2 Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, Chang-Gung University, Kwei-san, Tao-yuan, 333, Taiwan; 3 Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India; and 4 Department of Microbiology and Immunology, Chang-Gung University, Kwei-san, Tao-yuan, 333, Taiwan
Received on June 7, 2004; revised on August 15, 2004; accepted on August 24, 2004
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Abstract |
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Key words: Artocarpus lakoocha / carbohydrate specificities / glycoprotein binding / lectins / polyvalency
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Introduction |
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Data obtained from earlier studies provided information on the carbohydrate specificity of ALA, which was limited to the inhibition of monosaccharides and several T-related disaccharides (Chatterjee et al., 1988; Chowdhury and Chatterjee, 1993
). Further characterization according to the mammalian carbohydrate structural units and corresponding glycoconjugates may establish better understanding on the binding specificity of ALA for mammalian glycotopes in macromolecules. Such systematic analysis on the binding spectrum of a lectin is important particularly when used as a diagnostic tool. For decades, Agaricus bisporus agglutinin (ABA) has been recognized as an anti-T-specific lectin. However, results of more extensive analysis showed that this lectin also bound well to glycoproteins containing blood group precursor type I/II sequence glycotope (Galß1
3/4GlcNAc) (Wu et al., 2003a
). Therefore, in the present study, we systematically examined the glycan affinity of ALA at macromolecular level by enzyme-linked lectinosorbent assay (ELLSA) and our developed inhibition assay (Duk et al., 1994
; Lisowska et al., 1996
) using our structurally well-defined glycan/ligand collection. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens Tn and T
, but not with other common glycotopes on glycoproteins, such as ABH blood group antigens, Galß1
3/4GlcNAc (I/II) determinants, T/Tn covered by sialic acids, and N-linked plasma glycoproteins, suggesting that polyvalency of Tn/T glycotopes play an essential role in ALAglycoprotein binding.
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Results |
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Discussion |
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Interactions between carbohydrates on the surface of one cell type and proteins on the surface of another cell type are presumed to play critical roles in a wide variety of biochemical recognition processes. However, studies have shown that individual carbohydrateprotein binding is too weak to affect this specific interaction (Horan et al., 1999; Wu et al., 2003a
). This situation has been partially explained by the phenomenon of polyvalency. Indeed, numerous studies have demonstrated that polyvalent display of carbohydrates can lead to remarkably increase binding avidities (Liang et al., 1996
; Spevak et al., 1996
; Wu et al., 2003a
). Therefore, it becomes pivotal to estimate the polyvalent effects while establishing the binding profile of a lectin. However, due to the intrinsic difficulties in synthesizing branched carbohydrate derivatives, this kind of interaction was seldom investigated. As a consequence, natural polyvalent glyconconjugates bearing well-defined reactive glycotopes become materials of interest.
In this study, we examined the influence of polyvalency on the binding avidity of ALA. From the results of Table II, it is demonstrated that O-linked asialo HSM (Tn) generates an enhancement in affinity with ALA by about 6.7 x 105 times over Gal (curve 1 versus 12) and is about 1.3 x 105 times more active than monomeric Tn (curve 1 versus 10 in Table II). This increase in inhibitory potency from free monovalent sugars to glycoproteins with ALA suggests the importance of polyvalency. Based on the results of this study, the concept of glycoside cluster effect can be classified into two groups: (1) the multiantennary or simple glycoside cluster effect, as in galactosides with hepatic lectin (Lee, 1992; Lee and Lee, 2000
) and triantennary II sequences within the prototype chicken liver (CG-16) (Wu et al., 2001
) or Tn glycopeptides (curve 9 in Table II); and (2) the high-density polyvalent or complex glycoside cluster effect, as in macromolecular interaction of high density polyvalent Tn in asialo HSM and native ASG-Tn with ALA.
A similar phenomenon was also observed with some plant and animal lectins (Wu, 2004; Wu et al., 2002
, 2003a
, 2004a
). However, the effect of polyvalencies of glycotopes on the carbohydrateprotein binding does not always make such an important contribution. The theory on binding and the relation of the interaction of natural polyvalent glycotopes has not yet been well established. For example, the potency in the interaction of Pseudomonas aeruginosa II lectin with LFuc
1
polyvalent glycans is about as strong or weaker than the incremental increase in carbohydrate specificity of monomers (Mitchell et al., 2002
; unpublished data). Similarly, in Anguilla anguilla agglutinin (Wu et al., 2004b
), although greater affinity was seen for polyvalent ligands in glycoproteins than with monovalent haptens, the degree of enhancement was 1.5 x 104 times less compared to that found in other lectins, such as ABA (Wu et al., 2003a
) and galectin-4 from rat gastrointestinal tract (Wu et al., 2004a
). This indicates that the power of polyvalent effects on carbohydrateprotein interactions must be individually evaluated to establish its rules or theory.
Many reports revealed that lectins with the same mono- or oligosaccharide specificity may demonstrate completely different specificities for macromolecules and may even show a shift of binding specificity from one type of carbohydrate ligand to another when the surface density of the carbohydrate increases (Horan et al., 1999; Lee, 1992
; Lee and Lee, 2000
; Wu, 1988
, 2001
; Wu et al., 2003a
). Therefore, to provide a more satisfactory and realistic depiction of the carbohydrate specificity of a lectin, the following five criteria are suggested to be considered (Wu, 2001
): (1) monosaccharide specificity (Gal, GalNAc, GlcNAc, and/or Man); (2) expression of reactivities toward mammalian structural units in decreasing order (disaccharide and Tn specificity); (3) the most active ligand; (4) simple multiantennary or cluster effect, using either natural glycopeptides or synthetic neoglycoconjugates with branching sugar side chains; and (5) complex multivalent or cluster effects, displayed by macromolecules with characterized reactive glycotopes. In the current study, we provide almost all the information required for the comprehensive description of the carbohydrate specificity of ALA.
T/Tn antigen occurs on the surface of tumor cells as a mucin-associated antigenic marker and is one of the few chemically well-defined tumor antigens with a proven link to malignancy (Springer, 1995). They are good marker for several cancerous tissues, including the prognosis of colorectal cancer (Itzkowitz et al., 1990
; Ono et al., 1994
). From the remarkably strong affinity of ALA for Tn/T glycoproteins observed in this study without cross-reacting with other glycoproteins, one can reasonably assume that ALA may be an useful tool (1) as a structural probe to detect the aberrant expression of Tn/T epitopes in tissues and cells, (2) in the analysis of O-linked glycoproteins, and (3) for the identification and fractionation of glycopeptides and oligosaccharides.
Although some plant lectins have specificity for T/Tn glycoconjugates (Ahmed and Chatterjee, 1989; Medeiros et al., 2000
; Wu, 2004
; Wu et al., 1999
), plants do not appear to have T/Tn structures (Takeya et al., 1998
), suggesting that the functions of these lectins may act against the animals other than themselves. This is further supported by the fact that some insects express Tn structures on the mucins or other O-glycosylated glycoproteins in their digestive tract that act as receptors for a Tn antigenbinding dietary lectin (Wang et al., 2003
). If such a lectinglycan interaction provokes deleterious effects on cells carrying the Tn structures, the digestive system of the insect may be impaired, which in turn may lead to a more or less general toxic effect (Wang et al., 2003
), implying its role in plant defense (Peuman and Van Damme, 1995
). It is worth noting in this context that jacalin from Artocarpus integrifolia a species closely related to A. lakoocha, binds to T/Tn glycoproteins and possesses insecticidal properties (Czapla and Lang, 1990
). The possible toxicity of ALA against animals and invertebrates has not yet been studied. Nevertheless, ALA is capable of interacting with Tn/T glycoconjugates, which readily leads to the assumption that it can exert deleterious effects in the gastrointestinal tract of higher animals or insects, thus implying its role in plant defense.
It is of interest to examine the binding specificities of ALA with that of other Gal/GalNAc reactive lectins, such as jacalin and ABA (Wu et al., 2003a,b
; Table IV): (1) The three lectins have equal preference for poly Tn; (2) the reactivity for poly T can be ranked as ABA > jacalin > ALA; (3) ABA and jacalin reacted well with multiantennary II-containing N-glycans, whereas ALA binds weakly or not at all (Table IV); (4) the exposion of Tn, II is not required for binding when it reacts with ABA and jacalin because it also recognizes and reacts with cryptic forms of Tn/II glycotopes; on the other hand, ALA acts poorly or is completely inactive with crypto or sialylated glycoproteins, thus demonstrating that ABA and jacalin are excellent reagents to detect the presence of both exposed and crypto Tn/II, whereas ALA should be an ideal reagent to distinguish between exposed and sialylated Tn glycoproteins. These informations not only illustrate the concept that every lectin has its own binding character but also provide the essential knowledge for future selection or development of these structural probes in medical and biotechnological applications, such as differentiation and/or characterization of sugar mixtures.
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Materials and methods |
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Glycoproteins and polysaccharides
HSM, one of the simplest glycoproteins among mammalian salivary mucins containing sialyl-Tn (NeuAc-GalNAc) and Tn (GalNAc1
Ser/Thr) residues as carbohydrate side chains, was obtained by a modification of the methods previously described (Wu et al., 1994
). The sialyl-Tn- and Tn-containing glycoprotein isolated from hamster submaxillary gland contained 28% GalNAc, 35% NeuAc, and 33% total amino acids, in which six amino acids (Thr+Ser, 54%; Pro, 16.2%; Glu, 8.7%; Lys, 8.3%; and Ala, 6.7%) constituted 94 mole % of the protein core.
Ovine, bovine, and porcine submandibular/salivary glycoproteins were purified according to the method of Tettamanti and Pigman (1968) with modifications (Herp et al., 1979
, 1988
). About 75% of the carbohydrate side chains of asialo OSM were Tn. Asialo PSM contains T
together with Tn and GalNAc
1
3Gal (A) sequences, as most of the outer fucosyl residues and sialic acids are cleaved by mild acid hydrolysis. Native ASG-Tn (Wu et al., 1994
), a salivary glycoprotein of nine-banded armadillo (Dasypus novemcinctus mexicanus) containing only Tn as carbohydrate side chains, was isolated from 0.01 M PBS, pH 6.8, gland extract after removal of ASG-A, which is one of the sialoglycoproteins in armadillo glands (Wu and Pigman, 1977
).
Glycophorin A was prepared from the membranes of outdated human blood group O erythrocytes by phenol/saline extraction and purified by gel filtration in the presence of sodium dodecyl sulfate (Lisowska et al., 1987). Desialylation of glycophorin was performed as described later (Tettamanti and Pigman, 1968
; Wu and Pigman, 1977
). The Tn-type glycophorin was obtained by removing galactose residues from asialo-glycophorin by periodate oxidation and mild acid hydrolysis (Smith degradation) (Duk et al., 1994
).
Desialylation of sialoglycoproteins was performed by mild acid hydrolysis in 0.01 N HCl at 80°C for 90 min and dialyzed against distilled water for 2 days to remove small fragments (Tettamanti and Pigman, 1968; Wu and Pigman, 1977
).
The antifreeze glycoprotein from the Antarctic fish (Trematomus borchgrevinki), which contains only T as carbohydrate chains (De Vries et al., 1970
) was provided by Dr. R. E. Feeney (Department of Food Science and Technology, University of California, Davis) through the late Dr. E. A. Kabat (Columbia Medical Center, New York).
Cyst blood group active glycoproteins (e.g., 9, 19, Mcdon, Beach, Tighe, JS, etc.) were prepared from human ovarian cyst fluid as described previously (Wu, 1988; Wu et al., 1982
, 1984
). In general, the P-1 fractions (e.g., Cyst Mcdon P-1, or Tighe P-1) represent the nondialyzable portion of the blood group substances after mild hydrolysis at pH 1.52.0 for 2 h, which removes most of the L-fucopyranosyl end groups, as well as some blood group A and B active oligosaccharide side-chains (Allen and Kabat, 1959
; Kabat et al., 1948
; Leskowitz and Kabat, 1954
). P-1 fractions from human ovarian cyst fluid glycoproteins, which expose the internal structures equivalent to those on the blood group precursors are defined as precursor equivalent glycoproteins.
The human blood group P1-active substance, purified from sheep hydatid cyst glycoprotein (Cory et al., 1974; Morgan and Watkins, 1964
), was kindly provided by late Dr. W. M. Watkins (Imperial College School of Medicine, Hammersmith Hospital London).
Human 1-acid glycoprotein (Sigma) contains tetra-, tri-, and diantennary complex type glycans in the ratio of 2:2:1 (Fournet et al., 1978
; Fournier et al., 2000
). Fetuin (Gibco Laboratories, Grand Island, NY), which is the major glycoprotein in fetal calf serum and has six oligosaccharide side chains per molecule, three O-glycosidically linked to Ser/Thr and three N-glycosidically linked to Asn (Nilsson et al., 1979
), contains tri- and diantennary complex type glycans in the ratio of 1:2.
RSL was prepared by the method of Moschera and Pigman (1975). The established carbohydrate side chains were found to be composed of 9, 10, 12, 13, and 15 sugar residues, respectively, and contain sialic acid, N-acetylglucosamine, galactose, and N-acetylgalactosaminitol (Slomiany and Slomiany, 1978
). RSL may also contain Tn-reactive determinants (Wu et al., 1995
).
Yeast high-mannose type glycan (mannan) and pectins from apple and citrus fruits were purchased from Sigma.
Sugars used for inhibition studies
Mono-, di-, and oligosaccharides were purchased from Dextra (Berkshire, UK) and Sigma. Carbohydrate structural units were purchased or prepared by Dextra Laboratories. The Tn clusters used for this study were mixtures of Tn-containing glycopeptides from OSM in the filterable fraction (molecular mass cutoff < 3000) (Wu et al., 1997).
The microtiter plate lectinenzyme binding assay
ELLSA was performed according to the procedures described by Duk et al. (1994). The volume of each reagent applied to the plate was 50 µl/well, and ALA incubations, except for coating, were performed at room temperature (20°C). The reagents, if not indicated otherwise, were diluted with TBS containing 0.05% Tween 20 (TBS-T). TBS buffer or 0.15 M NaCl containing 0.05% Tween 20 was used for washing the plate between incubations.
The 96-well microtiter plates (Nunc, MaxiSorp, Vienna) were coated with glycoproteins at 5 to less than 0.1 ng (Figure 1a) and 500 to less than 20 ng per well (Figure 1d) in 0.05 M carbonate buffer, pH 9.6, and overnight at 4°C. After washing the plate, biotinylated lectins (25 ng) were added to each well and incubated for 30 min. The plates were washed to remove unabsorbed lectin and the ExtrAvidin/alkaline phosphatase solution (Sigma, diluted 1:10,000) was added. After 1 h, the plates were washed at least four times and incubated with p-nitrophenyl phosphate (Sigma 104 phosphatase substrate 5-mg tablets) in 0.05 M carbonate buffer, pH 9.6, containing 1 mM MgCl2 (1 tablet/5 ml). The absorbance was read at 405 nm in a microtiter plate reader after 2 h incubation with the substrate.
For inhibition studies, serially diluted inhibitor samples were mixed with an equal volume of lectin solution containing a fixed amount of lectin. The control lectin sample was diluted twofold with TBS-T. After 30 min incubation at 20°C, samples were tested in the binding assay as described. The inhibitory activity was estimated from the inhibition curve and is expressed as the amount of inhibitor (ng or nmol/well) giving 50% inhibition of the control lectin binding.
All experiments were done in duplicates or triplicates, and data are presented as the mean value of the results. The standard deviation did not exceed 12%, and in most experiments was less than 5% of the mean value. For the binding experiment, the control wells, where coating or addition of biotinylated lectin was omitted, gave low absorbance values (below 0.1). It showed that blocking the wells before lectin addition was not necessary when Tween 20 was present in the TBS. On the other hand, for the inhibition experiment, two controls were set up. The first control was treated under exactly the same conditions as the experimental group except that the inhibitor was left out; the absorbance value recorded at 2 h was between 2.8 and 3.0. For the second control (coating only or negative control), both lectin and inhibitors were left out and other conditions were kept the same as in the experimental group, and the absorbance value of this control was below 0.1, which was used as the subtracted background value.
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Acknowledgements |
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Abbreviations |
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References |
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