[Advances in Experimental Medicine and Biology] The Molecular Immunology of Complex Carbohydrates-3 Volume 705 || Novel Concepts About the Role of Lectins in the Plant Cell

271A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_13, © Springer Science+Business Media, LLC 2011

Keywords Classification • Nucleocytoplasmic protein • Plant lectin • Physiological role • Specificity

The history of plant lectins dates back to 1888 when Stillmark published his dissertation Über Ricin ein giftiges Ferment aus den Samen von Ricinus communis L. und einigen anderen Euphorbiaceen, in which he linked the toxicity of castor beans to the presence of a proteinaceous hemagglutinating factor called ricin [1]. Only in 1952, it was shown that the agglutination properties of lectins are based on a specific sugar-binding activity [2]. Since then, a lot of carbohydrate-binding pro-teins have been reported in plants. For a long time, research was concentrated on those plant tissues that contain readily detectable amounts of lectin by agglutination assays. As such, a selection was made for plant lectins occurring in high concentra-tions, mostly in seeds and vegetative tissues. In recent years, evidence has accumu-lated of a different class of plant lectins occurring in the nucleus and the cytoplasm of the cell in low concentrations. This chapter aims to give an overview of the most recent findings and the impact thereof on our understanding of the physiological role of lectins in plants.

13.1 Plant Lectins: A Group of Bioactive Plant Proteins

Many plants, including important food plants such as wheat, potato, tomato, and bean, contain carbohydrate-binding proteins commonly referred to as lectins, agglutinins, or hemagglutinins. This group of proteins comprises all plant proteins possessing at least one noncatalytic domain that binds reversibly to specific

E.J.M. Van Dammes (*) Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium e-mail: [email protected]

Chapter 13Novel Concepts About the Role of Lectins in the Plant Cell

Els J.M. Van Dammes, Elke Fouquaert, Nausicaä Lannoo, Gianni Vandenborre, Dieter Schouppe, and Willy J. Peumans

272 E.J.M. Van Dammes et al.

mono- or oligosaccharides [3, 4]. Hitherto, about 500 different plant lectins have been isolated and (partially) characterized [5]. At first glance, all these lectins form a heterogeneous group of proteins because of the obvious differences in structure, specificity, and biological activities. However, structural and molecular studies have revealed the existence of a limited number of plant lectin families. Hitherto, different sugar-binding domains/motifs have been identified with certainty in plants (Table 13.1). Using these domains as basic structural units, a relatively simple sys-tem was elaborated that allows classifying virtually all known plant lectins into families of structurally and evolutionarily related proteins. Most plant lectins belong to one of the seven common families, which are (in alphabetical order) the amaranthins, the Cucurbitaceae phloem lectins, the Galanthus nivalis agglutinin family, the lectins with hevein domain(s), the jacalin-related lectins, the legume lectins, and the ricin-B family [3, 4]. In addition, two families with a narrow taxo-nomic distribution have been identified only recently, namely, orthologs of the fungal Agaricus bisporus agglutinin [6] and catalytically inactive homologs of class-V chitinases [7]. Furthermore, recent studies have provided information on the existence of a new motif with lectin activity, referred to as the Euonymus lectin (EUL) domain.

Although there are still a lot of unanswered questions regarding the physiologi-cal role of plant carbohydrate-binding proteins, during the past decade, some sub-stantial progress has been made in our general understanding of the role of those plant lectins that are constitutively expressed in reasonable quantities. Biochemical and molecular studies of numerous lectins eventually demonstrated that only a

Table 13.1 Overview of different lectin families occurring in the vacuole and/or the nucleus and cytoplasm of plant cells

Lectin family Vacuolar lectins Nucleocytoplasmic lectins

Amaranthins No examples known Documented in Amaranthus spp and Prunus spp

Cucurbitaceae phloem lectins/lectins with Nictaba domain

No examples known Wide taxonomic distribution

Galanthus nivalis agglutinin family

Wide taxonomic distribution Found in diverse taxa

Lectins with hevein domains

Wide taxonomic distribution No examples known

Jacalin-related lectins Only documented in a few Moraceae spp

Ubiquitous

Legume lectins Common in Fabaceae and Lamiaceae No examples knownRicin-B family Wide taxonomic distribution No examples knownOrthologs of Agaricus

bisporus agglutininNo examples known Documented in Marchantia

polymorpha and Tortula ruralis

Homologs of class-V chitinases

Only documented in a few legumes No examples known

Lectins with Euonymus lectin domain

No examples known Ubiquitous

27313 Novel Concepts About the Role of Lectins in the Plant Cell

limited number of carbohydrate-binding motifs evolved in plants [8]. Since the specificity of these binding motifs is primarily directed against foreign glycans, it is generally accepted now that many plant lectins are involved in the recognition and binding of glycans from foreign organisms and accordingly play a role in plant defense. Animal and insect feeding studies with purified lectins and experiments with transgenic plants confirmed that at least some lectins enhance the plant’s resis-tance against herbivorous higher animals or phytophagous invertebrates. To recon-cile the presumed defensive role with the high concentration, the concept was developed that many plant lectins are storage proteins that can be used as nonspe-cific defense proteins in case the plant is challenged by a predator [8, 9]. Evidently, such a defense/storage role applies only to lectins that are present in relatively high concentrations. This role of plant lectins for defense against foreign attack is in marked contrast with the role of animal lectins; most of these lectins are believed to recognize and bind endogenous receptors and, accordingly, are involved in rec-ognition mechanisms within the organism itself [10, 11]. Furthermore, there is increasing evidence that protein–carbohydrate interactions are very important for the normal development and functioning of animal organisms. In addition, it was shown that different types of lectins are the mediators of these protein– carbohydrate interactions. Therefore, the question arises whether protein–carbohydrate interac-tions are also important in signal transduction in plants.

13.2 Classical and Inducible Plant Lectins

During the past 5 years, evidence has accumulated that plants synthesize well-de-fined carbohydrate-binding proteins upon exposure to stressful situations such as drought, high salt, wounding, treatment with some plant hormones, or pathogen attack. Localization studies demonstrated that, in contrast to the “classical” plant lectins, which are typically found in vacuoles, the “inducible” lectins are exclu-sively located in the cytoplasm and the nucleus. Based on these observations, the concept that lectin-mediated protein–carbohydrate interactions in the cytoplasm and the nucleus play an important role in the stress physiology of the plant cell was developed [12, 13]. The development of this novel concept was founded primarily on the results obtained with the inducible lectins discovered in rice [14] and tobacco [15]. Since then, firm evidence has been obtained that plants express several fami-lies of nucleocytoplasmic lectins that, according to the available sequence informa-tion, are definitely unrelated to each other (Table 13.1). Since most of these inducible lectins are synthesized only as a response to specific physical, chemical, and biotic stress factors and occur in low but physiologically relevant concentra-tions, it can be assumed that they play a specific role in the plant. Taking into consideration that any physiological role of plant lectins most likely relies on their specific carbohydrate-binding activity and specificity, the discovery of the novel stress-related lectins provides strong evidence for the importance of protein– carbohydrate interactions in plants.


Hitherto, five families of nucleocytoplasmic lectins have been identified (Table 13.1). Some of these lectin families have been known for several years but have only now been shown to reside in the cytoplasm and/or nucleus of the plant cells (e.g., amaranthin-like sequences and Nictaba-related sequences). Other lectin families, such as the mannose-binding lectins related to Galanthus nivalis aggluti-nin (GNA) and the jacalin-related lectins, comprise both vacuolar and nucleocyto-plasmic homologs. Finally, a new lectin family of nucleocytoplasmic lectins has been discovered for which no vacuolar homologs have been reported until now. The latter family is characterized by the presence of a so-called EUL domain. Each of these nucleocytoplasmic lectin families is discussed in more detail below.

13.2.1 Jacalin-Related Lectins

The family of jacalin-related lectins groups all proteins with one or more domains that are structurally equivalent to jacalin, a galactoside-binding lectin from jack fruit (Artocarpus integrifolia) seeds [16]. In recent years, several lectins related to jacalin but with specificity toward mannose have been discovered and characterized in detail [17, 18]. Therefore, the family of jacalin-related lectins is now divided into two subfamilies with a distinct specificity and molecular structure. The galactose-specific jacalin-related lectins are built up of four cleaved protomers comprising a small (b) (20 amino acid residues) and a large (a) (133 amino acid residues) sub-unit and exhibit a clear preference for galactose over mannose [19]. In contrast, mannose-specific jacalin-related lectins are built up of either uncleaved protomers of approximately 150 amino acids (Fig. 13.1a) or protomers comprising two to seven tandemly arrayed (uncleaved) jacalin domains that exhibit an exclusive speci-ficity toward mannose. The first unambiguous evidence for the cytoplasmic loca-tion of a mannose-specific jacalin-related lectin came from localization studies in rhizomes of Calystegia sepium [20]. It was shown that the localization pattern for the mannose-specific Calystegia lectin definitely differed from the vacuolar loca-tion of the galactose-specific jacalin from Artocarpus integrifolia. Based on these observations, the hypothesis was put forward that the galactose-specific jacalin-related lectins evolved from their mannose-specific homologs through the acquisition of vacuolar targeting sequences [12, 13, 20].

The first inducible jacalin-related lectin was purified and characterized from rice. This protein, called Oryza sativa agglutinin or Orysata, was already described in 1990 as SalT (a salt-inducible protein) [21] but was identified as a lectin belong-ing to the family of “mannose-specific jacalin-related lectins” in 2000 [14, 22]. Orysata cannot be detected in untreated plants but is rapidly expressed in roots and sheaths after exposing the whole plant to salt or drought stress, or jasmonic acid and abscisic acid treatment [21, 23, 24] (Table 13.2). The lectin is also expressed in excised leaves after infection with an incompatible Magnaporthe grisea race [25, 26] and during senescence [27]. Irrespective of the inducing agent, the lectin level remains very low. Orysata is synthesized without signal peptide on free


ribosomes and, based on the analogy of other mannose-specific jacalin-related lectins with a known location, probably remains in the cytoplasmic/nuclear com-partment. We have recently confirmed the nucleocytoplasmic location of Orysata using confocal microscopy of tobacco BY-2 cells expressing a lectin sequence fused to enhanced green fluorescent protein (EGFP) (Fig. 13.2a). Fluorescence was seen in the nucleus and cytoplasm of the tobacco cells, whereas the vacuole was completely devoid of any signal.

A search for proteins and genes comprising domain(s) equivalent to Orysata revealed that these sequences are widespread. Jasmonate-inducible orthologs of Orysata have been identified in several other Gramineae species, as well as in Helianthus tuberosus [28] and Ipomoea batatas [29]. Moreover, according to tran-scriptome analysis, all higher plants (Tracheophyta) studied thus far apparently express (low levels of) one or more of these lectins. An extended family of genes encoding proteins with single or multiple jacalin domains was found in Arabidopsis. One of these proteins (RTM1) is involved in the restriction of long-distance move-ment of tobacco etch virus, whereas others correspond to wounding/jasmonate-induced myrosinase-binding proteins [30, 31]. Within the Gramineae family, several inducible stress/defense-related proteins containing a jacalin domain fused

Fig. 13.1 Schematic representation of the structural relationships between nucleocytoplasmic lectins and their vacuolar homologs. Comparison of sequences for (a) vacuolar galactose-binding and cytoplasmic mannose-binding jacalin-related lectins from jack fruit (jacalin) and Calystegia sepium (Calsepa), respectively; (b) Nictaba and Cucurbita phloem lectin PP2; and (c) vacuolar GNA and cytoplasmic GNA-related sequence from maize (GNA

maize)


to an unrelated domain have been found. One of these proteins (called VER2) is specifically expressed during vernalization [32, 33]. The VER2 protein in rice and wheat was shown to be jasmonate inducible. Expression of wheat VER2 is also inducible by gibberellins [33]. Characterization of the recombinant rice protein (expressed in Escherichia coli) revealed inhibition of the agglutination activity by mannose [34], confirming that VER2 is indeed a lectin. Recent studies have also showed that overexpression of the gene in rice suppresses coleoptile and stem elon-gation, indicating that this lectin plays an important role in rice growth and develop-ment [35]. Another structurally similar protein containing a jacalin domain occurs in maize, where it is known as a b-glucosidase-aggregating factor [36–38]. Judging from the available sequence information, we can conclude that the jacalin domain is widespread in plants.

13.2.2 Proteins with a Nictaba Domain

In 2002, Chen et al. reported that jasmonic acid methyl ester induces lectin activity in leaves of Nicotiana tabacum (var. Samsun NN) [15]. This lectin (called Nicotiana tabacum agglutinin or Nictaba) cannot be detected in untreated tobacco plants but is specifically induced in leaves after treatment with jasmonic acid and other

Table 13.2 Overview of stress conditions inducing lectin activity

Lectin or Lectin family Plant species Biotic or abiotic factor References

Mannose-specific jacalin-related lectins

Rice Abscisic acid [21]Rice Salt stress [21]Rice Drought stress [21]Rice Jasmonate [23]Rice Wounding [24]Rice Magnaporthe grisea

infection[25, 26]

Rice Leaf senescence [27]Rice Gibberellins [33]Wheat, Rice Vernalization, jasmonate [32, 33]Barley Light [116]Barley Jasmonate [117]Barley Salt stress [118]Oilseed rape Jasmonate [119]Sweet potato Jasmonate [29]Jerusalem artichoke Jasmonate [120]

Nictaba Tobacco Jasmonate [15]Insect herbivory [39]

Proteins with EUL domain

Rice Abscisic acid [121]Rice Salt stress [62]Maize Drought stress [122]Banana Desiccation [123]


jasmonates (Fig. 13.3). It was shown that jasmonate treatment of a single leaf of a tobacco plant results in lectin expression not only in the treated leaf but also in the leaves below and above the treated leaf, indicating that some signal is transported between different leaves and triggers lectin expression. In addition, Nictaba was recently also shown to be induced by insect herbivory [39] (Table 13.2). Nictaba and its corresponding gene have been isolated and characterized. In its native form, Nictaba is a homodimer consisting of two identical unglycosylated subunits of approximately 19 kDa. The deduced amino acid sequence of the complementary

Fig. 13.2 Confocal images of living, transiently transformed BY-2 cells expressing EGFP-Orysata (Genbank Accession No. CB632549) (a); EGFP-Nictaba 24 h after transformation (AF389848) (b); EGFP-Nictaba 48 h after transformation (c); EGFP-Nictaba mutated in NLS (d); EGFP-GNA

maize (GNA-related sequence from maize, BM351398) (e); EGFP-EUL

Arabidopsis (Arabidopsis

sequence related to EUL, AF411801; [114, 115]) (f); AmaranthinPrunus

-EGFP (Amaranthin-like sequence from Prunus, DN554056) (g). Free EGFP (h) was used as a control for nuclecytoplasmic localization, whereas the expression of sporamin-EGFP (U12436) [95, 96] was a control for vacuolar targeting (i). Scale bars represent 25 nm


DNA (cDNA) clone encoding Nictaba revealed a 165-amino acid sequence containing a putative nuclear localization signal (NLS) sequence (102KKKK105).

Immunocytochemical localization studies using polyclonal Nictaba-specific antibodies revealed that the lectin is located in the nucleus and cytoplasm of leaf cells. No labeling could be detected in vacuoles or chloroplasts. All immunolabel-ing could be detected in the leaf parenchyma cells and not in vascular tissues [15]. More recently, the nucleocytoplasmic location of Nictaba was confirmed using confocal microscopy of tobacco BY-2 cells expressing a Nictaba sequence fused to EGFP (Fig. 13.2b, c) [40]. Furthermore, it became evident that the lectin is not uniformly distributed over the nucleus or the cytoplasm of BY-2 cells. Confocal microscopy of transiently transformed BY-2 cells revealed that 24 h after biolistic delivery, the expressed EGFP-Nictaba is predominantly located in the nucleus and, to a lesser extent, in the cytoplasm surrounding the central vacuole and the strands of cytoplasm transversing the vacuole (Fig. 13.2b). However, 48 h after DNA deliv-ery, the ×48 nucleus still contained most of the EGFP-Nictaba but showed a rim-like staining pattern, suggesting that the fusion protein was now concentrated at the periphery of the nucleus (Fig. 13.2c). Very similar staining patterns were observed in BY-2 cells stably transformed with the EGFP-Nictaba construct. Similar experi-ments with a fusion protein of EGFP and a Nictaba mutant, in which the presumed NLS (102KKKK105) was changed into (102KTAK105), provided evidence for the involvement of an NLS-dependent transport mechanism. Confocal microscopy of the transiently transformed BY-2 cells clearly demonstrated that the mutant protein is exclusively located in the cytoplasm and cannot be detected in the nucleus (Fig. 13.2d). The distribution pattern seen 24 h after biolistic delivery of the DNA

2

Nictaba

2

Internalcontrol

RNA levelProtein levela b c

µg Nictaba/g FW

12

treated leaf : 0

−1−2

−2 −1 0 1 2

a

b

c

0 30 60 90 120

-2

-1

0

1

2

Fig. 13.3 Induction of Nictaba expression in tobacco plants after treatment with jasmonates. (a) Using semiquantitative agglutination assays, Nictaba expression was determined at the protein level in different leaves of a tobacco plant, of which only one leaf was treated with methyl jas-monate. Lectin activity was analyzed 72 h after treatment and calculated as ug lectin per gram fresh weight (FW). (b) The treated leaf was assigned as leaf 0; the two leaves above the treated leaf are referred to as leaf 1 and 2, whereas the two lower leaves are referred to as leaf 1 and 2. (c) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on different tobacco leaves for lectin messenger RNA (mRNA). After 12 h of treatment of leaf 0, mRNA for Nictaba could already be detected in the treated leaf (a). To clearly show mRNA for Nictaba in the systemic leaves, a nested PCR was performed (b). The quality of the RNA samples was assured by an internal control for ribosomal RNA (rRNA) (c). Samples were analyzed after 12 h of treatment


did not change during the subsequent 36 h, indicating that the NLS is required and sufficient for transport of Nictaba from the cytoplasm into the nucleus.

Based on the results of hapten inhibition assays, the lectin was originally classi-fied as a chitin-binding protein. However, more detailed specificity studies with glycan arrays revealed that Nictaba preferentially recognizes high-mannose as well as complex N-glycans and strongly interacts with glycoproteins carrying such gly-cans [40]. A detailed analysis of the binding studies suggests that the binding site of Nictaba is most complementary to (Man)

3b1-4GlcNAcb1-4GlcNAcb-N-Asn.

The tobacco lectin was originally discovered in Nicotiana tabacum L. cv. Samsun NN leaves. To check whether and, if so, to what extent the specific induc-tion of this lectin applies to related tobacco species, a collection of 19 Nicotiana species, covering 12 Nicotiana sections and eight Nicotiana tabacum cultivars, was screened for their capability to synthesize the jasmonate-inducible lectin. Protein analyses by agglutination assays and Western blot confirmed that only nine out of the 19 species examined synthesize lectin after jasmonate treatment. Remarkably, all cultivars tested of the allotetraploid species N. tabacum L. express the lectin after jasmonate treatment. Polymerase chain reaction (PCR) analyses demonstrated that all responsive species possess one or more lectin genes, whereas no lectin gene(s) could be traced in the nonresponding tobacco species. These findings provide the first firm evidence for a striking intragenus difference with respect to the activation of a well-defined jasmonate-inducible gene that can be correlated with the presence/absence of orthologous genes in the genomes of closely related species [41].

Searches in the databases revealed that many flowering plants contain sequences encoding putative homologs of the tobacco lectin, suggesting that Nictaba is the prototype of a widespread or possibly ubiquitous family of lectins with a specific endogenous role. These database searches also revealed sequence homology with the family of Cucurbitaceae phloem lectins, a small group of chitin-binding agglu-tinins found in the phloem exudates of a number of Cucurbitaceae species [42]. These lectins, also called the phloem proteins 2 (PP2 proteins), are homodimers consisting of unglycosylated subunits of 17–25 kDa that show a high affinity toward oligomers of GlcNAc [43]. Nictaba shares 33% and 51% sequence identity and similarity, respectively, with PP2. However, alignment of the sequences of Nictaba and the Cucurbita maxima PP2 revealed several striking differences. First, the Nictaba sequence is 53 amino acid residues shorter than the sequence of PP2 (Fig. 13.1b). A more detailed sequence analysis shows that Nictaba lacks the C-terminal cysteine-rich pentapeptide of PP2 and 65 amino acid residues at the N-terminus of PP2. Second, the NLS found in the Nictaba sequence is missing in the PP2 sequence. Another important difference concerns the localization of both lectins in the plant cell. As already mentioned above, Nictaba could be detected in all leaf cells except in the vasculature. In contrast, the Cucurbitaceae phloem lectins are typically present in phloem exudates of cucurbit species [44]. By virtue of their cysteine-rich pentapeptide, the Cucurbitaceae phloem lectins can establish intermo-lecular disulfide bridges with the phloem protein PP1. Finally, it should also be mentioned that, in contrast to Nictaba, the Cucurbitaceae phloem lectins are consti-tutively expressed.


In view of the sequence similarity and sugar specificity, it is believed that Nictaba and the Cucurbitaceae phloem lectins are closely related to each other, but taking into account the differences mentioned above, both types of lectin probably do not fulfill the same role. Though it is possible that the extra N-terminal sequence determines ligand specificity, it is more likely that the sugar-binding module is not the equivalent of PP2 but rather of Nictaba. Since Nictaba is a potent hemaggluti-nin, it must comprise a complete carbohydrate-binding domain. Accordingly, the extra sequences found in the PP2 proteins can be considered accessory domains with an unrelated function.

Other Nictaba homologs could be found in many flowering plants, among them Solanum tuberosum (potato), S. esculentum (tomato), Glycine max (soybean), Lotus japonicus, Hordeum vulgare (barley), and Oryza sativa (rice). Arabidopsis thaliana contains the largest number of Nictaba homologs [45]. Most of these Arabidopsis proteins are chimeric proteins consisting of a C-terminal Nictaba domain and an unrelated N-terminal domain. Amongst these N-terminal domains F-box, TIR (Toll-interleukin 1-resistance) domains and AIG1 (avirulence-induced gene) domains could be identified, which are believed to function in protein degradation and defense signaling, respectively [46–48]. Taking into account the widespread occur-rence of proteins with Nictaba domains, we propose that Nictaba is the prototype of a lectin family much larger than the Cucurbitaceae phloem lectins. Since, in addi-tion, Nictaba can by no means be considered a phloem lectin, we refer to these Nictaba-like proteins as the “superfamily of proteins with a Nictaba domain.”

13.2.3 Cytoplasmic Galanthus nivalis Agglutinin (GNA)-Related Lectins

In 1987, a lectin with exclusive specificity toward mannose was isolated and char-acterized in snowdrop (Galanthus nivalis) bulbs [49]. GNA is a homotetramer of noncovalently linked 12-kDa monomers. Originally, this group of lectins was referred to as the “monocot mannose-binding lectins” [5], since similar mannose-binding lectins were found in numerous monocot plant families (e.g., Alliaceae, Liliaceae, Orchidaceae, Araceae, Bromeliaceae, Ruscaceae, and Iridaceae) [3, 5, 50, 51]. However, in recent years, very similar lectins have been identified in plants other than Liliopsida (e.g., in the liverwort, Marchantia polymorpha) [52]. Therefore, this group of lectins is now referred to as “GNA-related lectins” after the first identified member.

All GNA-related plant lectins are synthesized as preproproteins with an N-terminal signal peptide and a C-terminal propeptide [3, 53] and accordingly are thought to be located in the vacuolar compartment. However, recent findings revealed that some plants (Triticum aestivum, Zea mays, and Medicago truncatula) express proteins that closely resemble the vacuolar GNA-related lectins but lack the signal peptide and C-terminal propeptide [12] (Fig. 13.1c). Analysis of the sequences of the GNA-related proteins from Zea mays indicated that they lack


specific sorting sequences such as a signal peptide. Transient expression of the fusion construct of the coding sequence of the GNA orthologs with EGFP in tobacco BY-2 cells revealed that the GNA homolog from maize is located in the nucleus and cytoplasm of the cell (Fig. 13.2e).

Until now, no information is available on the carbohydrate-binding specificity of the cytoplasmic GNA-related lectins, since none of these lectins have been purified. However, molecular modeling studies using the three-dimensional structure of GNA as a model have shown that all amino acids known to be involved in the carbohydrate-binding site of GNA are conserved in most of the cytoplasmic GNA-related lectins. Hence, it can be envisaged that these cytoplasmic lectins will have very similar binding properties as their vacuolar homologs.

During the past few years, evidence has accumulated for the occurrence of GNA-like proteins outside the plant kingdom. A first nonplant protein was identi-fied in Dictyostelium discoideum [54] as comitin, a bifunctional actin-binding protein with a mannose-binding GNA domain. More recently, a lectin-like bacterio-cin with high sequence similarity to the GNA domain has been isolated from the bacterium Pseudomonas putida [55]. Furthermore, it was demonstrated that the skin and intestine mucus of the Japanese puffer fish (Fugu rubripes) produce mannose-binding lectins that share striking sequence similarity with GNA [56, 57]. Finally, genome and transcriptome sequencing programs revealed the occurrence of expressed GNA-like proteins in several fungi [58] and in the freshwater sponge Lubomirskia baicalensis [59]. These observations leave no doubt that the GNA-like plant lectins represent only a subgroup of a more extended family of proteins.

Biochemical analyses of the so-called comitin from Dictyostelium discoideum [54] and puffer fish from Fugu rubripes [56, 57] provided firm evidence that these proteins contain a functional mannose-binding domain. Importantly, cloning of the corresponding genes revealed that comitin and puffer fish lack a signal peptide and accordingly do not follow the secretory pathway but are synthesized on free ribo-somes in the cytoplasm. Expression analysis of fusion proteins with EGFP revealed very similar location patterns for GNA orthologs from the fish and the fungus as was observed for maize [60].

The identification of these nonvacuolar GNA-like plant proteins sheds a new light on the molecular and functional evolution of plant lectins. It is suggested that the newly identified cytoplasmic GNA homologs are regulatory/signaling plant proteins functionally well different from the vacuolar lectins that are thought to play a role in plant defense and storage. In addition, it was proposed that the cytoplasmic lectins may have served as templates for the development of their vacuolar homologs through the insertion of a signal peptide and a C-terminal propeptide [12].

13.2.4 Cytoplasmic Lectins with an EUL Domain

For many years, it has been known that the Euonymus europaeus (spindle tree) contains an agglutinin, referred to as Euonymus europaeus agglutinin [61].


Since no sequence information was available, this lectin could not be classified yet in one of the currently known families of plant lectins. Recently, molecular cloning of EUL and detailed analysis of the sequence indicated that this lectin shares a marked sequence identity (46%) and similarity (62%) with a family of rice proteins that were found to be induced in roots by abscisic acid and salt stress, and presum-ably play a role in the adaptation of the roots to a hyperosmotic environment [62]. This family of so-called OSR40 proteins comprises four different proteins. Two of them consist of a single OSR40 domain of approximately 150 amino acid residues preceded by a short glycine-rich (osr40g3) or a long histidine-rich N-terminal pep-tide (unnamed osr40g3homolog). The two other proteins are built up of two OSR40 domains separated by a short linker and preceded by a short histidine-rich N-terminal peptide (osr40g2 and osr40c1, respectively). Hitherto, no specific bio-logical activity could be attributed to this OSR40 domain. However, taking into consideration the marked sequence similarity with the EUL, it seems likely that this sequence represents a new carbohydrate-binding domain and can be referred to as the EUL domain. According to the available sequence data, all rice proteins with OSR40 domain(s) are synthesized on free polysomes and are presumed to be located in the cytoplasm (and possibly also in the nucleus). This implies that the rice OSR40 proteins represent a family of cytoplasmic proteins, the expression of which is abscisic acid and salt-stress responsive. Confocal microscopy of a fusion construct of an Arabidopsis thaliana protein containing an EUL domain linked to EGFP confirmed its localization in the nucleus and cytoplasm (Fig. 13.2f).

A preliminary screening of the databases further revealed that proteins comprising one or two EUL domains occur not only in flowering plants but also in gymnosperms and mosses [63], suggesting that the EUL domain probably plays a universal role in stress-related physiological processes. At present, one can only speculate about the working mechanism of proteins containing EUL domain(s). Taking into consideration (1) that all proteins with EUL domain(s) are synthesized in the cytoplasm and (2) that the EUL domain apparently possesses lectin activity, it seems reasonable to expect that the activity of these proteins relies on their bind-ing to cytoplasmic and/or nuclear glycoconjugates.

13.2.5 Amaranthin-Like Sequences

The Amaranthin family is a rather small family of closely related lectins found in different Amaranthus species. This group is called after the first lectin of this family isolated from Amaranthus caudatus seeds. All known amaranthins are homodimers built up of 33-kDa subunits. Detailed specificity studies have shown that amaran-thin preferentially recognizes the T-antigen disaccharide Galb(1,3)GalNAc [64]. Recently, amaranthin-like sequences have also been reported outside the family Amaranthaceae [65, 66].

Until now, no data have been reported on the biosynthesis, processing, or subcel-lular location of amaranthin. However, some predictions can be made on the basis


of the sequence of the mature amaranthin and the deduced sequence of the presumed Amaranthus hypochondriacus lectin [67]. Both sequences are almost identical except that the deduced sequence of the Amaranthus hypochondriacus lectin has four extra residues at its C-terminus, indicating that a short C-terminal propeptide may be cleaved from the primary translation product of the amaranthins. Analysis of the amino acid sequence data further revealed the absence of a signal peptide, suggesting that the lectin is synthesized on free polysomes and possibly located in the nucleus or the cytoplasm. To analyze the location of this group of lectins, a cDNA clone encoding an amaranthin-like sequence from Prunus persica, found in the EST database prepared from leaves infected with the plum pox virus, was used to make a construct encoding a fusion protein of the amaranthin-like sequence with EGFP and expressed in tobacco BY-2 cells. Confocal images indi-cated that the amaranthin-like lectin is expressed mainly in the nucleus and partially in the cytoplasm (Fig. 13.2g). The fluorescence pattern did not change over time. These findings meet the evidence that the amaranthin-like lectin is synthesized without signal peptide.

13.3 Physiological Role of Inducible Plant Lectins

As indicated above, evidence has recently accumulated that plants synthesize well-defined carbohydrate-binding proteins upon exposure to stressful situations. In contrast to the classical plant lectins, which are typically found in vacuoles, the inducible lectins are exclusively located in the cytoplasm and nucleus. Therefore, it can be envisaged that this new class of lectins might play a specific role within the plant cell. In addition, the observation that many of these inducible lectins are apparently widespread in the plant kingdom makes them interesting tools to study the importance of protein–carbohydrate interactions in the plant cell.

Although there is good evidence for the carbohydrate-binding properties of at least some of the inducible lectins, there are at present few indications for the pos-sible receptors for these lectins in the plant cell. In the case of Orysata, specificity studies indicated that this cytoplasmic jacalin-related lectin has a relatively poor affinity for mannose but binds strongly to oligomannosides and high-mannose N-glycans [14]. For Nictaba, it was shown that the lectin reacts well with GlcNAc oligomers but exhibits a higher affinity for high-mannose N-glycans [40]. The pref-erence of both Orysata and Nictaba for high-mannose N-glycans indicates that N-glycosylated glycoproteins are the most likely glycan-receptors for these lectins. At present, the possible occurrence in the cytoplasmic/nuclear compartment of glycoproteins with N-linked glycans is still controversial. However, several research groups have already reported the presence of nuclear and cytoplasmic N-glycosylated glycoproteins in animal systems [68–73].

Irrespective of the exact nature of the receptor glycans, conclusive evidence was obtained for both in vitro and in situ interactions between Nictaba and nuclear/cytoplasmic tobacco proteins. Far Western blots clearly demonstrated that Nictaba


reacts in a GlcNAc oligomer-inhibitable manner with many proteins present in a crude extract from purified nuclei [40]. PNGase treatment of the proteins almost completely abolished the interaction with Nictaba, suggesting that Nictaba reacts with N-glycans. Therefore, one can reasonably assume that Nictaba interacts through its carbohydrate-binding activity with endogenous glycoprotein receptors in the cytoplasmic/nuclear compartment. This, taken together with the nucleocyto-plasmic location and the induction by jasmonate, strongly argues for a specific role of Nictaba in jasmonate-inducible or jasmonate-dependent physiological processes [12, 15]. To get a better insight into the physiological role of these lectins in plants, it is important to understand what the possible receptors for these lectins are.

13.3.1 Role of Lectins in Nucleocytoplasmic Transport

It has been shown that transport of proteins as well as RNA molecules between the cytoplasm and the nucleus plays an important role in animal and plant cells [74]. Several studies unambiguously demonstrated that glycoproteins substituted with O-glycans play an important role in nuclear transport. It has been shown, for example, that the import of proteins into the nucleus of animal cells is inhibited by antibodies directed against O-GlcNAc-modified proteins of the nuclear pore com-plex (NPC) as well as by an intracellular application of wheat germ agglutinin, a GlcNAc-specific lectin that binds to the O-GlcNAc-residues of glycoproteins in the NPC [75].

Microscopical analysis of EGFP-Nictaba expression in tobacco cells revealed a strong staining of the nuclear rim, indicating that Nictaba may interact with pro-teins in the NPC. Taking into account the carbohydrate-binding specificity of Nictaba for GlcNAc oligomers and N-glycans, it can be envisaged that Nictaba could interact with O-GlcNAc-modified NPC proteins. Previous reports have also shown that some NPC proteins are glycosylated [76–78].

Based on all these observations and the presence of Nictaba both in the cyto-plasm and the nucleus, the hypothesis that Nictaba could be a shuttle protein between the nucleus and the cytoplasm was put forward [12, 13, 15].

13.3.2 Role of Inducible Lectins in Plant Defense

Plants possess several constitutive as well as inducible defense mechanisms to pro-tect themselves against insect attack and other threats. Over the last few years, it has become clear that insect herbivory also influences the expression of several lectins or proteins containing lectin domains.

As already indicated above, a jasmonate-inducible lectin was identified in tobacco leaves. It was shown that insect herbivory (e.g., by cotton leaf worm Spodoptera littoralis) induces lectin expression in tobacco plants, most probably


through activation of the jasmonate pathway. When larvae were allowed to feed on a single leaf, systemic induction of lectin activity was observed in all leaves of the plant. In addition, preliminary experiments showed that the tobacco lectin exerts a repellent effect on chewing insects [79].

Evidence for the expression of lectin-like sequences was also obtained from molecular studies showing that wheat plants respond to insects by the expression of lectin genes [80]. These wheat plants react to the initiation of first-instar larval feed-ing of specific biotypes of Hessian fly (Mayetiola destructor) with rapid changes in the expression of several mRNA transcripts at the feeding site, among which was an mRNA encoding Hfr-1, a protein containing a C-terminal domain with sequence similarity to jacalin-related lectins [80, 81]. More recently, two other Hessian fly-responsive wheat genes, called Hfr-2 and Hfr-3, containing a lectin domain similar to amaranthin and hevein, respectively [65, 82], have been reported. Sequence analysis of Hfr-2 revealed an N-terminal lectin domain similar to amaranthin fused to a region similar to hemolytic lectins and channel-forming toxins. The Hfr-3 sequence contains four predicted chitin-binding hevein domains. All these data sug-gest the involvement of a set of lectins in the resistance of wheat to Hessian fly.

The fact that insect herbivory stimulates the expression of lectins or proteins with lectin domains suggests that plants respond to insect herbivory through the synthesis of proteins with carbohydrate-binding activity. Certainly in the case of the tobacco lectin, it has been shown that the lectin is fully active and is able to recog-nize and bind carbohydrates. Future experiments will have to show whether these lectins induced by insects are expressed in sufficient amounts to exert a toxic effect on the insect.

13.4 Biomedical Applications

At present, only a limited amount of information is available with regard to the carbohydrate-binding properties and specificity of this novel class of nucleocyto-plasmic lectins. However, despite the relatively short history, some of these new lectins have already proven to be useful tools.

Nictaba was shown to be highly inhibitory to human immunodeficiency virus, similar to the chitin-binding lectin from stinging nettle (Urtica dioica, [83]) (Balzarini J, unpublished results). These results are in good agreement with the strikingly similar carbohydrate-binding properties of both lectins.

Amaranthin and EUL have intensively been studied for a long time but have only recently been recognized as nucleocytoplasmic lectins. It has been shown previ-ously that amaranthin is a valuable, highly specific tool for the detection of T- and cryptic T-antigens [64] and accordingly is very useful in cancer diagnosis [84–86]. EUL was proven useful to detect M-cells [87] but was reported unsuitable for deliv-ering vaccines to M-cells [88].

The jacalin-related lectin from Morus nigra, called Morniga M, was shown to exhibit a strong interaction with high-mannose N-glycans [89]. It was suggested


that the capability of Morniga M to recognize the N-linked structure makes this lectin a powerful tool for the purification of oligomannosyl residues containing N-glycans and glycoproteins, and as a selective marker to identify N-glycans. Recently, it has been shown that Morniga M binds glycotopes of mammalian retinal neurons [90].

Biomedical applications of many other nucleocytoplasmic lectins are hampered at the moment because the proteins have not been purified yet. It can be envisaged that once sufficient amounts of the (recombinant) lectins have been made available, detailed studies of the carbohydrate-binding properties of the new lectins will reveal more new interesting specificities that possibly can be exploited, e.g., for purification of particular glycoproteins or for the detection of changes in the glyco-sylation pattern on cell surfaces.

13.5 Nucleocytoplasmic Lectins Outside the Plant Kingdom

Extensive studies with animal lectins have shown that most of them recognize endogenous glycoconjugates and accordingly are involved in specific recognition processes within the organism itself. Depending on their location (extracellular, surface exposed, or intracellular), animal lectins mediate either cell–cell interactions or intracellular protein–glycoconjugate interactions. In the past, there have also been several reports of nucleocytoplasmic lectins in animal cells [91]. In particular, the b-galactoside-binding lectins, referred to as galectins, have been studied in detail, and at least eight galectins have been documented in the nucleus and cyto-plasm. It should be mentioned that, for some galectins, a dual localization was reported in that these lectins were found both intracellularly (in nucleus and cyto-plasm) as well as in the extracellular compartment (cell surface). Each individual galectin is expressed in a tissue-specific or developmentally regulated fashion. Galectins are involved in many biological processes, such as morphogenesis, con-trol of cell death, immunological responses, and cancer. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus and interact with Gemin4 [92]. Some reports suggest that the splicing activity of galectins depends on their carbohydrate-recognition domain [93, 94]. However, other reports indicate that the binding of galectin-3 to DNA and RNA is carbohydrate independent [95, 96]. Detailed analyses have further provided evidence to show that galectin-3 serves as a shuttle between the nucleus and the cytoplasm [97–100]. The expression of at least one of the galectins was shown to be inducible. Ovga11 is a lectin in the cytoplasm and nucleus of the upper epithelial cells of the gastrointestinal tract, the mRNA of which is greatly upregulated in tissues infected with the nematode parasite Haemonchus contortus [101]. The lectin is believed to play an immunomodulatory role. Until now, no galectin-like sequences have been reported in plants.

Certain members of the annexin family may interact with carbohydrates and like the galectins are predominantly intracellular proteins [91]. Another glycosylated nuclear lectin, called CBP70, was reported as a GlcNAc-binding lectin interacting


with an 82-kDa nuclear glycoprotein [69, 102]. In addition, it was shown that CBP70 can interact with laminin, suggesting a role in the interaction with the cytoskeleton [103].

In addition, several intracellular animal lectins have been reported in the endo-plasmic reticulum (ER) where they play an important role in folding of glycopro-teins (calnexin, calreticulin), intracellular routing of glycoproteins (ERGIC-53, VIP-36), and targeting of misfolded glycoproteins to ER-associated degradation (ERAD) [104–107]. Once misfolded glycoproteins have been targeted into the cytoplasm, they are recognized by sugar-binding F-box proteins and tagged with ubiquitin to be degraded by the proteasome [108, 109]. Functional homologs for at least some of these lectins have also been found in plants [110].

Comitin is a cytoplasmic actin-binding protein that until now has exclusively been found in the slime mold Dictyostelium discoideum. The N-terminal core domain of comitin has lectin-like activity [54] and has been modeled from the X-ray coordinates of the mannose-binding lectin from snowdrop (Galanthus niva-lis). Docking experiments performed on the three-dimensional model showed that two of the three mannose-binding sites of the comitin monomer are functional. They are located at both ends of the comitin dimer, whereas the actin-interacting region occurs in the central hinge region where both monomers are noncovalently associated. This distribution is fully consistent with the bifunctional character of comitin, which is believed to link the Golgi vesicles exhibiting mannosylated mem-brane glycans to the actin cytoskeleton in the cell [111, 112]. Analyses with mutant Dictyostelium discoideum lines lacking comitin revealed some defects in phagocy-tosis and altered response to hyperosmotic shock [113].

13.6 General Remarks

Recently, evidence has accumulated that nucleocytoplasmic plant lectins also occur in plants and are far more abundant throughout the plant kingdom than was believed until a couple of years ago. As such, carbohydrate-binding motifs seem to be important molecules in the animal as well as the plant kingdom. Thanks to novel technologies, such as confocal fluorescence microscopy and life-time imaging, localization of lectins fused to fluorescent marker proteins such as the green fluo-rescent protein could be studied. It was unambiguously shown that these fusion proteins are expressed in the nucleocytoplasmic compartment. In addition, analyses of the new sequence data resulting from whole-genome sequencing projects and transcriptome analyses predict that more lectins or proteins with lectin domains might be present in the cytoplasm and nucleus of plant cells. Interestingly, a detailed study of a few lectin families for which vacuolar as well as nucleocytoplas-mic lectins have been reported revealed that both forms seem to be related evolutionarily.

At present, a lot of questions still exist regarding the possible function of these nucleocytoplasmic lectins. To get a better insight into the physiological role of


nucleocytoplasmic lectins, it is important to have detailed information on their carbohydrate-binding properties and the proteins they could interact with in the cell. Since these nucleocytoplasmic plant lectins are present in very low concentra-tions, the challenge in plant lectin research is put nowadays on recombinant protein technology. As soon as a small amount of recombinant protein is available, the new glycan array technology will allow one to study or refine the carbohydrate-binding properties of these lectins. In addition, interaction studies of the recombinant pro-tein with putative receptors for the lectin in the cell will help to identify the binding partners for the nucleocytoplasmic lectins.

Acknowledgments This work was supported in part by grants from the Research Council of Ghent University and the Fund for Scientific Research-Flanders (FWO grants G.0201.04 and 3G.0163.06). Gianni Vandenborre acknowledges the receipt of a scholarship from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

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[Advances in Experimental Medicine and Biology] The Molecular Immunology of Complex Carbohydrates-3 Volume 705 || Novel Concepts About the Role of Lectins in the Plant Cell