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Am J Physiol Gastrointest Liver Physiol
278: G915–G923, 2000.
Accessibility of glycolipid and oligosaccharide epitopes
on rabbit villus and follicle-associated epithelium
NICHOLAS J. MANTIS, ANDREAS FREY, AND MARIAN R. NEUTRA
Department of Pediatrics, Harvard Medical School, and Gastrointestinal Cell Biology Laboratory,
Children’s Hospital, Boston, Massachusetts 02115
Peyer’s patch; glycocalyx; adhesion; pathogen; M cells
ENTERIC PATHOGENS SELECTIVELY infect specific epithelial cell types in the gastrointestinal tract (1, 6, 25). For
example, rotavirus replicates within villus enterocytes
(39), whereas reovirus selectively binds to, and is
endocytosed by, M cells (51). Adherence, the initial step
in the infection process, is postulated to be mediated by
lectinlike microbial adhesins that recognize defined
glycoproteins and/or glycolipid epitopes on the apical
surfaces of intestinal epithelial cells (6, 22, 25, 27).
Lectinlike adhesins have been identified on a wide
array of enteroviruses and pathogenic bacteria (1, 6, 19,
27). For example, rotavirus binds the glycosphingolipid
asialo-GM1 and O-linked sialylglycoproteins in overlay
assays (49, 50), and reovirus attachment to mouse
fibroblasts in vitro depends on the presence of host cell
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sialoglycoconjugates (14). However, because most of
these glycolipids and galactosyl- or sialic acid-containing oligosaccharides are widely distributed on intestinal epithelial cells, receptor distribution alone cannot
explain the cell type-specific tropisms of viral and
bacterial pathogens that are observed in vivo. Thus
additional factors must exist that influence pathogen
attachment to specific cells or regions of the gastrointestinal mucosa (26, 27).
The intestinal epithelium is comprised primarily of
absorptive villus enterocytes (28). Enterocyte apical
surfaces in vivo are highly differentiated structures
consisting of rigid, closely packed microvilli whose
membranes contain stalked glycoprotein enzymes (33,
45). In addition, the tips of enterocyte microvilli are
coated with a 400- to 500-nm-thick meshwork called
the filamentous brush border glycocalyx (FBBG) (23),
which is composed of highly glycosylated transmembrane mucins (29, 30). The epithelium overlying
organized mucosal lymphoid nodules, the so-called
follicle-associated epithelium (FAE), is comprised of
enterocyte-like cells interspersed with M cells. The M
cell is a morphologically distinct epithelial cell type
whose primary function is the transport of macromolecules, particles, and microorganisms from the lumen
to underlying lymphoid tissue (11, 35). M cells generally lack an organized brush border and a well-defined
FBBG, although their apical membranes have a thin
(20–30 nm) glycoprotein coat (10).
We have recently shown that the FBBG on the apical
surfaces of rabbit villus and FAE enterocytes is a
size-selective barrier that can prevent particles from
gaining access to membrane glycolipids (10). This was
demonstrated using the B subunit of cholera toxin
(CTB), which specifically binds to ganglioside GM1, a
glycolipid whose carbohydrate head extends just 2.5
nm from the plasma membrane (47). When applied in
soluble form to rabbit mucosa, CTB (diameter 6.8 nm)
had free access to GM1 on all epithelial cell types and
bound to villus enterocytes, FAE, and M cells (10).
When CTB was coupled to colloidal gold to form a
particulate probe 28 nm in diameter, it was no longer
able to penetrate the FBBG and was therefore unable
to bind to enterocytes, but the particles did adhere to
the apical surfaces of M cells. The thin glycoprotein
coat on M cells can prevent larger particles from
gaining access to ganglioside GM1, however, because
CTB-coated, 1-µm particles were unable to bind. Our
previous studies did not test whether the M cell glyco-
0193-1857/00 $5.00 Copyright r 2000 the American Physiological Society
G915
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Mantis, Nicholas J., Andreas Frey, and Marian R.
Neutra. Accessibility of glycolipid and oligosaccharide epitopes on rabbit villus and follicle-associated epithelium. Am J
Physiol Gastrointest Liver Physiol 278: G915–G923, 2000.—
The initial step in many mucosal infections is pathogen
attachment to glycoconjugates on the apical surfaces of
intestinal epithelial cells. We examined the ability of virussized (120-nm) and bacterium-sized (1-µm) particles to adhere to specific glycolipids and protein-linked oligosaccharides on the apical surfaces of rabbit Peyer’s patch villus
enterocytes, follicle-associated enterocytes, and M cells. Particles coated with the B subunit of cholera toxin, which binds
the ubiquitous glycolipid GM1, were unable to adhere to
enterocytes or M cells. This confirms that both the filamentous brush border glycocalyx on enterocytes and the thin
glycoprotein coat on M cells can function as size-selective
barriers. Oligosaccharides containing terminal b(1,4)-linked
galactose were accessible to soluble lectin Ricinus communis
type I on all epithelial cells but were not accessible to lectin
immobilized on beads. Oligosaccharides containing a(2,3)linked sialic acid were recognized on all epithelial cells by
soluble Maackia amurensis lectin II (Mal II). Mal II coated
120-nm (but not 1-µm) particles adhered to follicle-associated
enterocytes and M cells but not to villus enterocytes. The
differences in receptor availability observed may explain in
part the selective attachment of viruses and bacteria to
specific cell types in the intestinal mucosa.
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GLYCOCONJUGATE ACCESSIBILITY ON INTESTINAL EPITHELIUM
MATERIALS AND METHODS
Chemicals and reagents. All lectins, lectin derivatives, and
fluorophore-conjugated streptavidin reagents were purchased from Vector Laboratories (Burlingame, CA). Biotinylated CTB was purchased from List Laboratories (Campbell,
CA). BSA, biotinylated BSA, and biocytin were from Sigma
(St. Louis, MO). Neutravidin-coated, fluorescent, carboxylatemodified polystyrene particles (FluoSpheres) were purchased
from Molecular Probes (Eugene, OR). Liquid paraformaldehyde (16% solution) was obtained from Electron Microscopy
Sciences (Fort Washington, PA).
Preparation of ligand-coated particles. The fluorescent red
or green virus-sized particles used in this study had an
estimated diameter of 0.093 µm 6 7.5%. The manufacturer
coated them with Neutravidin, resulting in ,10 nmol biotin
binding sites per milligram of polystyrene. To derivitize
particles with ligands, 1 3 1012 particles were suspended in
500 µl of PBS (50 mM sodium phosphate, pH 7.4, 50 mM
NaCl) containing 0.02% Tween-20, 0.5% BSA, 2 mM NaN3,
and 50 µg/ml of biotinylated ligand or biocytin. The particle
mixtures were gently rocked overnight at 4°C and then
transferred to a 0.5-ml Dispodialyzer dialysis tube (Spectrum, Houston, TX) with a molecular weight cut-off of 300,000.
Excess unbound ligand was removed by dialysis against 4 l of
PBS containing 2 mM NaN3 changed daily for 4 days. In
control experiments, this dialysis protocol was sufficient to
remove .95% of soluble ligand. After dialysis, particles were
transferred to siliconized microcentrifuge tubes and stored at
4°C in the dark. The bacterium-sized (1.0 µm 6 2.5%)
particles were coated with biotinylated ligand as described
previously (10). Briefly, particles and biotinylated ligand (400
µg/ml) were combined in 1 ml of PBS and incubated with
gentle rocking overnight at 4°C. The particles were then
collected by centrifugation (3,500 g) and washed with PBS
several times to remove excess, unbound ligand. The particles
were resuspended in 400 µl PBS containing 2 mM NaN3 and
then stored in the dark at 4°C. Before use in adhesion assays,
particles were vortexed for 2 min at room temperature and
then bath sonicated (Laboratory Supply, Hicksville, NY) in an
ice slurry for 10 min. Ligand-coated, fluorescent green particles and biocytin-quenched (or BSA-coated), fluorescent red
particles were diluted at a 1:1 ratio as determined by fluorescence microscopy into sterile PBS containing BSA (0.1%
wt/vol) and stored on ice until ready to use. BSA-coated and
biocytin-coated particles were used interchangeably as controls. The final diameter of the virus-sized, CTB-coated
particles was estimated to be 120 nm. This value was
calculated knowing that the diameter of the carboxylatemodified latex beads was 93 nm and assuming a uniform coat
of Neutravidin (7 nm) and CTB-biotin (6.5 nm), as described
previously (10). Although the hydrodynamic diameters of
RCA-I [relative molecular weight (Mr) 120,000] and Mal II
(Mr 140,000) are undetermined, we expect that they are
slightly greater than that of avidin (Mr 60,000).
Cell culture. The Caco-2BBe cell line was a generous gift
from Dr. Mark Mooseker (Yale University). Caco-2 cells were
grown in high-glucose DMEM (GIBCO BRL, Rockville, MD)
supplemented with 10% FCS (HyClone Labs, Logan, UT),
penicillin (100 IU/ml), streptomycin (100 µg/ml), and glutamine (2 µg/ml) at 37°C with an atmosphere containing 5%
CO2, as described previously (41). For particle adhesion
assays, cells were seeded at 2 3 105 cells/ml onto acetonewashed, 12-mm-diameter glass coverslips (Bellco Glass, Vineland, NJ) in 12-well plates (Costar, Cambridge, MA), as done
previously (10). Seven days after confluence, cells were washed
three times with PBS and then overlaid with soluble, biotinylated lectins (100 µg/ml) or particle mixtures and incubated
with gentle rocking for 1 h at room temperature. Cells on
coverslips were washed three times with PBS, fixed by
immersion in formaldehyde (4% vol/vol in PBS), then inverted and mounted using Mowiol 50.5 g/ml glycerol, 0.1 g/ml
Mowiol 4–88 (Calbiochem, San Diego, CA), 10 mg/ml
diazabicylo[2.2.2]octane (Sigma) in 0.1 M Tris · HCl (pH 8.5)6
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protein coat can exclude particles in the size range of
most viruses from contact with membrane glycolipids
in vivo. This is important because it has been shown
that HIV-1 (120 nm in diameter) attaches to intestinal
cells in culture via galactosylceramide (9, 12). HIV can
adhere to M cells in rabbit intestine (2) and may
possibly exploit M cell transport activity to enter the
rectal mucosa in humans (34).
Protein-linked oligosaccharides would be expected to
provide more accessible binding sites for microorganisms because they extend up to 500 nm from enterocyte
plasma membranes and 20–30 nm from M cell membranes (10). They would thus be the first structures
encountered by intestinal pathogens (25). Integral membrane mucins such as those of the enterocyte FBBG
consist of up to 80% heterogeneous O-linked carbohydrates that form dense, negatively charged gels thought
to function in cytoprotection (48). Oligosaccharide side
chains on both mucin and nonmucin membrane glycoproteins are notoriously heterogeneous, and the exact
composition, structure, and topology of only a small
minority have been defined (25). There is evidence that
glycosylation patterns not only of M cells but of the
entire FAE differ from those of villus epithelium (5, 13,
17, 18, 46), but the exact topology or accessibility of
specific oligosaccharide structures on epithelial cells of
small intestinal villi and FAE has not been explored.
Such information could be important for understanding
microbial tropism in the intestine and for designing
strategies for targeting of particulate vaccines and
vaccine vectors to the FAE and M cells.
For this study we selected three ligands that recognize glycolipid or protein-linked oligosaccharide epitopes that are known to be exploited by enteric pathogens. We examined the ability of the ligands immobilized
on virus-sized (120-nm) and bacterium-sized (1-µm)
particles to adhere to apical surfaces of rabbit Peyer’s
patch villus enterocytes, FAE, and M cells. Particles
were coated with CTB to probe accessibility of the
glycolipid GM1, the lectin Ricinus communis agglutinin type I (RCA-I) specific for terminal b(1, 4)-linked
galactose, and Maackia amurensis lectin II (Mal II),
which recognizes a hierarchy of oligosaccharides containing a(2,3)-linked sialic acid. Data presented indicate that the relatively thin glycoprotein coat on the
apical surfaces of M cells is sufficient to prevent virussized particles from gaining access to glycolipids like
GM1. In addition, we found that protein-linked oligosaccharides vary in their accessibility to particulate ligands and that differences in accessibility exist between villus and FAE. We conclude that successful
attachment of pathogens to glycolipids and glycoproteins on apical surfaces of intestinal epithelial cells is
dependent on both receptor availability and receptor
accessibility.
GLYCOCONJUGATE ACCESSIBILITY ON INTESTINAL EPITHELIUM
have a variable degree of nonspecific binding to intestinal
mucosa, 1:1 mixtures of ligand-coated and control particles
were applied in all experiments. For each experimental
condition we analyzed 40 tissue sections, determining the
numbers of ligand-coated particles (fluorescent green) and
control particles (fluorescent red) bound to villus-associated
epithelium and FAE. In specimens in which specific binding
occurred, there were on average .5,000 total particles bound
to 40 sections. For each tissue section we divided the number
of ligand-coated particles by the number of control particles to
obtain a relative index of binding. Binding indices were then
averaged. All experiments were done at least twice using two
independently prepared batches of ligand and ligand-coated
particles and two different rabbits.
RESULTS
CTB-coated, 120-nm particles did not adhere to live
Peyer’s patch mucosa but adhered selectively to M cells
on aldehyde-fixed tissues. To probe glycolipids on the
apical surfaces of intestinal epithelial cells, we applied
soluble, biotinylated CTB to rabbit Peyer’s patch mucosa. As observed previously (10), we found that CTB
bound to GM1 in apical plasma membranes of all
intestinal epithelial cells on live and formaldehydefixed Peyer’s patch mucosa (Fig. 1A). Biotinylated BSA
applied at identical concentrations to Peyer’s patch
mucosa did not label epithelial cells (Fig. 1B). To test
whether virus-sized particles have access to GM1, we
applied CTB-coated 120-nm particles (fluorescent green)
mixed 1:1 with biocytin-coated control particles (fluorescent red) to live Peyer’s patch explants. We observed
that very few particles bound to either villus or FAE
and that equal numbers of CTB-coated and control
particles were associated with the mucosal surface
(Table 1). Thus the FBBG on villus and FAE enterocytes as well as the thin glycoprotein coat on M cells
were sufficient to prevent these virus-sized particles
from gaining access to glycolipid receptors.
Tissue fixation with formaldehyde cross-links glycoproteins and may unmask certain glycoconjugate epitopes not normally available in vivo (20). We therefore
predicted that aldehyde fixation might sufficiently alter
the thin glycoprotein coat on M cells to render membrane glycolipids accessible to particles but that this
might not be the case for the thick FBBG on enterocytes. To test this hypothesis, CTB-coated 120-nm
particles and control particles were applied to aldehydefixed Peyer’s patches, and binding was visualized by
fluorescence microscopy. On fixed tissue we observed
that CTB-coated particles adhered in greater numbers
than control particles and that CTB-coated particles
adhered preferentially to M cells and not to enterocytes
on FAE or adjacent villi (Fig. 1C; Table 1).
RCA-I binding sites on intestinal epithelial cells were
inaccessible to 120-nm particles. Galactose-containing
glycoproteins are proposed to be important attachment
sites for enteric bacteria. To confirm the distribution of
carbohydrates containing terminal b(1,4)-linked galactose, we applied soluble, biotinylated RCA-I to live or
fixed Peyer’s patch mucosa. RCA-I labeled the surfaces
of all epithelial cells strongly and uniformly on live
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onto Fisher Superfrost microscope slides (Fisher Scientific,
Pittsburgh, PA) and viewed by fluorescence or phase-contrast
microscopy.
Application of ligand-coated particles to live or fixed rabbit
Peyer’s patch mucosa. New Zealand White female rabbits
weighing ,1.5 kg each were purchased from Charles River
Laboratories (Wilmington, MA) and maintained in the animal resource facility at the Children’s Hospital. All animal
procedures were conducted in strict compliance with Guidelines for Animal Experimentation established by Harvard
Medical School, the Children’s Hospital, and the National
Institutes of Health. Animals were fasted overnight with
water ad libitum before surgery. To obtain Peyer’s patches,
rabbits were sedated with an intramuscular injection of 0.5
ml acetopromazine maleate (10 mg/ml) followed 45 min later
by an intramuscular injection of ketamine (80 mg/kg) and
xylazine (20 mg/kg). Animals were laparotomized and jejunoileal Peyer’s patches were excised and treated as described
below. Rabbits were euthanized by a single intravenous
injection of pentobarbital sodium (100 mg/kg) into the marginal ear vein.
Freshly excised Peyer’s patches were washed in PBS
(25°C), and the luminal surfaces were blotted lightly with
Whatman paper (3 MM) to remove excess mucus. Peyer’s
patches were then pinned to surgical cork board, and the
mucosa was removed from the muscularis externa using a
single-edged razor blade. For experiments using live tissue,
the mucosae were cut into 2 3 2 mm pieces and each was
placed in a single well of a 12-well tissue culture plate
(Costar), which had been previously treated with RPMI 1640
(GIBCO BRL) and BSA (0.5% wt/vol). Taking special care not
to damage the epithelium, the mucosa was washed with
RPMI 1640 and overlaid with control and experimental
particles (4 3 1010 total particles in 0.5 ml) or soluble,
biotinylated lectins (100 µg/ml) and incubated at room temperature for 40 min. The tissue was carefully rinsed three
times with PBS and either fixed for cryosectioning by immersion in formaldehyde (4% vol/vol) in PBS or for plastic
embedding by immersion in formaldehyde (4% vol/vol) in 0.1
M cacodylate buffer (pH 7.2) (17). Lectin-treated tissue
destined for cryosectioning was washed with PBS, incubated
with glycine (0.1 M in PBS) to quench residual reactive
aldehydes, blocked in PBS containing BSA (0.5% wt/vol) for 1
h, and labeled with fluorophore-conjugated streptavidin (50
µg/ml). For experiments using prefixed tissue, freshly excised
mucosae were immersed in four volumes of formaldehyde (4%
vol/vol) in PBS for 12 h. The tissue samples were then cut into
2 3 2 mm blocks, washed with PBS, incubated in 0.1 M
glycine in PBS to quench residual reactive aldehydes, and
blocked in PBS containing BSA (0.5% wt/vol) for 1 h. Blocks of
tissue were then overlaid with lectins or particles for 40 min,
rinsed, fixed again, and processed as described above for live
tissue.
Tissue sectioning, microscopy, and quantitation of particle
binding. For cryosectioning, tissue samples were incubated
for 1 h in sucrose (15% wt/vol in PBS), followed by 10 min in
Tissue-Tek optimum cutting temperature embedding medium (Sakura Finetek, Torrance, CA), frozen in liquid nitrogen-cooled isopentane, and stored at 220°C. Frozen tissue
sections were cut at 8–10 µm using a Leica cryostat model
CM3050 (Nussloch, Germany). Sections were captured on
Fisher Superfrost microscope slides and mounted with coverslips using Mowiol. Slides were viewed under oil immersion
using a Zeiss Axiophot microscope (Carl Zeiss, Thornwood,
NY) equipped for epifluorescence and photographed using a
35 mm camera. Because protein-coated polystyrene particles
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G918
GLYCOCONJUGATE ACCESSIBILITY ON INTESTINAL EPITHELIUM
Fig. 1. Adhesion of soluble B subunit of cholera toxin (CTB) and
CTB-coated, 120-nm particles to Peyer’s patch epithelium. A: aldehyde-fixed rabbit Peyer’s patch explants were exposed to soluble,
biotinylated CTB (100 µg/ml) for 40 min, labeled with streptavidinrhodamine, and viewed by fluorescence microscopy. Soluble CTB
labeled apical surfaces of cells in follicle-associated epithelium (FAE)
(bottom) and villus epithelium (top). B: on same tissue, soluble,
biotinylated BSA (100 µg/ml) failed to label apical surfaces of
epithelial cells. C: aldehyde-fixed Peyer’s patch explants were exposed to an equal number of CTB-coated, 120-nm particles (green)
and biocytin-quenched particles (red), cryosectioned, and viewed by
fluorescence microscopy. Representative micrograph shows CTBcoated particles bound in greater numbers than control particles to
apical surface of M cells, which were identified by dark areas
representing M cell pockets. Scale bars 5 100 µm.
(Fig. 2, A and B) and fixed tissue (data not shown). To
test whether the galactose epitopes recognized by RCA-I
are accessible to virus-sized particles in vivo, we applied a 1:1 mixture of RCA-I-coated 120-nm particles
(fluorescent green) and biocytin-quenched control par-
Table 1. Binding of ligand-coated particles to rabbit
Peyer’s patch villus and follicle-associated epithelium
Live Tissue
Fixed Tissue
Particles,
Diameter
Villus
FAE
Villus
FAE
CTB, 120 nm
RCA-1, 120 nm
Mal II, 120 nm
Mal II, 1 µm
1.1 (0.08)
0.9 (0.06)
1.5 (0.2)
1.1 (0.8)
1.0 (0.08)
1.0 (0.1)
4.5 (0.3)
1.5 (0.9)
1.1 (0.05)
n.d.
37 (4.7)
25 (1.8)
2.9 (0.2)
n.d.
54 (7.1)
28 (5.0)
Live or fixed tissue explants were exposed to a one-to-one mixture
of ligand-coated and control particles for 40 min, washed, cryosectioned, and then viewed by fluorescence microscopy as described in
MATERIALS AND METHODS. Number of ligand-coated and control particles bound to either villus epithelium or follicle-associated epithelium (FAE) was determined for 40 sections. For each section, number
of ligand-coated particles was divided by number of control particles,
and these values were averaged to obtain a relative index of binding.
These indices are shown with standard errors in parentheses. CTB, B
subunit of cholera toxin; RCA-I, Ricinus communis type 1; Mal II,
Maackia amurensis lectin II; n.d., not determined.
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ticles (fluorescent red) to live rabbit Peyer’s patch
mucosa. The RCA-I particles did not show specific
binding to any epithelial cells (Table 1). Both RCA-I
and control particles adhered sparsely (Fig. 2C) and
RCA-I-coated particles did not bind to the intestinal
epithelium any better than control particles. On the
FAE, nonspecific particle binding occurred primarily on
the apical surfaces of M cells. This was not surprising
since rabbit Peyer’s patch M cells have been shown
previously to actively phagocytose uncoated polystyrene particles (10, 37). Aldehyde fixation did not enhance the availability of terminal b(1, 4) galactose sites
to RCA-I-coated particles (data not shown).
To exclude the possibility that the particle-coating
procedure destroyed the receptor-binding activity of
RCA-I, the same green-red particle mixture was applied to confluent monolayers of Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma (41). We previously established that within 7
days after confluence, the cloned Caco-2 cells used in
this study express RCA-I recognition epitopes on their
apical surfaces (15) but lack a thick FBBG (10). We
observed that RCA-I-coated, 120-nm particles adhered
to live or fixed Caco-2 cells 39 times better than control
particles (Fig. 2, E and F). RCA-I particle binding could
be inhibited by coadministration of 250 µg/ml soluble
RCA-I (data not shown). These data indicate that the
particles were sufficiently coated with active lectin to
impart RCA-I-dependent binding to cells. Moreover,
this result underscores the fact that the accessibility of
protein-linked oligosaccharide epitopes on epithelial
cells grown in vitro may not be the same as on
enterocytes in vivo.
Mal II-coated 120-nm particles preferentially adhered to the FAE. We next examined the accessibility of
carbohydrate structures containing a(2,3)-linked sialic
acid to virus-sized particles. On live and fixed tissue,
soluble, biotinylated Mal II adhered to the luminal
surfaces of villus enterocytes, FAE, and M cells (Fig. 3).
On the FAE, Mal II labeled the apical surfaces of
enterocytes somewhat more intensely than the apical
GLYCOCONJUGATE ACCESSIBILITY ON INTESTINAL EPITHELIUM
G919
surfaces of M cells, possibly reflecting an abundance of
lectin-binding sites in the thick FBBG (data not shown).
To examine to what degree the Mal II epitopes are
accessible to 120-nm particles, we applied a 1:1 mixture
of Mal II-coated particles (fluorescent green) and biocytin-quenched control particles (fluorescent red) to live
rabbit Peyer’s patch explants. On villus epithelium,
Mal II-coated particles bound in very low numbers and
the ratio of Mal II to control particles was ,1:1 (Table
1). However, on the FAE Mal II particles adhered 4.5
times better than control particles (Fig. 4A and Table
1). Fluorescence microscopy of plastic-embedded thin
sections confirmed that Mal II particles bound to both
FAE and M cells (data not shown). These data suggest
that although glycoconjugates recognized by Mal II
epitopes are present on the apical surfaces of all
intestinal epithelial cells in vivo, they are only accessible to virus-sized particles on the FAE.
To test whether cross-linking of the FBBG could
expose additional a(2,3)-linked sialic acid-containing
sites to virus-sized particles, Mal II-coated particles
were applied to aldehyde-fixed Peyer’s patch mucosa.
On fixed tissue, specific binding of Mal II particles to
FAE and M cells was enhanced (Fig. 4B and Table 1).
In addition, Mal II particles now adhered to villus
enterocytes. From these data we conclude that the
a(2,3)-linked sialic acid-containing oligosaccharides in
the FBBG of villus enterocytes, although inaccessible
to virus-sized particles on living tissue, are readily
exposed by fixation. This is in contrast to the glycolipids
that lie deep to the glycocalyx in the plasma membrane.
Accessibility of oligosaccharides to bacterium-sized
(1-mm) particles. In vitro binding assays have revealed
bacterial adhesins that recognize a diversity of oligosaccharides and glycolipids (1, 19, 22, 43). Adhesins are
often located on the tips of fimbriae and pili that extend
hundreds of nanometers from the bacterial surface,
suggesting that they may be able to penetrate the
surface coats of host cells. This could be particularly
important in the intestine, where the thick FBBG of
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Fig. 2. Binding of soluble Ricinus communis
type I (RCA-I) and 120-nm RCA-I particles to
Peyer’s patch epithelium and Caco-2 cell monolayers. Live Peyer’s patch explants were exposed to RCA-I biotin (A and B) or a 1:1
mixture of RCA-I particles (green) and biocytin particles (red) (C and D) for 40 min. A:
RCA-I biotin (100 µg/ml) visualized by streptavidin-rhodamine-labeled apical surfaces of all
epithelial cells present in FAE. B: RCA-I
biotin (100 µg/ml) visualized by streptavidinfluorescein-labeled apical surfaces of all villus
epithelial cells. C: RCA-I particles and biocytin particles (red) adhered in equal numbers
to M cells present in FAE (arrow) and occasionally to goblet cells on villi (top). D: phasecontrast image of C. Arrow indicates apical
surface of an M cell. E–F: same particle
mixture used in experiment shown in C was
applied to a monolayer of Caco-2 cells. E:
monolayer viewed by confocal laser scanning
fluorescence microscopy. RCA-I particles
(green) bound .20 times better than biocytin
particles (red). F: phase-contrast image of
Caco-2 monolayer used in experiment described in E. Scale bar 5 100 µm.
G920
GLYCOCONJUGATE ACCESSIBILITY ON INTESTINAL EPITHELIUM
cated that the a(2,3)-linked sialic acid-containing determinants present on the FAE that were accessible to
virus-sized particles were not accessible to particles as
large as bacteria. When the same particle mixture was
applied to fixed tissue, Mal II particles adhered to both
FAE and villus epithelium .25 times better than
control particles (Fig. 4D, Table 1). This confirmed that
cross-linking of cell surface glycoprotein coats resulted
in exposure of the normally sequestered sialic acidcontaining epitopes.
DISCUSSION
enterocytes faces an environment rich in microorganisms. On this basis one might predict that many of the
epithelial glycolipid and oligosaccharide structures that
are potential microbial binding sites might be inaccessible to lectins or toxins immobilized on the surfaces of
inert bacterium-sized particles that lack appendages.
Indeed, we previously showed that ganglioside GM1 on
live rabbit Peyer’s patch mucosa is not accessible to
CTB-coated, 1-µm beads (10).
To test the accessibility of oligosaccharides containing terminal b(1,4)-linked galactose or a(2,3)-linked
sialic acid, we coated 1-µm particles with RCA-I or Mal
II, mixed them with BSA-coated control particles at a
ratio of 1:1, and applied the mixture to live or fixed
rabbit Peyer’s patch explants. We found that 1-µm
RCA-I-coated particles failed to adhere to villus enterocytes, FAE, or M cells (data not shown), consistent with
our results obtained using 120-nm particles (Fig. 2 and
Table 1). Similarly, 1-µm Mal II-coated particles were
unable to bind specifically to any cell surface, including
villus epithelium or FAE (Fig. 4, Table 1). This indi-
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Fig. 3. Adhesion of soluble Maackia amurensis lectin II (Mal II) to
both villus and FAE cells. Live rabbit Peyer’s patch explants were
incubated with soluble, biotinylated Mal II (100 µg/ml) for 40 min and
then labeled with streptavidin-fluorescein. Mal II labeled apical
surfaces of epithelial cells in villus epithelium (A) and FAE (B). Scale
bar 5 50 µm.
For the majority of enteric pathogens, the primary
event in mucosal infection is attachment to glycoconjugates on apical surfaces of intestinal epithelial cells.
Indeed, bacteria and viruses express lectin-like adhesins on their surfaces (1, 19, 22, 27), and intestinal
absorptive cells are coated with a heterogeneous array
of glycolipids and protein-linked oligosaccharides (30,
45). The fact that glycoconjugates may extend from a
few nanometers to hundreds of nanometers from the
plasma membrane lead to the proposal that potential
glycoconjugate binding sites may not be equally accessible to pathogens present in the intestinal lumen (25).
In this study we have shown that certain glycolipid and
protein-linked oligosaccharide epitopes that are present on the surfaces of all villus and FAE cells are not
uniformly available for binding of ligands immobilized
on virus- and bacterium-sized particles. The regional
and cellular differences in receptor availability that we
observed may explain in part the selective attachment
of pathogenic viruses and bacteria to specific cell types
in the intestinal mucosa.
Membrane glycolipids such as GM1, whose carbohydrate head extends only 2.5 nm from the membrane
surface (47), should be relatively inaccessible to particles and most readily masked by membrane-associated glycoproteins. Since the thick FBBG on the microvilli of villus and FAE enterocytes had previously been
shown to exclude CTB-coated particles as small as 28
nm in diameter, it was not surprising that 120-nm
particles were also prevented from contacting enterocyte GM1. In contrast, the apical glycoprotein coat on
rabbit Peyer’s patch M cells is a relatively thin, irregular layer that extends only 20–30 nm from the membrane and allows binding of CTB-coated 28-nm particles to GM1 (10). This observation had raised hopes
that virus-sized or even larger particles coated with
CTB could be used to target vaccines to M cells and
enhance the efficiency of mucosal immunization (16,
24). Unexpectedly, we found that the glycoprotein coat
on live M cells was sufficient to prevent 120-nm particles from gaining access to GM1. This implies that
CTB may not be useful for M cell targeting of particulate vaccine carriers such as copolymer microspheres
but may be most effective as a component of macromolecular antigen complexes, as has been previously
observed (31).
Our results also have implications for understanding
rectal transmission of HIV-1. It was observed that
cell-free HIV-1 adheres to, and is endocytosed by, M
GLYCOCONJUGATE ACCESSIBILITY ON INTESTINAL EPITHELIUM
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cells in mucosal explants from experimental animals
(2) and humans (P. D. Smith, personal communication).
Although it has been proposed that HIV-1 initially
adheres to the glycosphingolipid galactosylceramide (9,
12), our data would suggest that it is unlikely that this
molecule would be accessible to virus particles. Rather,
we propose that the initial attachment of HIV-1 to the
apical surfaces of M cells may involve an interaction of
gp120 with more accessible membrane surface components (4, 32) or the interaction of host-derived adhesion
molecules in the viral envelope with relevant ligands on
M cells (21).
Mucinlike glycoproteins, including those that comprise the FBBG on the apical surfaces of intestinal
epithelial cells, contain abundant heterogeneous oligosaccharide side chains that tend to form thick, negatively charged gels (48). We hypothesized that the
accessibility of certain carbohydrate determinants in
the enterocyte glycocalyx to lectins immobilized on
virus- or bacterium-sized particles may vary, depending on their relative position within the FBBG. We
found that soluble RCA-I, which preferentially recognizes oligosaccharides containing terminal b(1,4)linked galactose, adhered to rabbit villus epithelium
and FAE, but RCA-I immobilized on 120-nm or 1-µm
particles did not. The particles did bind specifically to
our Caco-2 cells that have abundant terminal galactose
moieties and that lack a thick FBBG (10). Although the
exact topological locations of the epitopes recognized by
RCA-I on rabbit enterocytes and M cells are unknown,
our results suggest that they are sequestered within
the FBBG or perhaps located on smaller brush-border
membrane glycoproteins.
Since oligosaccharides are commonly terminated by
sialic acid, we expected that glycoconjugates containing
a(2,3)-linked sialic acid recognized by Mal II on both
FAE and villus epithelium would be readily available to
lectin immobilized on particles. Surprisingly, on live
tissue 120-nm particles had access to Mal II epitopes
only on the FAE and not on villus epithelium. Access to
the Mal II epitopes on the FAE was size restricted,
since 1-µm particles were unable to bind. Both 120-nm
and 1-µm Mal II-coated particles adhered to aldehydefixed tissue, indicating that the failure of the particles
to bind to villus epithelium was due to limited accessibility and not to a lack of Mal II binding sites. These
data add to the list of phenotypic differences that are
known to exist between the FAE and villus epithelium.
For example, all cells of the FAE lack expression of
polymeric immunoglobulin receptors (38) and show
reduced brush border hydrolase activity compared with
villus enterocytes (44). Lectin and antibody-binding
studies in humans, mice, and rabbits revealed that
FAE may differ from villus epithelium in glycosylation
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Fig. 4. Adherence of Mal II-coated
120-nm and 1-µm particles to FAE and
villus (v) epithelium. Live or aldehydefixed Peyer’s patch explants were exposed to a 1:1 mixture of Mal II-coated
particles (green) and biocytin-quenched
particles (red) for 40 min. A: on live
tissue, Mal II-coated 120-nm particles
bound preferentially to FAE, including
enterocytes and M cells. B: on fixed
tissue, Mal II-coated 120-nm particles
bound to both FAE and villus epithelium. C: on live tissue, Mal II-coated
1-µm particles bound to FAE, but this
binding was nonspecific since control
particles were present in equal numbers. D: on fixed tissue, Mal II-coated
particles bound to both FAE and villus
epithelium. Scale bar 5 100 µm.
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GLYCOCONJUGATE ACCESSIBILITY ON INTESTINAL EPITHELIUM
We thank Drs. Paul Giannasca, Karen Giannasca, and Larry
Silbart for their helpful comments.
N. J. Mantis gratefully acknowledges the receipt of a postdoctoral
fellowship from the Harvard AIDS Institute, Cambridge, MA [National Institutes of Health (NIH) Grant T32-A107387-07] and a NIH
Individual National Research Service Award (NIH F32-AI10009-02).
A. Frey was supported by a personal grant from the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie, and
research grant FR958/2-1 from the Deutsche Forschungsgemeinschaft. M. R. Neutra is supported by NIH research grants (HD-17557
and AI-34757) and by a NIH Center grant to the Harvard Digestive
Diseases Center (DK-34854).
Present address for A. Frey: Center for Molecular Biology of
Inflammation, University of Muenster, Germany.
Address for reprint requests and other correspondence: M. R. Neutra,
GI Cell Biology Laboratory, Enders 1220, Children’s Hospital, 300 Longwood Ave., Boston, MA 02115 (E-mail: [email protected]).
Received 24 June 1999; accepted in final form 14 January 2000.
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