Carbohydrate recognition factors of a T{alpha} (Galß1->3GalNAc{alpha}1->Ser/Thr) and Tn (GalNAc{alpha}1->Ser/Thr) specific lectin isolated from the seeds of Artocarpus lakoocha

Tanuja Singh2, Urmimala Chatterjee3, June H. Wu4, Bishnu P. Chatterjee3 and Albert M. Wu1,2

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


1 To whom correspondence should be addressed; e-mail: amwu{at}mail.cgu.edu.tw

Received on June 7, 2004; revised on August 15, 2004; accepted on August 24, 2004


    Abstract
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 Abstract
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 Results
 Discussion
 Materials and methods
 References
 
Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha fruit, is a galactose-binding lectin and a potent mitogen of T and B cells. Knowledge obtained from previous studies on the affinity of ALA was limited to molecular and submolecular levels of Galß1->3GalNAc (T) and its derivatives. In the present study, the carbohydrate specificity of ALA was characterized at the macromolecular level according to the mammalian Gal/GalNAc structural units and corresponding glycoconjugates by an enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens GalNAc{alpha}1->Ser/Thr (Tn) and Galß1->3 GalNAc{alpha}1->Ser/Thr (T{alpha}). It barely cross-reacts with other common glycotopes on glycoproteins, including ABH blood group antigens, Galß1->3/4GlcNAc (I/II) determinants, T/Tn covered by sialic acids, and N-linked plasma glycoproteins. Dense clustering structure of Tn/T{alpha}-containing glycoproteins tested resulted in 2.4 x 105–6.7 x 105-fold higher affinities to ALA than the respective GalNAc and Gal monomer. According to our results, the overall affinity of ALA for glycans can be ranked respectively: polyvalent Tn/T{alpha} glycotopes >> monomeric T{alpha} and simple clustered Tn >> monomeric Tn > GalNAc > Gal; while other glycotopes: Gal{alpha}1->3/4Gal (B/E), Galß1->3/4GlcNAc (I/II), GalNAc{alpha}1->3Gal/GalNAc (A/F), and GalNAcß1->3/4Gal (P/S) were inactive. The strong specificity of ALA for Tn/T{alpha} cluster suggests the importance of glycotope polyvalency during carbohydrate–receptor interactions and emphasizes its value as an anti-Tn/T lectin for analysis of glycoconjugate mixtures or transformed carbohydrates.

Key words: Artocarpus lakoocha / carbohydrate specificities / glycoprotein binding / lectins / polyvalency


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The T{alpha} (Galß1->3GalNAc{alpha}1->Ser/Thr) and Tn (GalNAc{alpha}1->Ser/Thr) determinants, normally a cryptic structure in the peptide core of O-glycoproteins, are expressed in an unmasked form in ~ 90% of human carcinomas (Itzkowitz et al., 1990Go; Springer, 1984Go, 1997Go). A direct link has been shown to exist between carcinoma aggressiveness and the density of these antigens, including extent of tissue spread and vessel invasion (Springer, 1995Go). Therefore, the T/Tn determinants have been considered the most specific human tumor-associated structures (Springer, 1995Go). Although T/Tn antigens have been characterized using monoclonal antibodies (Hirohashi et al., 1985Go; Nakada et al., 1993Go), human macrophage lectins (Iida et al., 1999Go), glycosyltransferases (Van den Steen et al., 1998Go), as well as some plant lectins (Medeiros et al., 2000Go; Tollefsen and Kornfeld, 1983Go; Wu, 2004Go; Wu et al., 1999Go), novel lectins recognizing T/Tn structures are still being discovered. An example of such a plant lectin is Artocarpus lakoocha agglutinin (ALA). Isolated from the seeds of A. lakoocha that belong to the family Moraceae, ALA has been characterized as a glycoprotein containing 11.7% carbohydrate in which D-xylose (6%) is the main sugar (Chowdhury et al., 1987Go). Results of biochemical analysis revealed that ALA is a dimer composed of two nonidentical subunits, and each of the dimeric lectin molecule contains two sugar-binding sites.

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., 1988Go; Chowdhury and Chatterjee, 1993Go). 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., 2003aGo). 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., 1994Go; Lisowska et al., 1996Go) using our structurally well-defined glycan/ligand collection. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens Tn and T{alpha}, 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 ALA–glycoprotein binding.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Lectin–glycan interaction
The avidity of ALA for various glycoproteins studied by ELLSA is summarized in Table I, according to the interaction profiles shown in Figure 1. Among the glycoproteins tested, ALA reacted best with asialo hamster submaxillary Tn glycoprotein (asialo HSM) and native Tn glycoprotein from armadillo salivary gland (ASG-Tn), requiring less than 1.0 ng glycoproteins to reach 1.5 A405 within 2 h (Figure 1a; Table I). These glycoproteins contain almost exclusively GalNAc{alpha}1->Ser/Thr (Tn) residues as carbohydrate side chains. Except for active antifreeze glycoprotein (MW 10.5 x 103–21.0 x 103; Figure 1c), ALA also reacted strongly with other Tn/T-containing glycoproteins. These include polyvalent Tn-containing glycoproteins (asialo ovine major fraction of ovine submandibular glycoprotein, asialo OSM in Figure 1a, bovine submandibular glycoprotein [asialo BSM], and porcine submandibular Tn glycoproteins [asialo PSM] in Figure 1b); human blood group precursor equivalent Tn-containing glycoproteins (Mcdon P-1 in Figure 1d); Tn-glycophorin prepared from human erythrocytes (Figure 1b); and T-containing asialo human glycophorin (Figure 1c; Table I). However, ALA reacted poorly with sialylated Tn/T{alpha} mammalian glycoproteins (HSM and OSM in Figure 1a; BSM and PSM in Figure 1b; human glycophorin and inactive antifreeze glycoprotein [MW 2.0 x 103–3.8 x 103] in Figure 1c), ABH glycosylated glycoproteins (Cyst 9, 19 Tighe, Mcdon, Beach, and JS phenol insoluble in Figures 1d and 1e), sheep hydatid cyst glycoprotein (Figure 1d), and multiantennary II-containing N-glycans (native and asialo human {alpha}1-acid, fetuin, and rat sublingual glycoprotein [RSL] in Figure 1f; Table I). Furthermore, ALA was found to be inactive with mannan (Figure 1c), and pectins (Figure 1e).


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Table I. Binding of ALA to human blood group A, B, H, P1, Lewis a– and Lewis b–active glycoproteins, sialoglycoproteins, and asialo glycoproteins by ELLSAa

 


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Fig. 1. Binding of ALA to microtiter plates coated with serially diluted human blood group A, B, H, Lewis a– and Lewis b–active glycoproteins, sialoglycoproteins, and asialoglycoproteins. The amount of lectin used was 25 ng per well. Total volume of the assay was 50 µl. A405 was recorded after 2 h incubation.

 
Inhibition of ALA–glycoform interaction by various glycoproteins
To exclude the possibility that the affinity differences of these glycoproteins were due to the plate adsorption discrepancies, the binding affinity was further confirmed by the inhibition assay described in Materials and methods. The ability of various glycans to inhibit the binding of ALA to a Tn-containing glycoprotein (asialo OSM) was analyzed by ELLSA. The results of inhibition assay are summarized in Table II, and details of the inhibition by individual glycoproteins are shown in Figure 2. Among the glycans tested for inhibition, asialo HSM was the best inhibitor. A mere amount of 0.6 ng was sufficient to inhibit 50% of the lectin–glycan binding, which was 2.4 x 105 and 6.7 x 105 times more potent than GalNAc and Gal, respectively (Figure 2 and Table II; curve 1 versus 11 and 12). The ALA–glycan interaction was also strongly inhibited by most of the high-density polyvalent Tn/T-containing glycoproteins but not by their cryptoforms masked with blood group determinants or sialic acids. The activities were 2.8 x 104 and 8.0 x 104 times higher than monomeric GalNAc and Gal, respectively (curves 2 to 8 versus 11 and 12). Tn glycopeptides (MW <3000) from ovine salivary glycoprotein was 66 and 117 times more active than monomeric Tn and GalNAc (curve 9 versus 10 and 11) and was 2.0 x 103 less active than the corresponding high-density polyvalent Tn glycans (curve 9 versus 1). These results imply that a high density of polyvalent Tn/T glycotopes is required for strong ALA–carbohydrate binding. The sialylated or glycosylated Tn glycotopes of O-glycans, such as blood group ABH active glycoproteins and human salivary glycoproteins, and multiantennary II-containing N-glycans were tested from 278 ng to 556 ng but did not reach 50% inhibition (Table II). The results of both interaction and inhibition results showed consistency.


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Table II. Amount of various glycoproteins giving 50% inhibition of binding of ALA (12.5 ng/50 µl) to a Tn-containing glycoprotein (asialo OSM-50 ng/50 µl)a

 


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Fig. 2. Inhibition of ALA binding to a Tn-containing glycoprotein (asialo OSM) coated on ELLSA plates by various glycoproteins. The quantity of glycoprotein in the coating solution was 50.0 ng per well. The quantity of lectin used for the inhibition assay was 12.5 ng per well. Total volume: 50 µl. A405 was recorded after 2 h incubation. The amount (ng) of glycoprotein required to induce 50% inhibition of binding was determined.

 
Inhibition of ALA–glycoform interaction by mono- and oligosaccharides
To confirm that these bindings are not nonspecific interactions but epitope-specific, the interaction of ALA with glycoproteins was further analyzed by inhibition of specific glycotope units and monosaccharide derivatives. The ability of various sugar ligands to inhibit the binding of ALA to a Tn-containing glycoprotein (asialo OSM) was determined by ELLSA. The amount required for 50% inhibition of the binding of ALA to asialo OSM is listed in Table III. Details of the inhibition by individual mono- and oligosaccharides are indicated in Figure 3. Among the oligosaccharides and mammalian glycoconjugates tested, T{alpha}- (curve 17) and Tn-containing glycopeptides from OSM (MW < 3000) (curve 15) were the best inhibitors, being 500 and 92 times more active than monomeric Gal and GalNAc, repectively. Tn-containing glycopeptides were 41 times more active than Tn (curve 16), indicating that cluster forms of Tn contributed significantly to binding. T{alpha} was about 47 times more active than T (curve 17 versus 18). Furthermore, the amount of T{alpha} and Galß1->3GalNAcß1->O->Methyl (Tß) required for 50% inhibition was 28 times greater than that of T{alpha} (curves 19 and 20 versus 17) alone, which was found inactive. These results suggest the preference of T disaccharide for a more bulky aromatic aglycon than the small methyl group for ALA binding. Moreover, other mammalian disaccharide structural units such as Galß1->3GlcNAc (I), Galß1->4GlcNAc (II), Gal{alpha}1->3Gal (B), Gal{alpha}1->4Gal (E), Galß1->4Glc (L), GalNAc{alpha}1->3GalNAc (F), GalNAcß1->4Gal (S), GalNAcß1->3Gal (P), and GalNAc{alpha}1->3Gal (A) (curves 21–29 in Table III) failed to yield 50% inhibition even when the amount used was increased to as high as 1.7 x 103 times of that of T{alpha} (required for 50% inhibition), demonstrating that the conformation of the hydroxyl group at carbon-4 and the carbon-2 acetamido group associated with the T{alpha}-disaccharide are important for binding with ALA.


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Table III. Amount of various saccharides giving 50% inhibition of binding of ALA (12.5 ng/50 µl) to a Tn-containing glycoprotein (asialo OSM-50 ng/50 µl)a

 


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Fig. 3. Inhibition of ALA binding to a Tn-containing glycoprotein (asialo OSM) coated on ELLSA plates by various saccharides. The amount of glycoprotein in the coating solution was 50.0 ng per well. The lectin (25.0 ng per well) was preincubated with an equal volume of serially diluted inhibitor. The final ALA content was 12.5 ng per well. Total volume: 50 µl. A405 was recorded after 2 h incubation.

 
Of the monosaccharides studied, p-NO2phenyl {alpha}Gal and GalNAc (Figure 2b, curves 3 and 1), were the best inhibitors, being 606 and 333 times more active than Gal (curve 9), respectively. Moreover, p-NO2phenyl {alpha}- and ß-Gal and GalNAc were more active than their corresponding methyl {alpha}- and ß-Gal and GalNAc derivatives, suggesting that the hydrophobicity surrounding Gal and GalNAc is essential for binding. ALA has a preference for the {alpha}-anomers of Gal and GalNAc; the {alpha}-derivatives of p-NO2phenyl and methyl were more active than the ß-anomers (curves 1 versus 2, 3 versus 4, 6 versus 7, and 5 versus 14 in Table III), indicating that the {alpha}-anomer is important for binding.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In the present study, we examined the binding activity of ALA using a series of mammalian glycotopes and polyvalent structural units in glycoproteins by our established ELLSA method (Duk et al., 1994Go; Wu et al., 1997Go, 2000Go, 2003aGo). Results of both binding and inhibition assays revealed a remarkable affinity of ALA for polyvalent Tn/T-containing glycoproteins (Tables I and II). ALA reacted best with all high-density Tn-containing glycoproteins (asialo HSM, native ASG-Tn, asialo OSM, and asialo BSM), indicating that the high density of polyvalent Tn plays an essential role in binding (Tables I and II). Mcdon P-1, the nondialyzable fraction of human blood group A active glycoprotein (cyst Mcdon phenol insoluble after mild acid hydrolysis; Wu, 1988Go), was also active. It is reasonable to assume that the reactivity increment of this glycoprotein is due to the exposure of a large number of Tn structures following mild acid hydrolysis. The weak binding of ALA with the active antifreeze glycoprotein can be ascribed to the absence of accessible 2-acetamido groups in GalNAc for reaction, in which the hydrophobic side of the disaccharide faces inward and interacts with the peptide backbone (Bush and Feeney, 1986Go). The situation is similar to the result obtained for another T-specific lectin, amaranthin, present in the seeds of Amaranthus caudatus (Rinderle et al., 1989Go). The poor or negative reactivities of ALA with sialylated glycoproteins and human ABH blood group antigens can be attributed to the densely glycosylated or sialylated effects of Tn/T structures.

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 carbohydrate–protein binding is too weak to affect this specific interaction (Horan et al., 1999Go; Wu et al., 2003aGo). 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., 1996Go; Spevak et al., 1996Go; Wu et al., 2003aGo). 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, 1992Go; Lee and Lee, 2000Go) and triantennary II sequences within the prototype chicken liver (CG-16) (Wu et al., 2001Go) 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, 2004Go; Wu et al., 2002Go, 2003aGo, 2004aGo). However, the effect of polyvalencies of glycotopes on the carbohydrate–protein 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{alpha}1->polyvalent glycans is about as strong or weaker than the incremental increase in carbohydrate specificity of monomers (Mitchell et al., 2002Go; unpublished data). Similarly, in Anguilla anguilla agglutinin (Wu et al., 2004bGo), 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., 2003aGo) and galectin-4 from rat gastrointestinal tract (Wu et al., 2004aGo). This indicates that the power of polyvalent effects on carbohydrate–protein 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., 1999Go; Lee, 1992Go; Lee and Lee, 2000Go; Wu, 1988Go, 2001Go; Wu et al., 2003aGo). 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, 2001Go): (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, 1995Go). They are good marker for several cancerous tissues, including the prognosis of colorectal cancer (Itzkowitz et al., 1990Go; Ono et al., 1994Go). 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, 1989Go; Medeiros et al., 2000Go; Wu, 2004Go; Wu et al., 1999Go), plants do not appear to have T/Tn structures (Takeya et al., 1998Go), 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 antigen–binding dietary lectin (Wang et al., 2003Go). If such a lectin–glycan 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., 2003Go), implying its role in plant defense (Peuman and Van Damme, 1995Go). 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, 1990Go). 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., 2003aGo,bGo; 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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Table IV. Comparison of binding specificity of ALA with T/Tn- and II-specific lectins (Wu et al., 2003aGo,bGo)

 
In conclusion, our work demonstrates that (1) Tn- and T{alpha}-containing glycoproteins are the most potent carbohydrate ligands for ALA; (2) Tn/T glycotopes should be present in exposed instead of cryptic forms (Tables I and II); (3) glycotope polyvalency (high-density polyvalent glycoside effect), should be the most important factor for ALA binding; (4) the {alpha}-anomers of Gal and GalNAc were more active than their ß-anomers, and hydrophobicity is essential for binding (Figure 3b); (5) the binding affinity of ALA for ligands can be summarized as polyvalent Tn/T{alpha} glycotopes >> monomeric T{alpha} and simple clustered Tn >> monomeric Tn > GalNAc > Gal; whereas other glycotopes: Gal{alpha}1->3/4Gal (B/E), Galß1->3/4GlcNAc (I/II), GalNAc{alpha}1->3Gal/GalNAc (A/F) and GalNAcß1->3/4Gal (P/S) were inactive. The strong specificity of ALA for cluster Tn/T suggests the importance of glycotope polyvalency during carbohydrate–receptor interactions and supports the concept that every lectin with unique amino acid sequence has its own binding characteristics (Wu, 2001Go, 2003Go). The oligomeric nature of ALA in relation to its preference to clusters will be tested when the reagents become available.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Lectin and biotinylation of lectin
ALA was purified by a combination of two methods (Chatterjee et al., 1988Go; Chowdhury et al., 1987Go). That is the saline extract of the seeds was precipitated by 40% saturated ammonium sulfate followed by 0.4% rivanol precipitation. The active protein was subsequently subjected to affinity chromatography on melibiose-agarose column. The minimum concentration of ALA for erythro-agglutination was 3.57 ng/ml. For ALA biotinylation by biotinamido-caproate-N-hydroxy-succinimide ester (biotin ester; purchased from Sigma Chemical Company, St. Louis, MO), the lectin (200 µg/250 µl phosphate buffered saline [PBS; 0.14 M NaCl, 0.027 M KCl, 0.081 M Na2HPO4, 0.0014 M KH2PO4, pH 7.3]) was mixed with 400 µl of the biotin ester solution (100 µg biotin ester per 200 µg lectin) for 30 min at room temperature. The biotinylated lectin was then dialyzed for 2–3 h against ddH2O and overnight against Tris-buffered saline (TBS; 0.05 M Tris HCl, 0.15 M NaCl, pH 7.35). After dialysis, the sample volume was adjusted to 1 ml with TBS and 20 µl 5% sodium azide (equivalent to 200 µg/ml lectin in 0.1% NaN3) (Duk et al., 1994Go; Lisowska et al., 1996Go).

Glycoproteins and polysaccharides
HSM, one of the simplest glycoproteins among mammalian salivary mucins containing sialyl-Tn (NeuAc-GalNAc) and Tn (GalNAc{alpha}1->Ser/Thr) residues as carbohydrate side chains, was obtained by a modification of the methods previously described (Wu et al., 1994Go). 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)Go with modifications (Herp et al., 1979Go, 1988Go). About 75% of the carbohydrate side chains of asialo OSM were Tn. Asialo PSM contains T{alpha} together with Tn and GalNAc{alpha}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., 1994Go), 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, 1977Go).

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., 1987Go). Desialylation of glycophorin was performed as described later (Tettamanti and Pigman, 1968Go; Wu and Pigman, 1977Go). 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., 1994Go).

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, 1968Go; Wu and Pigman, 1977Go).

The antifreeze glycoprotein from the Antarctic fish (Trematomus borchgrevinki), which contains only T{alpha} as carbohydrate chains (De Vries et al., 1970Go) 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, 1988Go; Wu et al., 1982Go, 1984Go). 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 1.5–2.0 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, 1959Go; Kabat et al., 1948Go; Leskowitz and Kabat, 1954Go). 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., 1974Go; Morgan and Watkins, 1964Go), was kindly provided by late Dr. W. M. Watkins (Imperial College School of Medicine, Hammersmith Hospital London).

Human {alpha}1-acid glycoprotein (Sigma) contains tetra-, tri-, and diantennary complex type glycans in the ratio of 2:2:1 (Fournet et al., 1978Go; Fournier et al., 2000Go). 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., 1979Go), contains tri- and diantennary complex type glycans in the ratio of 1:2.

RSL was prepared by the method of Moschera and Pigman (1975)Go. 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, 1978Go). RSL may also contain Tn-reactive determinants (Wu et al., 1995Go).

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., 1997Go).

The microtiter plate lectin–enzyme binding assay
ELLSA was performed according to the procedures described by Duk et al. (1994)Go. 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.


    Acknowledgements
 
This work was supported by Grants from the Chang-Gung Medical Research Project (CMRP No. 1028), Kwei-san, Tao-yuan, Taiwan; the National Science Council (NSC 92-2311-B-182-005, 92-2320-B-182-045, 92-2320-B-182-046), Taipei, Taiwan; and the Indian Council of Medical Research (No. 62/1/99-BMS) New Delhi, India.


    Abbreviations
 
ABA, Agaricus bisporus agglutinin; ALA, Artocarpus lakoocha agglutinin; ASG-Tn, Tn glycoprotein from armadillo salivary gland; BSM, Bovine submandibular glycoprotein; ELLSA, enzyme-linked lectinosorbent assay; HSM, hamster submaxillary Tn glycoprotein; OSM, major fraction of ovine submandibular glycoprotein; PBS, phosphate buffered saline; PSM, porcine salivary glycoprotein; RSL, rat sublingual glycoprotein; TBS, Tris-buffered saline; TBS-T, Tris–HCl-buffered saline with 0.05% Tween 20


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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