1 Introduction

Glycosylation is the most common posttranslational modification of proteins and plays diverse roles in numerous biological processes, including fertilization, development, differentiation, inflammation, cancer metastasis, and host–pathogen/­parasite interactions. A number of glycosylated proteins are bioactive molecules of medical/therapeutic or other commercial interest and are currently produced by recombinantly transformed cells and organisms. Among non-animal expression systems, plant cells and transgenic plants are considered an attractive alternative system for recombinant human and animal glycoproteins. The advantages of using plants for the production of commercially important glycosylated proteins include lower manufacturing costs and a reduced risk of transmitting mammalian pathogens [11, 27]. However, a major roadblock in the use of plants for this purpose is the lack of available information on N- and O-linked glycans in plants and specifically those in the endogenous plant glycosylation pathways [9, 31]. Thus, gathering detailed structural information on plant-derived glycoproteins is of utmost importance.

The traditional technologies used for the structural analysis of glycans, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, are highly sophisticated and precise in their analyses. These techniques are not only time and labor intensive but, also require expensive equipment, expert personnel, and relatively pure and large sample quantities [16]. Furthermore, the emerging field of glycomics, i.e., the functional assignment of glycans and lectins [21, 29], requires rapid analysis of the glycosylation status of proteins and cell surfaces in a systematic manner. Therefore, for rapid and high-throughput structural analysis, carbohydrate-binding proteins, especially lectins, are thought to offer great potential [14, 19, 26]. Various lectins, which are capable of binding a diverse array of glycan structures, are a major means by which glycosignatures on cell surfaces and of various molecules are decoded and the dynamics of carbohydrate structural variations in diseases interpreted [4, 7]. Thus, for the past few decades, lectin-based assays such as agglutination, mitogen stimulation, histology, blotting, affinity chromatography, and flow cytometry have been popular tools for glycan detection and characterization [22, 24, 32]. More recent developments in lectin-based technologies have included lectin arrays and biosensors for uses such as differentiating terminal glycans [5, 10, 15].

The characteristics of lectin–carbohydrate interactions for mammalian glycoconjugates have been well established, and the information is utilized by researchers to obtain further details on animal glycan structure [1, 23]. In contrast, the specificity of lectins (many of which in common usage are plant-derived) for nonmammalian glycoconjugates, particularly those from plants, has not been critically evaluated, although this information is crucial for structure–function data interpretation [31]. To fully realize the potential of lectin-based rapid glycan analyses in the field of plant glycomics, a more complete understanding of lectin–plant glycoprotein specificity is necessary. We have been utilizing lectin-based technologies, in addition to techniques such as high-performance liquid chromatography (HPLC) and MS, to investigate glycosylation of proteins in plants such Arabidopsis thaliana, tobacco, and rice.

2 Investigation of the Glycoproteins in A. thaliana

A. thaliana has been widely investigated and has several advantages as an experimental system. These include its small genome, which has been sequenced; an established suspension cell culture system with a rapid life cycle; and its popular and widespread use in molecular, genetic, and transfection studies. Previous research in our laboratory revealed a strong interaction of several lectins with cultured A. thaliana cells and their protoplasts, prepared by treatment with cell wall-solubilizing enzymes [Shah M (2005) Dissertation, Arizona State University]. Of special interest was the binding of Sambucus nigra-I (SNA-I) and Maackia amurensis (MAA) lectins with specificity for sialic acid and Vicia villosa agglutinin (VVA), Arachis hypogaea (peanut) agglutinin (PNA), and Artocarpus integrifolia (AIA, jacalin) with specificity for GalNAc-Ser/Thr-linked structures. This observation prompted us to investigate the structures of the potential glycoproteins in A. thaliana suspension-cultured cells, which may have interacted with the above-mentioned lectins. The cells were grown in suspension at room temperature in a medium made from MS519 salts (Sigma), vitamins, sucrose, α-naphthalene acetic acid, and kinetin. After seven days, the cells were harvested and delipidated with acetone followed by a mixture of chloroform and methanol (2:1 and 1:2). The delipidated cells were suspended in phosphate-buffered saline (PBS) containing protease inhibitor cocktail (Sigma) and sodium azide (0.02%), disintegrated using a French press, and the suspension was stirred overnight at 4°C. The extract was centrifuged (10,000 RPM, 30 min), the supernatant saved, and the residue extracted again as above but with PBS containing 0.1% Triton X-100. The PBS and PBS-Triton extracts were analyzed for protein content by the micro bicinchoninic acid (BCA) assay and the remainder was stored in aliquots at −20°C until required.

2.1 Enzyme-Linked Lectin Assay (ELLA)

A common and readily available method to quantitate the interaction of lectins with macromolecular glycoconjugates is the microtiter plate ELLA [6, 17]. Accordingly, 96-well microtiter plates were coated with PBS and PBS-Triton extracts of A. ­thaliana cells, and the binding of various biotinylated lectins to the bound plant proteins was determined using avidin-alkaline phosphatase and p-nitrophenyl phosphate substrate (Sigma). The assays were performed in triplicate and the mean values of the results were plotted. The results of typical experiments, as illustrated in Fig. 12.1, show that several lectins bound strongly to both extracts. Concanavalin A (ConA), SNA-I, AIA (jacalin), and VVA showed high binding to components in the PBS extract, whereas VVA, Lotus tetragonolobus lectin (LTA), and AIA bound maximally to components in the PBS-Triton extract. Experiments were then done to determine the nature of the glycoconjugates present in these extracts that interacted with the lectins. Since the cells were delipidated prior to the extraction, it was considered unlikely that glycolipids and lipids were contributing to the binding. While proteins and glycoproteins are insoluble in perchloric acid, polysaccharides and glycoproteins with very high carbohydrate content (mucins) remain in solution [2]. Therefore, to distinguish between glycoproteins/proteins and mucins/polysaccharides, the interaction of jacalin with PBS extract, which was pretreated with perchloric acid to remove mucins and polysaccharides, was tested. The results illustrated in Fig. 12.2 demonstrate that jacalin binding was not significantly influenced. Furthermore, the binding of jacalin was abolished if the extract was exhaustively (overnight) treated with Pronase. These results suggest that the lectin-binding components in the PBS extract of A. thaliana are protein-based molecules and are likely to be glycoproteins.

Fig. 12.1
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Binding of biotinylated lectins to ELLA plates coated with PBS (left) and PBS-Triton (right) extracts of delipidated Arabidopsis thaliana cells

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Fig. 12.2
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Binding of biotinylated jacalin before (─●─) and after perchloric acid precipitation (∙∙∙○∙∙∙) or Pronase treatment (─■─) of PBS extract of delipidated Arabidopsis thaliana cells

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To determine the specific nature of the binding of the biotinylated lectins to the putative glycoproteins, a series of experiments were conducted to test the ability of saccharides to inhibit the binding. Surprisingly, the binding of biotinylated VVA to A. thaliana cell proteins was not inhibited by a variety of monosaccharides (d-GalNAc, d-Gal, d-ManNAc, l-Rha, l-Ara, d-Xyl, d-GalN, d-Man, d-Glc, and α- methylmannoside) up to the highest concentration (100 mM) tested (Fig. 12.3). It should be noted that the saccharides tested included typical plant sugars such as l-Rha, l-Ara, d-Xyl, and d-Glc. In control experiments, the binding of VVA to asialo ovine submaxillary mucin was inhibited by 10 mM d-GalNAc and d-Gal, which are reported to be haptenic sugars for VVA [20, 28]. Similarly, the binding of biotinylated SNA-I to A. thaliana cell proteins was also not inhibited by 100 mM of either typical animal sugars (NeuNAc, lactose, d-Gal, d-GalNAc, d-glucuronic acid) or plant sugars (ketodeoxyoctulosonic acid [KDO], d-galacturonic acid) (not illustrated). In control experiments, the binding of SNA-I to fetuin was inhibited by lactose, d-GalNAc, and d-Gal, which are reported to be haptenic sugars for SNA-I [3, 25]. In addition, VVA and jacalin conjugated to alkaline phosphatase gave results similar to those obtained with biotinylated VVA and jacalin, respectively, which eliminated the possibility that the observed phenomenon was a peculiarity of the biotin/avidin system.

Fig. 12.3
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Effect of monosaccharides on the binding of biotinylated VVA (left) and biotinylated SNA-I (right) to PBS extract of delipidated Arabidopsis thaliana cells. GalNAc (filled circle), ManNAc (open circle), Gal, (x), rhamnose (filled diamond), lactose (filled square), NeuNAc (open diamond). VVA binding was also not inhibited by d-arabinose, d-xylose, or d-galactosamine, and SNA-I binding was not inhibited by d-glucuronic acid, d-galacturonic acid, or KDO

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2.2 Lectin Blotting After SDS-PAGE and Transfer to PVDF Membranes

To gather more information on the nature of the components of the A. thaliana extract that interacted with the biotinylated lectins, blotting experiments were carried out. Lectins that interacted with either intact A. thaliana cells or cell extracts were also found to bind to several individual components, which were well resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The results of the binding of biotinylated VVA, SNA-I, and jacalin to components in PBS and PBS-Triton extracts of A. thaliana cells are illustrated in Fig. 12.4.

Fig. 12.4
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Binding of biotinylated lectins to Arabidopsis thaliana proteins after 5–15% SDS-PAGE and transfer to PVDF membranes. Lane 1, proteins extracted from delipidated A. thaliana cells; lane 2, proteins extracted by boiling A. thaliana cells in SDS sample buffer

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2.3 Lectin Affinity Chromatography of the A. thaliana Extracts

To isolate larger quantities of the lectin-binding components, PBS extract of the cells was subjected to affinity chromatography on sepharose-immobilized lectins (VVA, SNA-I, and jacalin). After extensive washing to remove unbound material, the columns were eluted first with a 100 mM solution of the appropriate haptenic sugar, followed by a 50 mM glycine buffer, pH 2.2, to elute any material that is bound to the lectin either specifically or non-specifically, respectively. Macromolecules eluted from the column were recovered by exhaustive dialysis against distilled water, followed by lyophilization.

2.4 Analysis of the Monosaccharide Composition of Lectin-Binding Material

Portions of the isolated material were hydrolyzed with either 2 N trifluoroacetic acid at 100°C for 6 h to release neutral monosaccharide and hexosamines or with 0.1 N sulfuric acid at 80°C for 1 h to release sialic acids. In parallel, some major bands, shown in Fig. 12.4, were excised from preparative PVDF membrane blots and subjected to hydrolysis. The hydrolysates, after appropriate treatment to remove the acid, were analyzed by high-performance anion-exchange chromato­graphy with pulsed amperometric detection (HPAEC-PAD). The results of the ­analysis of material eluted from VVA–sepharose with glycine buffer, which were typical of other preparations, or excised PVDF, are illustrated in Fig. 12.5. Some of the preparations contained significant quantities of plant sugars, specifically d-Glc, d-Man, and d-Xyl, and several unidentified peaks. It should be noted that the expected ligands of the lectins VVA and SNA-I (e.g., GalNAc and/or Gal for VVA or Jacalin and NeuNAc for SNA-I) were either absent from the monosaccharide analysis or present only in minute, insignificant quantities.

Fig. 12.5
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Monosaccharide analysis by HPAEC-PAD of Arabidopsis thaliana glycoproteins isolated by affinity chromatography on VVA-Sepharose (top panel) as described in the text and the major jacalin reactive band excised from PVDF blots (bottom panel)

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In summary, microtiter plate assays and probing of blots revealed strong binding of several lectins to proteins extracted from A. thaliana cells. This binding was not inhibited by haptenic sugars. HPAEC-PAD analysis of bands excised from blots and lectin-affinity purified material revealed amounts of the expected monosaccharides, which were insufficient in quantity to explain the extent of the lectin–plant protein interaction.

3 Interaction of Lectins with Glycoproteins in Tobacco Seeds and Seedlings

Tobacco (Nicotiana spp) is another model system that is widely used for the ­investigation of plant biochemistry. Thus, it was of interest to examine the interactions of endogenous plant proteins with lectins to verify that the observed results were not unique to A. thaliana or, indeed, suspension cultured cells. Nicotiana sylvestris seeds were ground to a fine powder and then delipidated by treatment with acetone. The lipid-free powder was extracted with 50 mM Tris–HCl, pH 8.0, containing 200 mM NaCl and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). The extract was centrifuged, the supernatant analyzed for protein, and aliquots were stored at −20°C, as in the case of the A. thaliana cell extracts.

3.1 Lectin Blotting After SDS-PAGE and Transfer to PVDF Membranes

Aliquots of the extract were subjected to SDS-PAGE, transferred to PVDF membrane, and the blots probed with biotinylated lectins. The results of the binding of biotinylated VVA, SNA-I, MAA, and PNA to proteins extracted from tobacco seed and five-day-old seedlings, as well as their staining with Coomassie Blue, are illustrated in Fig. 12.6.

Fig. 12.6
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Binding of biotinylated lectins to proteins extracted from Nicotiana sylvestris seeds (0) or five-day-old seedlings (5) subjected to 4–12% SDS-PAGE and transferred to PVDF membranes. For comparison, staining of proteins with Coomassie Blue (R250) is shown in the first panel

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3.2 Effect of Sialidase Treatment on the Binding of MAA and SNA-I

Both MAA and SNA-I showed significant interactions with components in the tobacco extracts. Since these lectins are known to recognize ligands containing sialyl residues, we examined their binding after overnight treatment of the tobacco seed extracts with sialidases. The results illustrated in Fig. 12.7 show that exhaustive treatment of the extract with Arthrobacter ureafaciens or Clostridium perfringens sialidases had no effect in the binding of MAA to the tobacco proteins. The above treatments also had no effect on the binding pattern of SNA-I (not illustrated). Many sialidase preparations, particularly those from C. perfringens, are known to contain some protease activity. Therefore, the sialidase treatments were done in the presence of the protease inhibitor PMSF.

Fig. 12.7
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Staining of Nicotiana sylvestris seed proteins with biotinylated MAA after overnight incubation with increasing concentrations of Clostridium perfringens sialidase (0, 10, 15, and 20 mU) in the presence of protease inhibitor (a). Staining of N. sylvestris seed proteins with biotinylated MAA before (1) and after (2) overnight incubation with Arthrobacter ureafaciens sialidase (10 mU) (b)

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3.3 Effect of Various Pretreatments of VVA–Biotin on Its Interaction Ability

The following experiments were carried out to obtain information on the specificity of the interaction of biotinylated VVA with tobacco proteins. VVA–biotin was first preincubated with 10 mM GalNAc, a haptenic sugar for the lectin, or with glycine buffer, pH 2.2, which should affect the secondary structure of the lectin and, therefore, eliminate its specific binding. These treatments did not influence the binding of VVA to tobacco proteins, while binding to asialoagalacto fetuin (ASGF) was abolished (Fig. 12.8). Similarly, treatment of VVA-biotin at 100 °C for 12 min (Fig. 12.8) and incubation with 1M sodium chloride or 1% Tween 20 also had no effect on its binding to the tobacco proteins, but did abolish binding of the lectin to the control (data not shown). These results demonstrate that the binding of VVA to tobacco proteins was non-carbohydrate mediated, since the haptenic sugar did not affect the binding. Furthermore, denaturation of the lectin by heat treatment or exposure to acidic pH also had no effect on its binding. Finally, the binding observed is not mediated by ionic or hydrophobic interaction, since neither salt nor detergent treatment had any influence on the binding.

Fig. 12.8
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Staining of Nicotiana sylvestris seed protein (TSP) and ASGF with biotinylated VVA (lane A), biotinylated VVA in the presence of 10 mM GalNAc (B), heat-denatured biotinylated VVA–biotin (C), and biotinylated VVA in the presence of 50 mM glycine buffer, pH 2.2

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In summary, proteins extracted from tobacco seeds strongly interacted with various lectins. Pretreatment of the tobacco proteins with sialidases or preincubation of the lectins with haptenic sugar, heat, or acidic buffer, all of which would be expected to abolish specific binding, did not significantly affect binding. While these results suggest a non-carbohydrate-based binding between tobacco seed components (presumably protein-based, judging by SDS-PAGE and accompanying Coomassie Blue Staining) and the lectins, it is surprising that even reagents that disrupt ionic and hydrophobic interaction had no effect on the binding.

4 Interaction of Rice Prolamin with Jacalin

SDS-PAGE analyses of alcohol-soluble (prolamin) rice (Oryza japonica) protein revealed bands in the molecular weight range of 14–16 kDa, which interacted strongly with VVA, PNA, and wheat germ agglutinin. These proteins were purified and analyzed by ELLA using biotinylated jacalin before and after treatment with endo-α-N-acetylgalactosaminidase (O-glycanase) [30]. While Galβ1, 3GalNAc was released on digestion with the endoglycosidase, as confirmed by isolation and MS/MS analysis, the binding of jacalin to the untreated and treated prolamin was not significantly different (Fig. 12.9). Furthermore, the total monosaccharide content of prolamin, consisting of galactose, galactosamine, and glucosamine, accounted for only about 0.01% by weight [12]. These findings demonstrated that the binding of the lectins to rice prolamins is primarily mediated by non-carbohydrate ligands, inferred to be protein-protein interaction.

Fig. 12.9
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Binding of biotinylated jacalin to rice prolamin before (─●─) and after (∙∙∙○∙∙∙) treatment with endo-alpha-N-acetylgalactosaminidase

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In summary, prolamin purified from rice still interacted with jacalin after treatment with O-glycanase to release the small amount of Galβ1-3GalNAc associated with the protein.

5 Summary

The characteristics of lectin–carbohydrate interactions with respect to animal glycoconjugates have been well established and have thus been reliably utilized by researchers. In contrast, the specificity of the interaction of lectins with plant ­glycoconjugates had not been critically evaluated previously. The above-discussed results, as well as other studies carried out in our laboratory, provide evidence that there is considerable non-carbohydrate-mediated interaction between lectins and as-yet-unidentified plant proteins. Similar results obtained with A. thaliana and Nicotiana spp, two widely used plant model systems, suggested that our observations are probably a general phenomenon involving lectins and components in the plant, tentatively identified as proteins. The interaction of lectins with various hydrophobic plant molecules, such as adenine derivatives, cytokinins, and porphyrins, has been previously reported [8, 13, 18]. These findings, in addition to our own, provide evidence of molecular mimicry in the context of lectin–plant protein interaction. Therefore, it is clear that lectins are not just simple probes, as has been proposed, and results from lectin microarrays, particularly in the case of plant material, should be interpreted with caution. The long-term goal of our research is to understand their nature and thereby minimize non-carbohydrate-mediated binding events that may lead to a false-positive identification of plant glycans, making improved and robust high-throughput analysis of the plant glycome possible.