood Transf Immunohaematol. 1987; 30:685-692.
12. Koprowski H, Steplewski Z, Mitchell K, et al. Colorectal carcinoma antigens detected by hybridoma antibodies. Somatic Cell Genet. 1979; 5:957-971.
13. Candelier JJ, Mollicone R, Mennesson B, et al. a-3Fucosyltransferases and its glycoconjugate antigen
products in the developing human kidney. Lab Invest.
1993;69:449-459.
14. Mollicone R, Gane P, Rouger P, Oriol R. Inhibition of
haemagglutination with Synsorbs and salivas of anti-B
and anti-AB monoclonal antibodies. Blood Transf Immunohaematol. 1987; 30:570-587.
15. Chester MA, Johnson U, Lundblad A, et al, eds. Pro-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/08/2017
Mucin Antigens of Normal Human Conjunctiva
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
ceedings of the Second International Workshop and Symposium on Monoclonal Antibodies Against Human Red Blood
Cells and Related Antigens. Lund, Sweden: 1990:208219.
26.
Oriol R, Samuelsson BE, Messeter L. Workshop 1:
ABO antibodies—serological behaviour and immunochernical characterization. J Immunogenet. 1990; 17:
27.
279-299.
Graham RC, Lundholm U, Karnovsky MJ. Cytochemical demonstration of peroxidase activity with 3-amino9-ethylcarbazole. J Histochem Cytochem. 1965; 13:150- 28.
152.
Le Pendu J, Lemieux RU, Dalix AM, Lambert F, Oriol
R. Competition between ABO and Le gene specified
enzymes. Vox Sang. 1983;45:349-358.
BaraJ, Gautier R, Daher N, Zaghouani H, Decaens C.
29.
Monoclonal antibodies against oncofetal mucin Ml
antigens associated with precancerous colonic mucosae. Cancer Res. 1986;46:3983-3989.
30.
Daher N, Gonzales J, Gautier R, BaraJ. Evidence of
mucin Ml antigens in seminal plasma and normal cells
31.
of human prostatic urethra in relation to embryonic
development and tumors. Prostate. 1990; 16:57-69.
BaraJ, Herrero-Zabaleta ME, Mollicone R, Nap M,
32.
Burtin P. Distribution of GICA in normal gastrointestinal and endocervical mucosae and mucinous ovarian
cysts using antibody NS 19-9. Am J Clin Pathol.
1986;85:152-159.
Yamamoto FI, McNeill PD, Hakomori SI. Human
33.
histo-blood group A2-transferase coded by A2-allele,
one of the subtypes, is characterized by a single base
deletion in the coding sequence, which results in an
additional domain at the carboxyl terminal. Biochem
Biophys Res Commun. 1992; 187:366- 374.
Wright P, Mackie IA. Mucus in healthy and diseased
34.
eye. Trans Ophthalmol Soc UK. 1977; 97:1-7.
Holly FJ. Tear film physiology. Int Ophthalmol Clin.
1987;5:149-159.
1191
Tseng TSG, Huang AJW, Sutter D. Purification and
characterization of rabbit ocular mucin. Invest Ophthalmol VisSci. 1987;28:1473-1482.
Huang AJ, Tseng TSG. Development of monoclonal
antibodies to rabbit ocular mucin. Invest Ophthalmol
VisSci. 1987;28:1483-1497.
Greiner JV, Weidman TA, Korb DR. Histochemical
analysis of secretory vesicles in non goblet conjunctival epithelial cells. Ada Ophthalmol. 1985;63:89-92.
Yamabayashi S, Tsakahara S. Histochemical studies on
the conjunctival goblet cells: I. (Alcian blue) A.B., (periodic Acid-Schiff) P.A.S. staining and P.A.S-A.B.
staining (with 2 color plates). Ophthalmic Res.
1987;19:137-140.
Me Ghee CNJ, Lee WR. Quantification and enzymatic
identification of conjunctival surface mucus. Trans
Ophthalmol Soc UK. 1985; 104:446-449.
Moore JC, Tiffany JM. The ocular mucus: chemical
properties. Exp Eye Res. 1981;33:203-212.
Kawano K, Uehara F, Ohba N. Lectin-cytochemical
study on epithelium mucus glycoprotein of conjunctiva and pterygium. Exp Eye Res. 1988;47:439-447.
Versura P, Maltrello MC, Cellini M. Detection of
mucus glycoconjugates in human conjunctiva by using
the lectin-colloidal gold technique in TEM: II. a quantitative study in dry-eye patient. Acta Ophthalmol.
1986;64:451-455.
Versura P, Maltrello MC, Cellini M, et al. Detection of
mucus glycoconjugates in human conjunctiva by using
the lectin-colloidal gold technique in TEM: III. a
quantitative study in asymptomatic contact lens
wearers. Acta Ophthalmol. 1987; 65:661-667.
Greiner JV, Kenyon KR, Henriquez AS, et al. Mucus
secretory vesicles in conjunctival epithelial cells of
wearers of contact lenses. Arch Ophthalmol.
1980;98:1843-1846.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/08/2017