©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
High Affinity Binding of the Entamoeba histolytica Lectin to Polyvalent N-Acetylgalactosaminides (*)

(Received for publication, November 9, 1994; and in revised form, December 30, 1994)

Pablo Adler (1) Sheila J. Wood (2) Yuan C. Lee (3) Reiko T. Lee (3) William A. Petri Jr. (4)(§) Ronald L. Schnaar (1)(¶)

From the  (1)Department of Pharmacology and Molecular Science, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, (2)U. S. Army Chemical Research Development Command, Aberdeen Proving Ground, Aberdeen, Maryland 21010, the (3)Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, and the (4)Departments of Medicine, Pathology and Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-GalNAcBSA to E. histolytica membranes (K = 10 ± 3 nM, B(max) = 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). GalNAcBSA was 200,000-fold more potent than monovalent GalNAc in blocking radioligand binding. Among synthetic saccharide-derivatized linear polymers, the GalNAcbeta and GalNAcalpha3Galbeta derivatives were the most potent, with GalNAcalpha and GalNAcalpha3(Fucalpha2)Galbeta 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.


INTRODUCTION

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 times 10^5 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 < 10M)(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, GalNAcBSA, (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Monosaccharides were from Pfanstiehl (Waukegan, IL) or Sigma. Disaccharides, trisaccharides, p-nitrophenyl glycosides, fetuin, heparin, hyaluronic acid, chondroitin sulfate, dextran sulfate, and porcine stomach mucin (Type III) were from Sigma. Glycolipids were from Matreya (Pleasant Gap, PA) or EY Laboratories (San Mateo, CA). Saccharide-derivatized polyacrylamide polymers were from Synthesome, GmbH (München, Germany). Neoglycoproteins were synthesized by the method of Lee et al.(12) . Synthetic glycosides and cluster glycosides were synthesized as described previously(13, 14, 15, 16, 17, 18, 19) . Fetuin and porcine stomach mucin were chemically desialylated and degalactosylated by the methods of Kim et al.(20) . Phosphate-buffered saline (PBS) was a modification of Dulbecco's formulation, containing 154 mM NaCl, 8.1 mM Na(2)HPO(4), 2.7 mM KCl, 1.5 mM KH(2)PO(4), 0.7 mM CaCl(2), and 0.5 mM MgCl(2).

Membrane and Lectin Preparation

E. histolytica strain HM1:IMSS trophozoites were grown as reported previously(21) . Amoebae from 3-4 250-ml flasks were collected by centrifugation (150 times g, 15 min, 4 °C), resuspended in 75 mM Tris, 65 mM NaCl (pH 7.2), collected as above, and resuspended in lysis buffer containing 10 mM sodium phosphate (pH 8), 2 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 0.1 M 4-(2-aminoethyl)-benzenesulfonyl fluoride, and 2 mMp-hydroxymercuribenzoic acid. After incubation for 5 min at 37 °C to promote lysis, the suspension was chilled on ice and sonicated (3-5 10-s bursts, microtip, maximum energy, Sonifier Cell Disruptor). Membranes were collected by centrifugation (50,000 times g, 1 h, 4 °C). The pellets were resuspended in 10 ml of lysis buffer and membranes collected by centrifugation (100,000 times g, 1 h, 4 °C). The resulting pellets were stored for up to 48 h on ice, then were resuspended in 10 ml of ice-cold 10 mM sodium phosphate buffer (pH 7.4) containing 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 1 mMp-hydroxymercuribenzoic acid and subjected to further sonication (10 10-s bursts over a 20-min period). Membranes were collected by centrifugation as above and resuspended in 10 mM Hepes buffer (pH 7.4) containing 1 mg/ml bovine serum albumin (BSA). Aliquots of the membrane preparation were stored at -20 °C until use. E. histolytica lectin was purified as described previously(21) . Rat liver plasma membranes were prepared by the method of Ray(22) .

Hemagglutination

Hemagglutination was performed as described previously(21) . Human Type A blood was drawn into heparinized tubes, diluted 1:1 with PBS, and 4 ml pipetted over 3 ml of Ficoll-Paque (Pharmacia Biotech Inc.) in a centrifuge tube. Erythrocytes were collected by centrifugation at 400 times g for 25 min at 4 °C, washed twice in 10 ml of PBS by centrifugation at 400 times g for 5 min at 4 °C, and diluted to 2.4 times 10^9/ml in PBS for storage at 0 °C for up to 30 days. For use in the hemagglutination assay, a suspension of 2 times 10^7 cells/ml in PBS containing 2 mg/ml bovine serum albumin was prepared. Hemagglutination was performed in V-bottom 96-well polystyrene plates (Costar Seroclusters catalog No. 3897). Test samples containing E. histolytica membranes (approx4 µg of membrane protein) in 100 µl of PBS were placed in replicate microwells and hemagglutination was initiated by adding 100 µl of erythrocyte suspension. After gentle agitation, plates were incubated undisturbed at 4 °C for 6-16 h, after which hemagglutination was detected as a uniform distribution of erythrocytes in the well, rather than a point concentration of cells at the well bottom in the absence of hemagglutination. A membrane concentration curve was performed for each E. histolytica membrane preparation, and a membrane concentration approx2-fold of that necessary to detect hemagglutination was used for glycoconjugate inhibition experiments.

Radioligand-Membrane Binding

GalNAcBSA was radioiodinated using carrier-free NaI and Iodo-Beads (Pierce Chemical Co.), and the radiolabeled product was purified by gel filtration chromatography on Sephadex G-25 (Pharmacia). For most experiments, the specific activity ranged from 250 to 500 µCi/nmol.

The standard radioligand binding reaction contained 10 mM Hepes buffer pH 7.4, 50 mM NaCl, 2 mM CaCl(2), 5 mg/ml BSA, and the indicated concentrations of I-GalNAcBSA, 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-GalNAcBSA 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(2), 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(2)), and radioligand remaining on the filters was quantitated.

Biosensor-based Lectin Binding

Real time detection of E. histolytica lectin binding to GalNAcBSA was performed using a BIAcore (Pharmacia) biosensor-based analytical system (24) . GalNAcBSA was covalently coupled, via its primary amines, to a derivatized carboxyl group within a dextran layer on the sensor chip surface. A 35-µl aliquot containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was flowed across the sensor chip under conditions designed to activate 30-40% of the immobilized carboxyl groups using the manufacturer's reagents and conditions. This was followed by 40 µl of GalNAcBSA (45-60 µg/ml) in 10 mM Hepes buffer (pH 6.0) injected at the same rate. Immobilization of GalNAcBSA was performed at approx25 °C using a flow rate of 5 µl/min, and resulted in an initial reading of approx4000 relative units corresponding to approx500 µM GalNAcBSA within derivatized dextran layer. Any remaining activated carboxyls were reacted with ethanolamine hydrochloride as described(24) . The derivatized sensor was washed by injection of 15 µl of 10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20 prior to use.

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(2), 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 approx450-500 µM GalNAcBSA, 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.


RESULTS

Saccharide Inhibition of E. histolytica Lectin-induced Hemagglutination

An initial screen for saccharides with high affinity for the E. histolytica membrane lectin was initiated using human red blood cell agglutination as a rapid assay for inhibitory carbohydrates and glycosides. Consistent with previous studies, saccharides with nonreducing terminal Gal or GalNAc residues inhibited hemagglutination, while similar saccharides with other nonreducing termini (Glc, GlcNAc, Man, etc.) were noninhibitory. Two novel results arose from these studies (Table 1Table 2). Small molecular weight divalent or trivalent ``cluster glycosides'' of Gal or GalNAc(18, 19) , which are high affinity inhibitors of binding to the mammalian hepatic lectin (IC < 1 nM) were relatively poor inhibitors of the E. histolytica lectin (minimum inhibitory concentration 0.1-1 mM). In contrast, polyvalent Gal- and GalNAc-derivatized neoglycoproteins were very high affinity inhibitors of the E. histolytica lectin, with GalNAcBSA having a minimum inhibitory concentration of 5 nM, which was >150,000-fold more potent than GalNAc or lactose for inhibition of E. histolytica membrane-induced hemagglutination. The monosaccharide GalNAc was modestly (4-fold) more potent than Gal in inhibiting hemagglutination, whereas GalNAcBSA was 33-fold more potent than GalBSA in the same assay, suggesting that appropriately spaced GalNAc residues are the most potent saccharide ligands for the E. histolytica lectin.





High Affinity Binding of GalNAcBSA to E. histolytica Membranes

Based on the initial hemagglutination screen, GalNAcBSA was chosen as a potential ligand to further probe the E. histolytica lectin. GalNAcBSA was radioiodinated and incubated with E. histolytica membranes, then the membranes were collected by filtration. At low input radioligand concentrations (1 nM), specific binding was readily demonstrated (Fig. 1). Specific binding increased linearly as a function of added membrane protein, while nonspecific binding (in the presence of 300 nM unlabeled GalNAcBSA) was relatively low and did not increase markedly with added membranes. Specific binding was saturable, while nonspecific binding was characteristically nonsaturable, such that at higher input radioligand concentrations (>10 nM) nonspecific binding was a substantial fraction of total binding (Fig. 2A). Binding was sufficiently consistent to generate an equilibrium binding isotherm. Although binding of the multivalent ligand to a multivalent membrane is inherently complex (see ``Discussion''), the binding data fit a single-class receptor-ligand binding equation (Fig. 2B), resulting in an apparent K(D) of 9.7 ± 3.1 nM and a B(max) of 0.93 ± 0.08 pmol/mg membrane protein.


Figure 1: I-GalNAcBSA 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 GalNAcBSA. Data are presented as mean ± S.D. for duplicate determinations.




Figure 2: Isotherm of I-GalNAcBSA 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-GalNAcBSA (9.8 Bq/fmol). A, total binding, squares; background binding (in the presence of 300 nM unlabeled GalNAcBSA), 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(max) (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-GalNAcBSA 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 GalNAcBSA binding was achieved whether sodium chloride, calcium chloride, or a combination of the two were used (Table 3). Maximum GalNAcBSA 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-GalNAcBSA binding was broad, with similar binding over the pH range 6-9 (Fig. 4).




Figure 3: Effect of calcium concentration on I-GalNAcBSA 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 GalNAcBSA. Data are presented as mean ± S.D. for duplicate determinations.




Figure 4: Effect of pH on I-GalNAcBSA 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(2) 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 GalNAcBSA. Data are presented as mean ± S.D. for duplicate determinations.



Saccharide Inhibition of GalNAcBSA Binding to E. histolytica Membranes

The affinity of various saccharides for the E. histolytica lectin was measured indirectly by their ability to inhibit I-GalNAcBSA binding to E. histolytica membranes. Small saccharides having a nonreducing terminal Gal or GalNAc inhibited half-maximally in the mM range ( Fig. 5and Table 4). The presence of a hydrophobic aglycon on p-nitrophenyl beta-N-acetylgalactosaminide increased affinity 10-fold. Both alpha and beta p-nitrophenyl glycosides of GalNAc were inhibitory, with the beta glycoside having modestly higher affinity (Table 4).


Figure 5: Inhibition of I-GalNAcBSA binding to E. histolytica membranes by saccharides. Radioligand binding was performed under standard conditions using 2.2 nMI-GalNAcBSA (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 GalNAcBSA (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. GalBSA was >100-fold less potent than GalNAcBSA, 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-GalNAcBSA binding to E. histolytica membranes by glycoproteins. Radioligand binding was performed under standard conditions using 1.1 nMI-GalNAcBSA (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: GalNAcBSA (filled squares), GalBSA (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-GalNAcBSA binding half-maximally at 2 µg/ml, 5-fold less potent than GalNAcBSA 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 GalNAcBSA), 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 approx30 kDa by gel filtration(25) . The beta-GalNAc derivative was a potent inhibitor of I-GalNAcBSA binding to E. histolytica membranes, with an IC of 11 µg/ml, corresponding to 3.1 µM in sugar residues or approx400 nM in polymer chains (Fig. 7). The alpha-GalNAc derivative was much less potent, resulting in <50% inhibition at 500 µg/ml. Polymers bearing glycosides of Gal, Glc, and GlcNAc (all beta ananomers) were very weak inhibitors. Among the disaccharide-derivatized polymers tested, only the GalNAc alpha1,3-Gal beta-derivatized polymer was a potent inhibitor, with an IC of 11 µg/ml (2.5 µM in sugar residues, approx370 nM in polymer chains). The closely related GalNAc alpha1,3-GalNAc alpha 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 (GalNAcalpha1,3(Fucalpha1,2)Galbeta) polymer causing half-maximal inhibition at 250 µg/ml (44 µM in sugar residues, >8 µM in polymer chains).


Figure 7: Inhibition of I-GalNAcBSA binding to E. histolytica membranes by multivalent saccharide-derivatized linear polyacrylamide. Radioligand binding was performed under standard conditions using 0.88 nMI-GalNAcBSA (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, GalNAcbetapropyl, filled circles; GalNAcalphapropyl, open squares; Galbetapropyl, open triangles; Glcbetapropyl, closed squares; and GlcNAcbetapropyl, closed triangles. Middle panel, GalNAcalpha3Galbetapropyl, closed circles; GalNAcalpha3GalNAcalphapropyl, open circles; Galalpha3GalNAcalphapropyl, triangles; and Galalpha3Galbetapropyl, squares. Lower panel: GalNAcalpha3(Fucalpha2)Galbetapropyl, squares; and Galalpha3(Fucalpha2)Galbetapropyl, circles. Data are presented as mean ± S.E. for duplicate determinations.



Binding of Purified E. histolytica Lectin to GalNAcBSA

Direct binding of the purified E. histolytica lectin to GalNAcBSA was demonstrated using a BIAcore biosensor-based analytical system. GalNAcBSA was covalently immobilized via amide linkage between the neoglycoprotein (which retains approx20 free lysine -amino groups per molecule) and carboxylated dextran on the sensor surface. When purified E. histolytica lectin was introduced into the sensor flow cell, binding was detected (Fig. 8, top). The association and dissociation rate constants were obtained directly from the relative response plots(24) . The calculated k(a), based on analysis of kinetics using three different lectin concentrations, was 3.2 times 10^4M s. Dissociation (Fig. 8, bottom) was analyzed according to a two-site model, resulting in dissociation rates of 2.5 times 10 s (rapid phase, 13% of bound lectin) and 3.2 times 10 s (slow phase). This results in K(D) values of 9.8 times 10M for the larger fraction of the bound lectin (slow dissociating) and 7.9 times 10M for the smaller fraction (rapid dissociating). Addition of 35 µM asialofetuin to the purified lectin prior to introduction into the sensor flow cell resulted in a marked decrease in the k(a) and an increase in the k(d) (Fig. 8), suggesting that binding of asialofetuin to the lectin inhibited its subsequent association with the immobilized GalNAcBSA on the sensor chip. A decrease in the k and an increase in the k were also detected when higher concentrations of low molecular weight inhibitors were added to the lectin prior to injection (data not shown).


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 GalNAcBSA (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.




DISCUSSION

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 GalNAcBSA (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)(3) has a 20,000-fold higher affinity for the MHL than the corresponding monosaccharide, or approx7,000-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 GalNAcBSA) 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(2), 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-GalNAcBSA 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-GalNAcBSA 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 approx100-fold more potent at blocking hemagglutination than blocking I-GalNAcBSA 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-GalNAcBSA. 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-GalNAcBSA 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 GalNAcBSA, 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(D) values of purified lectin binding to GalNAcBSA approached those of membrane binding. However, inhibition of binding of the lectin to immobilized GalNAcBSA 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 GalNAcBSA 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-GalNAcBSA binding to E. histolytica membranes by multivalent carbohydrate-derivatized polyacrylamide polymers (Fig. 7). The beta anomer of GalNAc linked directly to the spacer arm on the polymer was >40-fold more potent at blocking binding than the corresponding alpha anomer, while an alpha anomer of GalNAc spaced away from the polymer backbone (GalNAcalpha1,3Galbeta) was equipotent to the betaGalNAc-derivatized polymer. These data may reflect a preference for the GalNAc-Gal disaccharide, but may also suggest that the alpha 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 GalNAcalpha1,3Galbeta 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 approx50% 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 NeuAcalpha2,3Galbeta1,3GalNAc, 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.


FOOTNOTES

*
This work was supported by The U. S. Army, Chemical and Biological Defense Command, Edgewood Research Development and Engineering Center, Edgewood, MD (TCN93-094, DO 0685), The Council for Tobacco Research (to W. A. P.), and National Institutes of Health Grant AI-26649 (to W. A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Burroughs Wellcome Fund New Investigator in Molecular Parasitology.

To whom correspondence should be addressed: Dept. of Pharmacology, The Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-8392; Fax: 410-955-3023; rschnaar{at}welchlink.welch.jhu.edu.

(^1)
Neoglycoproteins are denoted by the type of sugar covalently attached, the average number of sugars attached per protein molecule (subscripted), and the protein carrier, in this case bovine serum albumin (BSA). Other abbreviations used are: MHL, mammalian hepatic lectin; PBS, phosphate-buffered saline; Mes, 2-(N-morpholino)ethanesulfonic acid. Abbreviations for multivalent synthetic saccharides are given as footnotes to Table 1and Table 4.


ACKNOWLEDGEMENTS

We are grateful to Lauren Ashley for preparation of E. histolytica membranes.


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