(Received for publication, November 9, 1994; and in revised form, December 30, 1994)
From the
Entamoeba histolytica trophozoites initiate pathogenic
colonization by adherence to host glycoconjugates via an amebic surface
lectin which binds to galactose (Gal) and N-acetylgalactosamine (GalNAc) residues. Monovalent and
multivalent carbohydrate ligands were screened for inhibition of E.
histolytica lectin-mediated human red cell hemagglutination,
revealing that: (i) the synthetic multivalent neoglycoprotein
GalNAcBSA (having an average of 39 GalNAc residues linked
to bovine serum albumin) was 140,000-fold more potent an inhibitor than
monovalent GalNAc and 500,000-fold more potent than monovalent Gal; and
(ii) small synthetic multivalent ligands which bind with high affinity
to the mammalian hepatic Gal/GalNAc lectin do not bind with high
affinity to the E. histolytica lectin. Radioligand binding
studies revealed saturable binding of
I-GalNAc
BSA to E. histolytica membranes (K
= 10 ± 3
nM, B
= 0.9 ± 0.08
pmol/mg membrane protein). Maximal binding required the presence of
calcium chloride (300 µM) or sodium chloride (50
mM), and had a broad pH maximum (pH 6-9).
GalNAc
BSA was 200,000-fold more potent than monovalent
GalNAc in blocking radioligand binding. Among synthetic
saccharide-derivatized linear polymers, the GalNAc
and
GalNAc
3Gal
derivatives were the most potent, with GalNAc
and GalNAc
3(Fuc
2)Gal
derivatives much weaker. The data
support a model in which a unique pattern of spaced multiple GalNAc
residues are the highest affinity targets for the E. histolytica lectin.
Amebiasis is a parasitic infection caused by Entamoeba histolytica, that results in 40-50 million cases of amebic colitis and liver abscess and 40,000-100,000 deaths annually worldwide (1) . An initial step in parasite colonization of the large bowel is adherence to the colonic wall. Adherence involves recognition of colonic mucin and epithelial glycoconjugates by an amebic cell surface lectin which recognizes nonreducing terminal Gal and GalNAc residues (reviewed in (2) ).
The E. histolytica adherence lectin is a heterodimer consisting of a 170-kDa heavy chain and a light chain of 31 or 35 kDa linked by disulfide bonds(3) . The heavy subunit is encoded by a gene family of which three members (89-95% identical) from strain HM1:IMSS have been sequenced(4) . Each contains a putative carboxyl-terminal cytoplasmic and transmembrane domain, and a large cysteine-rich extracellular domain which may be involved in carbohydrate binding, since monoclonal antibodies to this domain block amebic adherence(5) . The light subunits are nearly identical to each other in amino acid composition and CNBr fragmentation, although the 31-kDa subunit is unique in that it contains a glycosylphosphatidylinositol anchor(3) .
Previous studies
demonstrated that 5-10 mM galactose or N-acetylgalactosamine, but not other monosaccharides,
inhibited E. histolytica adherence to target
cells(6, 7) . The glycoproteins asialoorosomucoid or
asialofetuin, with polyvalent nonreducing terminal Gal (and GalNAc)
residues, were much more potent inhibitors than monosaccharides, with
half-maximal inhibition of amebic adherence occurring in the micromolar
range(8) . Partially purified large molecular mass (9
10
Da) rat and human colonic mucins, which are highly
polyvalent in nonreducing terminal Gal and GalNAc residues, were very
potent inhibitors of E. histolytica adherence to target cells
(IC
< 10
M)(9, 10) . These data are
characteristic of other carbohydrate recognition systems, where
multiple appropriately spaced nonreducing terminal carbohydrates are
required for high affinity binding to cellular lectins(11) .
The current study evaluates a series of multivalent glycoconjugates
with nonreducing terminal Gal and GalNAc residues as potential
high-affinity binding ligands for the E. histolytica lectin.
One of these, GalNAc
BSA, (
)was of sufficiently
high affinity to use as a ligand in binding experiments which allowed
further characterization of the binding requirements and properties of
the E. histolytica lectin.
The standard
radioligand binding reaction contained 10 mM Hepes buffer pH
7.4, 50 mM NaCl, 2 mM CaCl, 5 mg/ml BSA,
and the indicated concentrations of
I-GalNAc
BSA, E. histolytica membranes, and any potential saccharide inhibitor in a total
volume of 100 µl. Reactions were incubated with gentle agitation
for 4-5 h at 4 °C, then rapidly diluted with 10 mM Hepes buffer pH 7.4 and rinsed onto glass fiber filters (presoaked
in 10 mM Hepes buffer pH 7.4, 1 mg/ml BSA) using a Brandel
Harvester (Brandel, Gaithersburg, MD). Membrane-bound radioligand
remaining on the glass fiber filters was quantitated using a
-radiation counter.
Comparative I-GalNAc
BSA binding to rat liver membranes
was based on the method of Grant and Kaderbhai(23) . The
binding reaction contained 25 mM Tris-HCl buffer (pH 7.8), 150
mM NaCl, 0.02% (w/v) Triton X-100, 20 mM CaCl
, 10 mg/ml BSA, 400 pM radioligand, 5
µg of rat liver membrane protein, and potential saccharide
inhibitors. After incubation for 110 min at 4 °C, membranes with
bound ligand were collected on glass fiber filters (as above), washed
with buffer (25 mM Tris-HCl buffer (pH 7.8), 150 mM NaCl, 0.02% (w/v) Triton X-100, 20 mM CaCl
),
and radioligand remaining on the filters was quantitated.
Lectin or lectin/inhibitor combinations were flowed
over the GalNAcBSA-derivatized surface at 25 °C in
freshly prepared running buffer containing 10 mM Hepes, 2
mM CaCl
, 50 mM NaCl, and 0.005% P20
surfactant. Initial experiments were performed to determine the
concentrations which would allow measurable binding. On an immobilized
base of
450-500 µM GalNAc
BSA,
measurable binding was obtained using 300-600 nM soluble E. histolytica lectin. To determine association kinetics, 40
µl of purified E. histolytica lectin was injected at a
flow rate of 5 µl/min, and data points were collected at 5-s
intervals for 8 min. At the end of each run, the surface was
regenerated using 50 mM HCl. Three graded concentrations of
soluble lectin (288 nM, 385 nM, and 578 nM)
were used. Dissociation curves were obtained using a flow rate of 60
µl/min and an injection of 120 µl of lectin or lectin/inhibitor
combinations (association phase) followed by running buffer only, at
the same flow rate (dissociation phase). Binding was observed over a
period of 17 min, with association occurring during the first 2 min and
dissociation during the following 15 min. The curves suggested a
biphasic dissociation, so were analyzed using a two-site model. Kinetic
data analyses were performed based on published principles (24) using BIAcore software.
Figure 1:
I-GalNAc
BSA
binding as a function of E. histolytica membrane
concentration. Radioligand binding was performed under standard
conditions using the indicated concentrations of membrane protein per
assay and a radioligand concentration of 1.1 nM (9.2 Bq/fmol).
Specific binding (filled circles) is the difference between
total binding (open circles) and background binding (squares), the latter measured in the presence of 300 nM unlabeled GalNAc
BSA. Data are presented as mean
± S.D. for duplicate determinations.
Figure 2:
Isotherm of I-GalNAc
BSA binding to E. histolytica membranes. Radioligand binding was performed under standard
conditions using 110 µg of E. histolytica membrane protein
per assay and the indicated concentrations of
I-GalNAc
BSA (9.8 Bq/fmol). A, total
binding, squares; background binding (in the presence of 300
nM unlabeled GalNAc
BSA), circles. B, specific binding was calculated by subtracting background binding
from total binding. The line represents the least squares fit
of the data to a rectangular hyperbola, from which the K
(9.7 ± 3.1 nM) and B
(102 ± 8.8 fmol/assay) were calculated.
Data are presented as mean for triplicate determinations. Standard
deviations fall within the data points in all cases, so are not
indicated.
I-GalNAc
BSA binding to E. histolytica membranes was highly dependent on ionic
conditions. Initial binding studies were performed in PBS, the same
buffer used in hemagglutination experiments. In comparison, binding was
essentially absent in a base buffer of 10 mM Hepes pH 7.4 (Table 3). Binding was largely recovered if either calcium
chloride (0.7 mM) or sodium chloride (154 mM) was
added to the base buffer, while addition of magnesium chloride (0.5
mM) resulted in recovery of less than a third of the specific
binding activity found using PBS. The same maximum level of
GalNAc
BSA binding was achieved whether sodium chloride,
calcium chloride, or a combination of the two were used (Table 3). Maximum GalNAc
BSA binding required
50-100 mM sodium chloride, with 20 mM NaCl
supporting half-maximal binding (data not shown). In contrast, a free
calcium ion concentration of <300 µM supported maximum
binding; half-maximal binding occurred at 35 µM free
calcium ion (Fig. 3). The pH dependence of
I-GalNAc
BSA binding was broad, with similar
binding over the pH range 6-9 (Fig. 4).
Figure 3:
Effect of calcium concentration on I-GalNAc
BSA binding to E. histolytica membranes. Radioligand binding was performed under standard
conditions, except the binding buffer was 10 mM Hepes pH 7.4
supplemented with 1 mg/ml BSA and various calcium concentrations
ranging from 42 to 840 µM. The introduction of EDTA (from
the membrane preparation) at a constant concentration of 200 µM resulted in the indicated (calculated) free calcium
concentrations. Each assay contained 2.8 nM radioligand (1.8
Bq/fmol) and 40 µg of E. histolytica membrane protein.
Specific binding (filled circles) is the difference between
total binding (open circles) and background binding (squares), the latter measured in the presence of 300 nM unlabeled GalNAc
BSA. Data are presented as mean
± S.D. for duplicate determinations.
Figure 4:
Effect of pH on I-GalNAc
BSA binding to E. histolytica membranes. Radioligand binding was performed under standard
conditions except that the binding buffer consisted of 10 mM of sodium acetate (pH 4-5), Mes (pH 6), Hepes (pH
7-8), or Tris (pH 9). Each buffer also contained 1 mM CaCl
and 150 mM NaCl. Each assay contained
2.8 nM radioligand (2.1 Bq/fmol) and 40 µg of E.
histolytica membrane protein. Specific binding (filled
circles) is the difference between total binding (open
circles) and background binding (squares), the latter
measured in the presence of 300 nM unlabeled
GalNAc
BSA. Data are presented as mean ± S.D. for
duplicate determinations.
Figure 5:
Inhibition of I-GalNAc
BSA binding to E. histolytica membranes by saccharides. Radioligand binding was performed under
standard conditions using 2.2 nM
I-GalNAc
BSA (6.9 Bq/fmol) and 32 µg
of E. histolytica membrane protein. Assays were performed in
the presence of the indicated concentrations of the following
inhibitors: p-nitrophenyl GalNAc (filled circles),
GalNAc (filled squares), lactose (open squares), or
galactose (open circles). Data are presented as mean ±
S.D. for duplicate determinations. Horizontal lines represent
the mean (solid) and S.D. (dotted) of binding in the
absence of inhibitors (upper) or in the presence of 300 nM unlabeled GalNAc
BSA (lower).
The most potent inhibitor was
GalNAcBSA itself, with half-maximal inhibition at 5 nM (Fig. 6), in good agreement with equilibrium binding data
and with inhibition of hemagglutination. Gal
BSA was
>100-fold less potent than GalNAc
BSA, while the
glycoprotein fetuin and its asialo- and asialoagalacto-derivatives did
not inhibit radioligand binding at concentrations exceeding 10
µM.
Figure 6:
Inhibition of I-GalNAc
BSA binding to E. histolytica membranes by glycoproteins. Radioligand binding was performed
under standard conditions using 1.1 nM
I-GalNAc
BSA (8.2 Bq/fmol) and 55 µg
of E. histolytica membrane protein. Assays were performed in
the presence of the indicated concentrations of the following
inhibitors: GalNAc
BSA (filled squares),
Gal
BSA (filled circles), asialofetuin (open
squares), fetuin (open circles), or asialoagalactofetuin (triangles). Data are presented as mean ± S.E. for
triplicate determinations. Horizontal lines represent the mean (solid) and S.E. (dotted) of binding in the absence
of inhibitors.
Porcine stomach mucin inhibited I-GalNAc
BSA binding half-maximally at 2
µg/ml, 5-fold less potent than GalNAc
BSA on a weight
basis. Asialo-porcine stomach mucin was slightly less potent than the
parent molecule, and subsequent chemical degalactosylation reduced
inhibition potency >25-fold compared to the parent mucin (Table 4). Hyaluronoic acid inhibited radioligand binding
half-maximally at 10 µg/ml (25-fold less potent than
GalNAc
BSA), while chondroitin sulfate and heparin were
only weak inhibitors.
A series of commercially obtained polyvalent
carbohydrate-derivatized linear polyacrylamide polymers with Gal or
GalNAc nonreducing termini were tested as inhibitors. Each co-polymer
contained 75% N-(2-hydroxyethyl)acrylamide, 20%
sugar-derivatized acrylamide, and 5% biotin-derivatized acrylamide, and
was 30 kDa by gel filtration(25) . The
-GalNAc
derivative was a potent inhibitor of
I-GalNAc
BSA binding to E. histolytica membranes, with an IC
of 11 µg/ml, corresponding
to 3.1 µM in sugar residues or
400 nM in
polymer chains (Fig. 7). The
-GalNAc derivative was much
less potent, resulting in <50% inhibition at 500 µg/ml. Polymers
bearing glycosides of Gal, Glc, and GlcNAc (all
ananomers) were
very weak inhibitors. Among the disaccharide-derivatized polymers
tested, only the GalNAc
1,3-Gal
-derivatized polymer was a
potent inhibitor, with an IC
of 11 µg/ml (2.5
µM in sugar residues,
370 nM in polymer
chains). The closely related GalNAc
1,3-GalNAc
derivative
was a poor inhibitor, as were Gal-terminated derivatives. Polymers
bearing the blood group A and B trisaccharides were weak inhibitors,
with the more potent A-group (GalNAc
1,3(Fuc
1,2)Gal
)
polymer causing half-maximal inhibition at 250 µg/ml (44 µM in sugar residues, >8 µM in polymer chains).
Figure 7:
Inhibition of I-GalNAc
BSA binding to E. histolytica membranes by multivalent saccharide-derivatized linear
polyacrylamide. Radioligand binding was performed under standard
conditions using 0.88 nM
I-GalNAc
BSA (15 Bq/fmol) and 29 µg
of E. histolytica membrane protein. Assays were performed in
the presence of the indicated concentrations of polymers derivatized
with the following glycosides (see text for description of polymers). Upper panel, GalNAc
propyl, filled circles;
GalNAc
propyl, open squares; Gal
propyl, open
triangles; Glc
propyl, closed squares; and
GlcNAc
propyl, closed triangles. Middle panel, GalNAc
3Gal
propyl, closed circles;
GalNAc
3GalNAc
propyl, open circles;
Gal
3GalNAc
propyl, triangles; and
Gal
3Gal
propyl, squares. Lower panel: GalNAc
3(Fuc
2)Gal
propyl, squares; and
Gal
3(Fuc
2)Gal
propyl, circles. Data are
presented as mean ± S.E. for duplicate
determinations.
Figure 8:
Real time association and dissociation
kinetics of purified E. histolytica lectin binding to
GalNAcBSA. Top, association kinetics of lectin
(580 nM) binding to immobilized GalNAc
BSA (460
µM) in the absence of inhibitors is represented by circles; association of lectin (580 nM) and ligand
(490 µM) in the presence of asialofetuin (35
µM) is represented by squares. Bottom, dissociation kinetics after binding of lectin (580 nM) to
immobilized ligand (760 µM) in the absence of inhibitor is
represented by circles; dissociation of lectin (390
nM) binding to ligand (450 µM) in the presence of
asialofetuin (35 µM) is represented by squares. Zero time in the bottom panel represents the time of peak
binding after which lectin-free running buffer was injected and
dissociation was initiated.
Infection by E. histolytica requires adherence of
the amoebae via interaction of a cell surface lectin with terminal
GalNAc and/or Gal residues on glycoconjugates on the surface of target
tissues (2) . In both hemagglutination assays and radioligand
binding assays, GalNAc was the preferred carbohydrate determinant when
compared to Gal, requiring one-third to one-tenth the concentration for
effective blocking of lectin-mediated binding, depending on the assay
and the aglycon ( Table 1and Table 4). Saccharides having
other than Gal and GalNAc termini were ineffective inhibitors in all
assays. Carbohydrate recognition by cell surface lectins is often
dependent on precise spatial organization of target carbohydrate
determinants(11) . The same holds true for E. histolytica lectin, as indicated by the relative inhibitory potency of
GalNAcBSA compared to GalNAc. The polyvalent
neoglycoprotein was 150,000-200,000-fold more potent than the
monosaccharide, depending on the assay ( Table 1and Table 4). This corresponds to a 3,500- to 5,000-fold increase in
binding affinity per GalNAc residue, apparently due to appropriate
multivalent spacing. Bovine serum albumin has 60 lysine residues, most
of which are available for saccharide derivatization. Prior studies on
vertebrate lectins suggest that BSA-based multivalent conjugates
represent a variety of multivalent saccharide spacings and thus have a
high potential for mimicking natural ligands.
The above pattern of
carbohydrate recognition is similar to that of the mammalian hepatic
lectin (MHL), which binds GalNAc and Gal residues (26) and
binds with high affinity to multivalent glycoconjugates such as
GalBSA and GalNAc
BSA (Table 1).
However, several small synthetic multivalent neoglycoconjugates which
are high affinity probes for the MHL (19) were poor inhibitors
of the E. histolytica lectin. For example, the synthetic
trivalent glycoconjugate YEE(GalNAc)
has a 20,000-fold
higher affinity for the MHL than the corresponding monosaccharide, or
7,000-fold higher based on the concentration of GalNAc termini. In
contrast, it has equal affinity to the monosaccharide, per GalNAc
residue, for the E. histolytica lectin. These data indicate
that high potency polyvalent binding of Gal/GalNAc residues is a key
factor in binding of both lectins to their target glycoconjugates, but
that there are fundamental differences in the precise spacing required
to maximize that binding. Presumably, the inherently heterogeneous
neoglycoproteins (such as GalNAc
BSA) can satisfy the
spacing requirements for both mammalian and amebic lectins. The
apparent requirement of the E. histolytica lectin for more
widely spaced ligands than those required for the MHL is analogous to
the requirements of the mannose-binding proteins(27) , for
which the term ``macro-cluster'' was coined to discern wide
spacings from the ``mini-clusters,'' such as those found on
the amino acid-based scaffolds used in the current study. This
observation raises the possibility that by obtaining information on the
spacing of GalNAc binding sites on E. histolytica membranes,
novel and specific high affinity inhibitors of E. histolytica adherence could be designed which would interact poorly with the
MHL. To date such an amebic-selective saccharide ligand has not been
found. Finding better defined and more selective high affinity ligands
will require screening of other synthetic multivalent glycoconjugates,
and/or additional information about the quaternary structure of the
lectin binding sites on the amoeba surface.
The high affinity of
GalNAcBSA for the E. histolytica lectin allowed
development of a rapid radioligand binding assay, and further
exploration of the lectin's binding characteristics. The ionic
requirements for binding were unusual, in that binding was not
supported in 10 mM sodium Hepes buffer, but required the
presence of either a low concentration of calcium
(half-maximal binding at 35 µM) or a higher concentration
of sodium chloride (half-maximal binding at 20 mM NaCl). The
observation that the same level of radioligand binding was supported
when NaCl, CaCl
, or both were added (Table 3)
suggests that a single lectin has a high affinity site for calcium
ions, the functional requirement for which can be overcome by higher
concentrations of monovalent ions. Current structural data on the
lectin is of insufficient detail to suggest a mechanism for this
observation.
Binding of I-GalNAc
BSA to E. histolytica membranes using a filter binding assay resulted
in well behaved data fitting a single site binding model, even though
the multivalent radioligand was binding to multivalent lectins in the
membranes. This type of data has been reported for binding of
neoglycoproteins to certain vertebrate lectins(28) , and would
be expected if binding was essentially an ``all or nothing''
event with no intermediate binding events or affinities.
Detailed
carbohydrate recognition studies based on inhibition of I-GalNAc
BSA binding to E. histolytica membranes (Table 4) generally correlated with the less
precise data obtained from hemagglutination inhibition (Table 1).
Exceptions included the glycoprotein fetuin and porcine stomach mucin,
and their chemically modified asialo- and asialoagalacto- forms, which
were
100-fold more potent at blocking hemagglutination than
blocking
I-GalNAc
BSA binding mediated by the
same E. histolytica membranes. These data do not fit a simple
binding model for lectin and glycoconjugate targets, but may reflect
the high cooperativity involved in binding to
I-GalNAc
BSA. The difference in inhibitory
potencies may be due to steric exclusion of the extended fetuin or
mucin structures from the neoglycoprotein binding site. Kinetics may
also be involved, in that binding of the soluble
I-GalNAc
BSA is expected to be more rapid
than binding between cells and membranes. Since off-rates of
multivalent ligands are characteristically slow (Fig. 8), the
system may not reach true equilibrium in the time of the experiment,
and the relative binding affinities may reflect, in part, the relative
binding rates of the ligands and inhibitors.
Analysis of purified
lectin binding to GalNAcBSA was readily demonstrated using
a BIAcore biosensor-based analytical system. Binding of purified lectin
to GalNAc
BSA, in addition to the membrane binding, lends
strong credence to the concept of targeting adhesion factors to
microbial surfaces using synthetic ligands. Using the BIAcore system, K
values of purified lectin binding to
GalNAc
BSA approached those of membrane binding. However,
inhibition of binding of the lectin to immobilized
GalNAc
BSA in the BIAcore by asialofetuin was achieved at
concentrations less than one-tenth of those needed for inhibition of
membrane binding. This may be due to kinetic factors, since in the
BIAcore system the lectin and inhibitor are premixed, then flowed past
the immobilized GalNAc
BSA under nonequilibrium conditions.
The rapid dissociation from the immobilized ligand on the sensor in the
presence of inhibitor suggests that polyvalent binding was reduced by
the inhibitor.
Steric access to the carbohydrate binding site is an
important factor, as reflected by inhibition of I-GalNAc
BSA binding to E. histolytica membranes by multivalent carbohydrate-derivatized polyacrylamide
polymers (Fig. 7). The
anomer of GalNAc linked directly to
the spacer arm on the polymer was >40-fold more potent at blocking
binding than the corresponding
anomer, while an
anomer of
GalNAc spaced away from the polymer backbone (GalNAc
1,3Gal
)
was equipotent to the
GalNAc-derivatized polymer. These data may
reflect a preference for the GalNAc-Gal disaccharide, but may also
suggest that the
anomer close to the polymer backbone may have
critical hydroxyls which are unavailable for binding due to steric
hindrance. In this light, it is interesting to note that a polymer
containing the A-trisaccharide, which differs from
GalNAc
1,3Gal
by the additional presence of a fucose residue
on the 2-position of the Gal was >20-fold less potent than the
corresponding disaccharide polymer, suggesting that the fucose
restricts access of the lectin to key GalNAc hydroxyls. A more complete
understanding of the requirements for binding to this lectin will
require additional structural data on the lectin's carbohydrate
binding domain.
The nature of the endogenous ligand for the E.
histolytica lectin on the colonic epithelium, or on other target
tissues (such as liver) is not known. It has been suggested that mucins
are either high affinity binding sites for the amoeba or may act to
inhibit infection by blocking lectin binding to cell surface sites. The
current study supports mucin as a potential target of E.
histolytica binding, since it is a likely source of multivalent
nonreducing terminal GalNAc residues. Porcine stomach mucin was the
most potent inhibitor of hemagglutination (0.12 µg/ml compared to
0.38 µg/ml for GalNAcBSA, Table 1). Since the
preparation is heterogeneous in structure and molecular weight, there
may be substructures with terminal saccharide orientations which have
very high affinity for lectin binding. These structures are likely to
consist of spaced GalNAc residues on the polypeptide backbone, since
desialylation and degalactosylation had little effect on potency (Table 1). This conclusion is supported by data using fetuin
preparations as inhibitors (Table 1). The commercial preparation
of fetuin had
50% desialylated Gal termini based on its previously
published oligosaccharide structures and carbohydrate analysis (data
not shown). Further desialylation did not change its inhibitory
potency, but degalactosylation enhanced inhibition. Fetuin has
three N-linked complex oligosaccharides which would terminate
in noninhibitory GlcNAc residues after degalactosylation and three O-linked oligosaccharides, comprised primarily of
NeuAc
2,3Gal
1,3GalNAc, which would be converted to GalNAc
termini upon degalactosylation. Based on the other carbohydrate
specificity data presented, it is likely that the spaced GalNAc
residues on fetuin result in the enhanced inhibitory potency.
Whatever the endogenous carbohydrates are for E. histolytica attachment which lead to establishment of colonic or hepatic disease, the data presented here establish that: (i) multivalent terminal GalNAc residues are potent binding ligands for the E. histolytica lectin; and (ii) the spacing of the multivalent GalNAc residues required for optimal E. histolytica binding is distinct from that required for optimal binding to the mammalian hepatic lectin. This raises the possibility of designing a specific multivalent carbohydrate inhibitor of E. histolytica adherence.