Lectins are carbohydrate-binding proteins other than enzymes or antibodies, which may contain a second type of binding site specific for noncarbohydrate ligands (Barondes, 1988). Agaricus bisporus lectin (ABL) was first reported by Presant and Kornfeld (1972), exhibiting erythrocyte agglutinating activity. This lectin (Mr 64,000) is a tetramer with four apparently identical subunits (Mr 16,000). Moreover, four different isoelectric forms that have quite similar carbohydrate-binding specificities were reported previously (Sueyoshi et al., 1985).
The ABL specificity has been related to alcali-labile carbohydrate chains of glycoproteins (Presant and Kornfeld, 1972; Chatterjee et al., 1985; Sueyoshi et al., 1985). There has been a recently renewed interest in the post-translational modification of serine and threonine hydroxyl groups by glycosylation because the resulting O-linked oligosaccharide chains tend to cluster over short stretches of peptides and hence display multivalent carbohydrate antigenic or functional determinants for antibody recognition, mammalian cell adhesion, and microorganism binding (Hounsell et al., 1996; Taylor-Papadimitriou and Finn, 1997). The majority of the O-linked chains on serum and membrane glycoproteins are the sialylated tri- and tetrasaccharide type based on type I core (Gal[beta]1-3GalNAc[alpha]1-Ser/Thr) having sialic acid linked [alpha]2-3 to Gal and/or [alpha]2-6 to GalNAc. The nonsialylated type I core, also called T-disaccharide (Thomsen-Friedenreich disaccharide), was reported as a tumor-associated antigen of non-oncofetal origin and probably as one of the few chemically well-defined antigens with a proven link to malignancy (Springer, 1984; Cao et al., 1995; Yang and Shamsuddin, 1996). Hence, anti-T probes have an enormous potential in cancer research (Carneiro et al., 1994; Desai et al., 1995; Langkilde, 1996). In addition, T-disaccharide and related carbohydrates were assayed as antigens in active specific immunotherapy of tumor-bearing hosts (Toyokuni and Singhal, 1995; Koganty et al., 1996; Graham et al., 1996; Springer, 1997). Moreover, ABL, which mainly binds Gal[beta]1-3GalNAc, is a reversible noncytotoxic inhibitor of epithelial cell proliferation (Yu et al., 1993) unlike the classical T-disaccharide specific lectin, peanut agglutinin, which stimulates proliferation of these cells (Ryder et al., 1992).
The aim of the present work is to further understand the structural requirements of carbohydrates for ABL binding in an attempt to explain these interactions, and perhaps contribute to the design of new antigens for specific cancer therapies.
In preliminary experiments, the concentration of ABL-peroxidase (ABL-HRP) and human monoclonal IgA1k Pan (containing O-linked oligosaccharides) which proved to be optimal for hapten inhibition of competitive enzyme-lectin assays (CELA) was determined (data not shown). Optimal conditions for CELA were 0.04 µg/ml ABL-HRP and 10 µg/ml IgA1k Pan. We first sought to elucidate the relevancy of free monosaccharides and anomeric linkage in binding since free Gal and GalNAc were compared to the [alpha]- and [beta]-anomer of the methyl and p-nitrophenyl glycosides, respectively. As seen in Figure
Figure 1. Competitive enzyme-lectin assay (CELA) using carbohydrates as inhibitors of ABL interaction. The importance of free saccharides and anomeric linkage derivatives were examined. Wells coated with IgA1k Pan (which contains O-linked oligosaccharides) were assayed against ABL-HRP, previous incubation of the lectin with the following carbohydrates: (A) monosaccharides: Gal (solid squares), Me[alpha]Gal (open triangles), Me[beta]Gal (open squares), GalNAc (open circles), pNO2Ph[alpha]GalNAc (solid circles), pNO2Ph[beta]GalNAc (inverted open triangles) and GlcNAc (inverted solid triangles). (B) disaccharides: Gal[beta]1-4Glc (solid diamonds), Gal[alpha]1-6Glc (open circles), Gal[beta]1-3GlcNAc (inverted open triangles), Gal[beta]1-4GlcNAc (solid circles), Gal[beta]1-6GlcNAc (open diamonds), Gal[beta]1-3GalNAc (solid triangles) and Gal[beta]1-3GalNAc[alpha]pNO2Ph (open triangles). The washes and color reaction were developed as described under Material and methods.
Among the monosaccharides and disaccharides tested, Gal[beta]1-3GalNAc and Gal[beta]1-3GalNAc[alpha]pNO2Ph were the most powerful inhibitors (Figure
Table I.
In addition, lactose and Gal[beta]1-6GlcNAc have a significantly inhibitory activity on the binding of ABL. Both disaccharides are ~10-fold more powerful inhibitors than GalNAc. From minimum energy conformations of disaccharides obtained by theoretical studies as well as from the structures obtained by x-ray crystallography (Sastry et al., 1986; Sharma et al., 1996), it was found that the C-4 hydroxyl group of the reducing sugar moiety in T-disaccharide occupies a similar position regarding terminal Gal to that of the C-3 and C-4 hydroxyl groups in lactose and Gal[beta]1-6GlcNAc, respectively. The relevance of the C-4 hydroxyl group position in the reducing sugar moiety of T-disaccharide on the interaction with ABL, and the similar position of C-3 and C-4 hydroxyl groups in lactose and Gal[beta]1-6GlcNAc, respectively, could be an explanation of the interaction of ABL with both disaccharides. Moreover, melibiose (Gal[alpha]1-6Glc), which has no hydroxyl group in a position similar to that of the C-4 hydroxyl group of the reducing sugar moiety from T-disaccharide, shows no interaction with ABL. N-Acetyllactosamine (Figure
In an attempt to obtain additional information from the study of carbohydrate requirements on ABL interaction, we used glycolipids in binding analysis. Their relative mobility and binding to ABL were analyzed by chromatographic separation on HPTLC-silica gel plates and HPTLC-lectin staining, respectively (Figure
Figure 2. HPTLC-lectin binding with glycolipids. N3 (1), N4 (2), N5a (3), N5b (4), N6 (5), N7 (6), A5b (7), A6 (8), GA1 (9), and nLcose4 (10) glycolipids were assayed on HPTLC plates using chloroform/methanol/aqueous 0.2% CaCl2 (45:45:10) as the running solvent. The glycolipids were visualized either chemically (A) using orcinol-sulfuric acid spray reagent, for 5 min at 120°C; or according to ABL interaction (B) incubating with ABL-HRP (0.08 µg/ml) in PBS-t for 2 h at 23°C. After washes, the color reaction was developed by using 0.5 mg/ml 4-chloro-1-naphthol and 0.02% H2O2 in methanol-PBS (1:29) during 30 min. Reactions were stopped by washes with distilled water.
Figure 3. Reactivity of ABL binding with glycolipids by using direct enzyme-lectin assay (DELA). Glycolipids, N3 (solid circles), N4 (multiplication signs), N5a (dotted line), N5b (solid triangles), N6 (open squares), N7 (solid squares), A5b (open circles), A6 (open triangles), GA1 (plus signs), and nLcose4 (asterisks) were dissolved in methanol, pipetted in triplicated into the wells of a microtiter plate, and evaporated at 60°C. The plates were incubated with a concentration range of ABL-HRP in PBS-t during 2 h at 23°C. The washes and color reaction was developed as described under Material and methods.
Table II.
When comparing ABL specificity with other T-disaccharide specific lectins such as AOL (Arthrobotrys oligospora lectin), jacalin, and peanut agglutinin (Sastry et al., 1986; Gupta et al., 1992; Rosen et al., 1996; Sharma et al., 1996), one important difference is that the last two lectins, unlike ABL, significantly bind to monosaccharides related to T-disaccharide. This ABL feature may be due to a more extensive carbohydrate-binding site in comparison to jacalin and peanut agglutinin, and thus higher selectivity of binding is achieved by extending binding sites through contacts between oligosaccharides and the protein surface (Weis and Drickamer, 1996). While, the fungal lectin AOL has an apparent major similarity to ABL binding properties.
In conclusion, we find than ABL only interacts with certain regions and under defined stereochemical requirements of the carbohydrates, showing a particular binding nature different from that of other T-disaccharide specific lectins. Based on previous studies that involved T-disaccharide in epithelial tumor cells (Springer, 1984; Cao et al., 1995; Yang and Shamsuddin, 1996), the level of anti-T and anti-related carbohydrate antibodies with the progression and aggressiveness of malignancy (Chen et al., 1995; Desai et al., 1995), and the inhibitory effect of ABL on the growth of tumor cell lines (Yu et al., 1993), we hypothesize that the achievement of immune response with a carbohydrate-binding specificity similar to ABL could be a new approach to the inhibition of tumor cell proliferation. Thus, the use of an adequately processed T-disaccharide, exposing relevant regions of the disaccharide that mainly interact with ABL such as the major significance of GalNAc over Gal, could be an alternative antigen for active immunization in cancer therapy.
Figure 4. CPK models of terminal trisaccharide corresponding to N6 (a), N5b (b), and GA1 (c). The side view of trisaccharides show the common Gal[beta]1-3GalNAc terminal as well as the adjacent carbohydrate that carried it. The arrows show the C-4 hydroxyl group positions of Gal (left) and GalNAc (right) from terminal T-disaccharide and arrowhead shows the differential position of C-6 residue from carbohydrate carrier to common T-disaccharide. Materials
Carbohydrates: Gal, Me[alpha]Gal, Me[beta]Gal, GalNAc, pNO2Ph[alpha]GalNAc, pNO2Ph[beta]GalNAc, GlcNAc, Gal[alpha]1-6Glc, Gal[beta]1-4Glc, Gal[beta]1-3GalNAc, Gal[beta]1-3GalNAc[alpha]pNO2Ph, Gal[beta]1-3GlcNAc, Gal[beta]1-4GlcNAc, and Gal[beta]1-6GlcNAc were purchased from Sigma Chemical Co. (St. Louis, MO). Human monoclonal IgA1k Pan was a gift from Dr. P. Aucouturier (Hospital Necker, Paris, France). The glycolipids used in this study were obtained from the following sources: N3, N4, N5a, N5b, N6, N7, A5b, and A6 were obtained from Calliphora vicina as previously reported (Dennis et al., 1985; Nores et al., 1991); GA1 and nLcose4 were prepared by mild acid hydrolysis of cow brain GM1 and human red blood cell LM1, respectively. ABL was purified and conjugated to horseradish peroxidase (ABL-HRP) as described previously (Irazoqui et al., 1997). Competitive enzyme-lectin assays (CELA)
CELA was performed as described previously (Irazoqui et al., 1997). Briefly, polystyrene microtitration plates (Corning, NY) were coated with 10 µg/ml human IgA1k Pan (100 µl/well) in 0.1 M carbonate buffer pH 9.6 overnight at 4°C, and saturated with PBS (10 mM potassium phosphate, pH 7.2, 150 mM NaCl)-0.05% Tween 20 (PBS-t) for 1 h at 37°C. Carbohydrates were preincubated with 0.04 µg/ml ABL-HRP for 1 h at 23°C, before adding 100 µl/well. The plates were incubated for 2 h at 23°C and washed six times with PBS-t. The color reaction was developed by using 2 mg/ml o-phenylenediamine and 0.02% H2O2 in 0.1 M sodium citrate pH 5.0 at 23°C for 30 min. Reactions were stopped by adding 100 µl/well of 2.5 M sulfuric acid and absorbance values were read at 492 nm with a microplate reader (model 450, Bio-Rad). HPTLC-lectin staining
The glycolipids were separated on HPTLC silica gel 60 (Merck) in the running solvent chloroform/methanol/aqueous 0.2% CaCl2 (45:45:10) by using a tank to obtain highly reproducible chromatograms (Nores et al., 1994). After air-drying for 15 min, the plates were coated by dipping in a 0.5% solution of polyisobutylmethacrylate (Plexigum P 28, Rohm and Haas, Darmstadt, Germany) in hexane/chloroform (9:1) for 1 min and air-dried again for 10 min. The plates were incubated with 0.08 µg/ml ABL-HRP in PBS-t for 2 h at 23°C. After five washes with PBS-t during 5 min, the color reaction was developed by using 0.5 mg/ml 4-chloro-1-naphthol and 0.02% H2O2 in methanol-PBS (1:29) for 30 min. Reactions were stopped by washing with distilled water (Mizutamari et al., 1994). Direct enzyme-lectin assays (DELA)
Glycolipids dissolved in methanol were pipetted into the wells of a microtiter plate and evaporated at 60°C. The plates were incubated with a concentration range (0.036-4.6 µg/ml) of ABL-HRP in PBS-t (100 µl/well) for 2 h at 23°C. The final washes and color reaction were performed as CELA. The data of DELA were fitted with LIGAND soft v. 3.1 (Munson and Rodbard, 1980; Hendriks et al., 1987) for the measurement of affinity constants (Ka) between ABL and glycolipids. All assays were performed in triplicate. Molecular modeling
Minimum energy conformations of the carbohydrates were performed using molecular mechanic calculations with MM2 force field. Three-dimensional structures were constructed using CPK models (Harvard Apparatus, South Natick, MA).
We thank Dr. P. Aucouturier for his generous supply of human monoclonal IgA1k Pan, Dr. P. Munson for kindly providing us with LIGAND soft, Dr. B. Caputto and Dr. L. Castagna for their critical reading of the manuscript and Mrs. I. Orsingher for the language assistance. This work was supported in part by grants (to G.A.N.) from CONICET (PMT-PICT 0462), CONICOR and SeCyT (UNC), and by a grant (to M.A.V.) from CONICOR. F.J.I. acknowledges a fellowship assistance from CONICET.
ABL, Agaricus bisporus lectin; AOL, Arthrobotrys oligospora lectin; CELA, competitive enzyme-lectin assays; DELA, direct enzyme-lectin assays; Gal, d-galactose; GalNAc, N-acetyl-d-galactosamine; Glc, d-glucose; GlcA, glucuronic acid; GlcNAc, N-acetyl-d-glucosamine; GM1, Gal[beta]1-3GalNAc[beta]1-4(NeuAc[alpha]2-3)Gal[beta]1-4Glc[beta]-Cer; HRP, horseradish peroxidase; LM1, NeuAc[alpha]2-3Gal[beta]1-4GlcNAc[beta]1-4Gal[beta]1-4Glc[beta]-Cer; Ka, affinity constant, Me, methyl; NeuAc, neuraminic acid; O.D., optical density; pNO2Ph, p-nitrophenyl; SD, standard deviation;T-disaccharide, Thomsen-Friedenreich disaccharide.
2To whom correspondence and reprint requests should be addressed
Carbohydrates
Concentration (mM) required for 50% inhibition
Relative inhibitory potency
Gal
>200
<0.2
Me[alpha]Gal
>200
<0.2
Me[beta]Gal
>200
<0.2
GalNAc
40
1
pNO2Ph[alpha]GalNAc
40
1
pNO2Ph[beta]GalNAc
40
1
Gal[beta]1-4Glc
5
8
Gal[beta]1-6GlcNAc
4
10
Gal[beta]1-3GalNAc
0.02
2000
Gal[beta]1-3GalNAc[alpha]pNO2Ph
0.02
2000
Glycolipids
10-7 × Ka (M-1)b
N6
22 (±3.2)c
N7
20 (±3.1)
A6
7.3 (±0.61)
N5
b6.9 (±0.59)
GA1
N. B.
Materials and methods
Acknowledgments
Abbreviations
References
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