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First published online June 12, 2003; 10.1104/pp.103.023853 Plant Physiology 132:1322-1334 (2003) © 2003 American Society of Plant Biologists The Tn Antigen-Specific Lectin from Ground Ivy Is an Insecticidal Protein with an Unusual Physiology1Department of Molecular Biotechnology (W.W., E.J.M.V.D.), and Laboratory of Agrozoology, Department of Crop Protection (G.S.), Ghent University, Coupure Links 653, 9000 Gent, Belgium; Institute of Plant Biochemistry, P.O.B. 110432, D–06018 Halle, Germany (B.H.); Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Willem de Croylaan 42, 3001 Leuven, Belgium (W.J.P.); West of Scotland Blood Transfusion Centre, Gartnavel General Hospital, 25 Shelley Road, G12 0XB Glasgow, United Kingdom (A.M., R.F.)
Leaves of ground ivy (Glechoma hederacea) contain a lectin (called Gleheda) that is structurally and evolutionary related to the classical legume lectins. Screening of a population of wild plants revealed that Gleheda accounts for more than one-third of the total leaf protein in some clones, whereas it cannot be detected in other clones growing in the same environment. Gleheda is predominantly expressed in the leaves where it accumulates during early leaf maturation. The lectin is not uniformly distributed over the leaves but exhibits a unique localization pattern characterized by an almost exclusive confinement to a single layer of palisade parenchyma cells. Insect feeding trials demonstrated that Gleheda is a potent insecticidal protein for larvae of the Colorado potato beetle (Leptinotarsa decemlineata). Because Gleheda is not cytotoxic, it is suggested that the insecticidal activity is linked to the carbohydrate-binding specificity of the lectin, which as could be demonstrated by agglutination assays with different types of polyagglutinable human erythrocytes is specifically directed against the Tn antigen structure (N-acetylgalactosamine O-linked to serine or threonine residues of proteins).
Recently, a galactoside-binding lectin was isolated and characterized from leaves of ground ivy (Glechoma hederacea), which shares a high sequence similarity with the classical legume lectins and exhibits a very similar overall fold and three-dimensional structure (Wang et al., 2003  ). The
identification of the ground ivy agglutinin (Gleheda) as a fully active
ortholog of the classical legume lectins eventually solved the problem of the
classification of the Lamiaceae lectins, but at the same time, it raised
several important questions with respect to the evolution and physiological
role of the legume lectin family. It is evident that the apparent occurrence
of a typical legume lectin outside the family Fabaceae urges revision and
refinement of all previously proposed models of the molecular evolution of
this lectin family (Van Damme et al.,
1998; Barre et al.,
2002). In addition, the discovery of Gleheda puts the
physiological role of the classical and novel legume lectins in a new
perspective because one cannot take it for granted that the Lamiaceae lectins
have been designed for the same activities/functions as the Fabaceae lectins.
For example, the proposed involvement of—at least some—classical
legume lectins in the specific interaction between legumes and their symbiotic
nitrogen-fixing Rhizobia sp.
(Diaz et al., 1989;
Brewin and Kardailsky, 1997)
cannot be extrapolated to the Lamiaceae lectins because members of this plant
family do not establish a symbiotic relationship with nitrogen-fixing
bacteria. In contrast, other roles proposed for the classical legume lectins
like defense against herbivorous animals and/or phytophagous invertebrates
possibly combined with a role as a storage protein
(Peumans and Van Damme, 1995),
may well apply to Gleheda or other Lamiaceae lectins. However, because at
present virtually no information is available about the temporal and spatial
regulation of the expression of Gleheda and the possible effects of the lectin
on foreign organisms, it remains speculative to attribute a comparable
physiological function to the Lamiaceae and the classical Fabaceae lectins.
For the same reason, it cannot be precluded that the Lamiaceae lectins fulfill
a yet unidentified role differing from all functions previously attributed to
the legume lectins. Therefore, it seemed worthwhile to study some basic
physiological aspects of the newly discovered ground ivy lectin to find clues
about the role of this lectin and to establish possible functional
relationships—or the absence thereof—to the previously studied
classical legume lectins. This paper gives an overview of the occurrence of Gleheda in a population of ground ivy clones and the temporal and spatial regulation of the expression of the lectin and presents evidence that Gleheda is well different from all previously described legume lectins. Gleheda is not only unique for what concerns the extremely high expression level in certain clones but is also the first documented example of a protein that is predominantly expressed in a single layer of palisade parenchyma cells. In addition, Gleheda exhibits insecticidal activity toward larvae of the Colorado potato beetle (Leptinotarsa decemlineata), indicating that the lectin may be involved in plant defense against insects.
Evidence for Dramatic Differences in the Gleheda Content between Individual Ground Ivy Clones A preliminary screening revealed that some ground ivy plants exhibit a reasonably high agglutination activity, whereas others are apparently completely devoid of lectin. To corroborate the obvious differences in lectin content between individual plants, a more extended screening was set up. Ground ivy is a perennial weed with creeping stems that root at the nodes. Flowers develop only during spring on erect inflorescences, but the creeping offshoots keep growing throughout the entire growing season. As a result, ground ivy often forms a dense, rapidly expanding mat of vegetation. Although many patches of ground ivy consist of a single clone, others are a composite of multiple individuals. To ensure that individual plants were sampled, single offshoots were collected and analyzed. A total number of 41 ground ivy clones were sampled from six different locations (Table I). To avoid multiple sampling of a single genotype, plants were collected from patches separated from each other by at least 50 m. Because the lectin content of the leaves increases as a function of age (see below) care was taken to use fully expanded mature leaves of a comparable age for the preparation of the extracts used for the estimation of the lectin content.
Semiquantitative agglutination assays revealed dramatic differences between the lectin levels of the individual clones. Some plants contained more than 2.5 mg lectin g–1 leaf tissue (fresh weight), whereas in others, the threshold level for detection (0.5 µg g–1) was not surpassed (Table I). When calculated on a protein basis, Gleheda represented more than one-third of the total protein in some plants, whereas the lectin accounted for less than 0.01% of the total protein in others. An overview of the data shown in Table I indicates that Gleheda contributes for <0.01%, 0.01% to 0.1%, 0.1% to 1%, 1% to 10%, and >10% of the total soluble leaf protein in 5, 2, 4, 20, and 10 plants, respectively. The apparent high incidence of lectin-negative ground ivy clones raised the question of the sensitivity of the detection method. Taking into consideration the minimal concentration required for the agglutination of trypsin-treated rabbit erythrocytes (0.1 µg mL–1), the level of detection in a crude extract is approximately 0.5 µg mL–1 when tested according to the procedure described in "Materials and Methods" (i.e. in a reaction mixture consisting of 1 volume of extract and 4 volumes of a 1% [v/v] suspension of red blood cells). To lower the level of detection, the extracts that yielded negative results in the standard assay were dialyzed against 0.5 M ammonium sulfate and assayed by mixing 45 µL with 5 µL of a 20% (v/v) suspension of red blood cells (which allowed detection of Gleheda levels of 0.1 µg mL–1 in the extracts, provided that the extracts are dialyzed and incubation lasts for at least 6 h). Taking into consideration that the extracts were made by grinding the leaf tissue in 4 volumes of buffer, the overall detection limit corresponded to roughly 0.5 µg Gleheda g–1 leaf tissue (on a fresh weight basis). According to the results summarized in Table I, the concentration of Gleheda remained below this level of detection in five clones. Attempts to use serological techniques to reduce the detection level were unsuccessful because western-blot analysis of extracts was less sensitive than the agglutination assays and the specificity of an ELISA could not be guaranteed at low concentrations of the lectin. In an alternative approach, 500-g samples of leaves of two lectin-negative clones (CA 1 and LD 5) were extracted and processed as described for the large scale preparation of Gleheda. The fractions (25 mL in total) desorbed from the column (2 x 2.6 cm; approximately 10-mL bed volume) of Gal-Sepharose 4B after the first affinity chromatography step were checked for the presence of Gleheda. No agglutination activity could be detected, indicating that the affinity-purified fractions contained <0.1 µg lectin mL–1, which implies that the total lectin content of the leaves was less than 2.5 µg 500 g–1 (or <0.005 µg g–1 fresh weight). To check whether the extremely high agglutination activities of the leaf extracts from some plants were really due to a correspondingly high level of Gleheda, the extracts were also analyzed by SDS-PAGE and western blot. As shown in Figure 1A, the intensity of the (Coomassie Blue-stained) polypeptides corresponding to the 28- and 26-kD Gleheda subunits was in good accordance with the results of the semiquantitative agglutination assays. Two prominent bands comigrating with the 28- and 26-kD Gleheda subunits can be distinguished in the lanes loaded with extracts from plants with a high agglutination activity (lanes 1–3). Both polypeptide bands are far less prominent (although still distinguishable) in the extracts from plants with a lower agglutination activity (lanes 4–6) and are apparently absent in the lanes loaded with extracts from the nonagglutinating clones. Western-blot analysis of a comparable gel with antibodies raised against Gleheda confirmed that the polypeptides comigrating with the 28- and 26-kD Gleheda subunits correspond to the respective lectin subunits (Fig. 1B). Moreover, because direct sequencing of the 28- and 26-kD polypeptides present in the extract from plant EF 5 (corresponding to lane 1 of Fig. 1, A and B) yielded a single sequence (KTTHF AVPPA LTFQG DAFDP NDTSF IRLT) identical to the N terminus of purified Gleheda, there is no doubt that Gleheda is the most abundant protein in the leaves of some ground ivy clones. Densitometric analysis of the gel shown in Figure 1A indicated that the 28- and 26-kD Gleheda subunits together account for approximately 35% of the total protein in lanes 1 to 3, which is in good accordance with the values calculated on the basis of the results of the semiquantitative agglutination assays (shown in Table I).
To corroborate whether the apparent absence of Gleheda in lectin-negative plants was possibly due to some unknown environmental and/or biological factors, an apparent lectin-negative clone (CA 1) growing in its natural habitat was regularly checked for the presence of lectin. In addition, clone CA 1 was also grown in the greenhouse for 6 months and was regularly checked for the possible presence of lectin. No lectin activity could be detected at any time. Similar analyses of a high-lectin clone (EF 3) growing under the same conditions (both in the field and in the greenhouse) consistently yielded a very high agglutinating activity. Although not conclusive, these observations strongly indicate that the expression level of Gleheda is genetically determined.
Preliminary experiments indicated that extracts from old leaves exhibited a much stronger agglutination activity than extracts from young leaves of the same plant. To check whether there is a possible relationship between leaf development/age and lectin concentration/content, all pairs of leaves from a single offshoot were collected separately and extracted, and their agglutinating activity was determined. For this experiment, both an erect (flowering) stem and a (vegetatively growing) creeping stem from clone EF 3 were used. As shown in Figure 2, the lectin concentration strongly increases during early leaf formation/maturation. Starting from the distal end (youngest leaves) to the proximal end (older leaves), the lectin concentration increases approximately 10-fold within the first four and six pairs of leaves of the erect and creeping stem, respectively. Thereafter, the increase becomes less dramatic and eventually comes to a halt. When the total lectin content is considered, the increase is even more dramatic. Fully expanded leaves of the erect flowering stem contained approximately 100 times more lectin than the youngest leaves, and in leaves of the creeping stem, the total lectin content increased more than 2,000-fold. To check whether the observed increase in agglutinating activity was really due to a corresponding increase in Gleheda concentration, extracts from the different pairs of leaves were also analyzed by SDS-PAGE and western blot. As shown in Figure 3, the intensity of the polypeptides reacting with antibodies against Gleheda dramatically increases from the distal to the proximal leaves. It can be concluded therefore that Gleheda is present in relatively small quantities in the youngest leaves but accumulates very rapidly during early leaf formation and maturation. Once the leaves are fully expanded (which takes approximately 20–25 d), no further accumulation of the lectin occurs. It should be emphasized here that the maximal lectin content is reached long before the onset of senescence. Ground ivy leaves have a long lifetime that spans almost the entire growth season.
The final lectin concentration in leaves from erect and creeping stems is comparable. However, due to their much larger size, leaves from creeping stems contain approximately 10 times more lectin compared with leaves from the erect stems.
Gleheda was originally discovered and isolated from leaves. To address the question of the possible tissue-specific expression of Gleheda, extracts were made from leaves, petioles, stems, roots, calyces, petals, and ovary tissues from a single ground ivy plant (EF 3) and were tested for the presence of lectin by agglutination assays and western-blot analysis. Agglutination assays yielded positive results only for extracts from leaves and calyces. No activity could be detected in the extracts from the other tissues. Western-blot analysis of the extracts from the different tissues confirmed that Gleheda occurs only in leaves and calyces (data not shown).
To check the possible induction of Gleheda in a normally lectin-negative clone, isolated leaves were treated with the phytohormones methyl jasmonate, abscisic acid, and GA3. No lectin could be induced by these phytohormones in the lectin-negative clone CA 1. Wounding of leaves of intact plants of clone CA 1 did not result in the expression of detectable amounts of Gleheda neither in the wounded leaves nor in other leaves of the plant. Similarly, no agglutination activity could be detected in intact and insect-affected leaves of CA 1 and LD 5 plants grown in the field. Extensive checking of senescing leaves sampled from clones CA 1 and LD 5 at the end of the growing season (i.e. in October) yielded no positive agglutination results, suggesting that senescence does not induce the expression of Gleheda in lectin-negative plants. Similar experiments were done with leaves and intact plants of the lectin-positive clone EF 3. As far as could be concluded from semiquantitative agglutination assays, neither the phytohormones nor wounding or insect attack provoked an increased expression of Gleheda. Moreover, senescence also was not accompanied with an increased level of Gleheda (Fig. 4).
Because at present, no data have been published on the cellular and
subcellular location of a lectin in a species of the family Lamiaceae, it
seemed worthwhile to localize Gleheda by immunocytochemical techniques. In
addition, it was expected that immunolocalization studies could provide
additional information about the dramatic increase in lectin content during
leaf development. Both young and mature leaves were investigated to analyze
possible differences in tissue and cell specificity of the occurrence of
Gleheda in high-lectin and no-lectin plants. Staining of cross sections from
young leaves (diameter, <1 cm; corresponding to node 2) with anti-Gleheda
antibodies yielded no signal for the no-lectin plant (CA 1;
Fig. 5A) but resulted in a
heavy labeling of the palisade parenchyma of the high-lectin clone (EF 5;
Fig. 5B), suggesting that
Gleheda is confined to a single cell layer. A closer examination of the
high-magnification micrographs of the palisade cells indicates that the
cytoplasm (around the plastids) exhibits some label
(Fig. 5C). However, the label
is predominantly associated with "inclusions" that are located
within the vacuole near to the cytoplasm and possibly correspond to a sort of
protein bodies (Fig. 5C).
Microscopic analysis of cross sections from older leaves (diameter, 5 cm;
corresponding to node 5) yielded similar results as for young leaves for what
concerns the cellular distribution of Gleheda. No or virtually no lectin could
be detected in leaves from the no-lectin plant, whereas a very strong labeling
was observed in the palisade parenchyma cells of the high-lectin clone
(Fig. 5, E versus F). Examination of the high-magnification pictures further indicates that in the
older leaves, Gleheda is predominantly located in the vacuole and is only to a
small extent associated with the cytoplasm
(Fig. 5G). This apparent
vacuolar location of Gleheda is in good agreement with the results of the
molecular cloning because the presence of a signal peptide in the primary
translation product suggested that Gleheda is targeted to the secretory
pathway (Wang et al., 2003
Oral feeding of potato (Solanum tuberosum) leaves dipped in a 2% (w/v) solution of Gleheda in distilled water caused dramatic effects on the growth and survival of the potato beetle larvae. Soon after the onset of the experiment, larvae feeding on Gleheda-treated leaves showed a dramatic inhibition of feeding and weight gain (Fig. 7). None of the Gleheda-fed larvae reached the pupal instar, indicating that the lectin caused complete mortality at the dose administered in this experiment.
It was demonstrated previously that Gleheda is a
Gal/N-acetylgalactosamine (GalNAc)-binding lectin with a slight
preference for type A over type B human erythrocytes
(Wang et al., 2003
To check whether the observed insecticidal activity of Gleheda could be due to a general cytotoxic activity of the lectin, two human and two murine cell lines were challenged in vitro with increasing concentrations of purified Gleheda. L1210 and CEM cells were not affected by Gleheda at concentrations below 200 µg mL–1. For FM3A and Molt4CI8 cells, the concentration required to obtain 50% inhibition was 155 (±37) and 188 (±16) µg mL–1, respectively. These experiments indicate that Gleheda exhibits little or no general cytotoxic activity toward human and murine cells. To assess the possible antiretroviral activity of Gleheda, the inhibition of the infection of CEM cells by HIV-1 and HIV-2 was evaluated. No effect was observed at concentrations up to 100 µg mL–1, indicating that Gleheda is devoid of antiretroviral activity. Gleheda also failed to affect the in vitro growth and development of Neurospora crassa and Botrytis cinerea, suggesting that the lectin does not act as an antifungal protein (data not shown).
The present study of the physiology and biological activities indicates that Gleheda differs from all previously described classical legume lectins for what concerns its "general biology" and in several aspects, can be considered a unique lectin. A first remarkable observation is the apparent huge difference in lectin content between individual ground ivy plants and the high incidence of plants without any detectable lectin activity. In this respect, ground ivy resembles some legume species in which lectin-negative genotypes have been identified. It should be mentioned, however, that the lectin-negative genotypes identified in collections of soybean (Glycine max; Pull et al., 1978 ; Goldberg et al.,
1983; Vodkin and Raikhel,
1986), common bean (Phaseolus vulgaris;
Staswick and Chrispeels,
1984), and other legumes (Pull
et al., 1978) were only tested for the presence of the seed
lectin(s), and therefore are not necessarily devoid of lectins in their
vegetative tissues. On the contrary, some seed lectin-deficient soybean
varieties have been shown to express a lectin in roots
(Vodkin and Raikhel, 1986). A
second striking observation is the extremely high expression level of Gleheda
in leaves of some ground ivy clones (up to 35% of the total soluble protein),
which is far superior to that of any other documented leaf lectin. Most other
leaf lectins are minor proteins representing less than 1% or maximally a few
percent of the total leaf protein
(Kitagaki-Ogawa et al., 1986;
Hankins et al., 1987;
Van Damme and Peumans, 1990;
Smeets et al., 1997). A third
particularity concerns the exclusive expression of Gleheda in leaves and to a
lesser extent in calyces. In general, plant lectins that are found in leaves
occur in all vegetative tissues (e.g. petioles, roots, and petals) and are
usually far more prominent in vegetative storage tissues like bark, tubers,
rhizomes, and bulbs (Van Damme et al.,
1998). A fourth striking observation concerns the regulation of
the expression of Gleheda in leaves, which is characterized by a dramatic
increase during leaf expansion/maturation. No comparable increase in lectin
content has been reported for any other plant species. In general, leaf
lectins accumulate only during the earliest phases of leaf development.
Thereafter, the total lectin content remains more or less constant but due to
the increase of the leaf size the concentration, progressively decreases as a
result of the dilution of the lectin in the leaf. The fifth, and probably the
most spectacular feature of Gleheda, is the predominant location of the lectin
in a single layer of palisade parenchyma cells. To the best of our knowledge,
such a specific location pattern has not been reported for any plant lectin
(or plant protein in general). Detailed information about the distribution of
a typical legume lectin over the different leaf cells has been reported for
the so-called soybean vegetative lectin. This lectin, which is related to but
not identical with the soybean seed lectin, occurs in leaves, stems, petioles,
and cotyledons of seedlings but not in seeds
(Spilatro et al., 1996).
Within the leaves, the soybean vegetative lectin was found in bundle sheath
and paraveinal mesophyll cells but could not be detected in the palisade
mesophyll or spongy mesophyll. It is evident, therefore, that the cellular
location of Gleheda in the leaves is completely different from that of the
vegetative soybean lectin. In addition to the exclusive location in the
palisade parenchyma cells, the apparent concentration of Gleheda in inclusion
bodies within the vacuole also is rather unusual because other presumed
vacuolar leaf lectins are more or less evenly distributed over the
protein-storage vacuoles, as has been demonstrated for the leaf lectin of
Sophora japonica (Herman et al.,
1988) and Dolichos biflorus
(Bunker and Etzler, 1994).
These observations leave no doubt that the temporal and spatial regulation of
the expression and the cellular and subcellular location of Gleheda differ
from that of the classical legume lectins and, as a matter of fact, of all
other known plant lectins. It is unlikely, therefore, that the ground ivy
lectin fulfills the same function as its orthologs in legume species.
Accordingly, the search for the physiological role of Gleheda can hardly be
supported by extrapolations from functional research with other legume lectins
or members from unrelated lectin families, but it should be focused on the
specific expression pattern of the ground ivy lectin and its biological
activities.
Although only preliminary, the results of the insect feeding trials may
give an important clue to the unraveling of the role of Gleheda because they
demonstrate that the lectin concentration in the leaves of most ground ivy
clones is sufficiently high to exert a noxious effect on at least some
insects. At present, the mode of action of Gleheda on the herbivorous pest
insect potato beetle is still unknown, but it is unlikely that the observed
insecticidal activity can be ascribed to an aspecific cytotoxicity because the
lectin does not affect the viability of human and murine cells in vitro. Most
probably, the observed adverse effects on the potato beetle larvae are somehow
related to the pronounced specificity of Gleheda toward the Tn antigen
(
Although Gleheda is certainly not the first member of the family of legume
lectins and related proteins that is found to exhibit anti-insect activity, it
was not obvious to assume a priori that Gleheda was an insecticidal protein.
There have been several reports that some legume lectins inhibit the growth
and/or development of some insects and in some cases even exert lethal
effects. However, there are several caveats. First, in some instances, the
presumed anti-insect activity was not due to the lectin but to contaminating
insecticidal proteins (e.g. it has been shown that the toxicity of
phytohemagglutinin preparations toward insects was due to contaminating
Evidently, after it turned out that Gleheda was capable of killing larvae
of the Colorado potato beetle, it became clear that there might be some
analogy to the insecticidal or entomotoxic properties of some legume lectins.
However, to the best of our knowledge, no legume lectin has ever been reported
to exert a toxic effect on this insect, which indicates that Gleheda is quite
different from the legume lectins with respect to its entomotoxic properties.
Most probably, this difference is intimately linked to the differences in
carbohydrate-binding specificity between Gleheda and the legume lectins. It
should be emphasized that the extremely high specificity toward the Tn antigen
is unique for Gleheda and has not been observed for any legume lectin. As
already mentioned above, the high affinity for the Tn antigen may explain the
potent toxicity of Gleheda toward insects. This structurally simple
O-linked glycan is quite common in lower animals but is normally not
present in higher vertebrates because higher organisms possess
glycosyltransfereases that extend the Tn structure with additional sugar
residues (e.g. Gal and sialic acid). For example, in humans, the Tn antigen is
diagnostic for a genetic disorder or a pathological condition. On the basis of
the particular specificity, it is tempting to speculate that Gleheda (and the
other Lamiaceae lectins) are directed against lower animals (insects and other
invertebrates), whereas the legume lectins, which preferentially bind to
complex N- and O-glycans that typically occur in higher
animals, are believed to play a role in plant defense against vertebrate
herbivores (Peumans and Van Damme,
1995
According to the results of the induction experiments, Gleheda is not an
inducible protein but is constitutively expressed. However, the level of
expression strongly differs among individual genotypes. Southern-blot and PCR
experiments indicated that lectin-negative clones do not possess the Gleheda
gene (data not shown). Although the results from these experiments have to be
interpreted with some care, they may explain why no Gleheda can be detected in
the lectin-negative clones. The apparent absence of the Gleheda gene in some
individuals has important consequences for what concerns the possible
defensive role of the lectin. It is evident that Gleheda is not essential for
the survival of the plant because a wild population comprises a reasonable
fraction of lectin-negative individuals. As far as can be concluded from a
visual inspection in the field, there is no apparent phenotypical difference
between lectin-negative and -positive clones, and both types of clones show a
similar insect damage. However, it is risky to draw conclusions from such
observations because there is always a possibility that the target insect(s)
(or other pests) were not present at the time of the observations. In this
respect, we would like to draw the attention to the analogy of the arcelins in
bean. Although there is firm experimental evidence that arcelins act as
anti-insect proteins in planta, it is also well known that not all bean
accessions express these proteins, which implies that the arcelins are not
essential for survival of these lines
(Mirkov et al., 1994 The apparent analogy to the arcelins implies that there is no reason to preclude an in planta defensive role of Gleheda. On the contrary, this observation cannot but stimulate further research aimed to corroborate the role of this novel insecticidal lectin in ground ivy itself and exploit its possible use as an insect resistance factor in transgenic plants.
Plant Materials
Collection of Individual Ground Ivy (Glechoma hederacea) Clones
Mass Propagation of a High-Lectin Clone
Freshly harvested leaves (5 kg) were extracted in 20 L of 20 mM unbuffered 1,3-diaminopropane containing 0.01% (w/v) thiourea using a Waring blender. The homogenate was filtered through glass wool, solid CaCl2 was added to a final concentration of 20 mM, and the extract was centrifuged at 3,000g for 10 min. After adjusting the pH to 7.5 with 1 N H3PO4, the crude extract was kept overnight in the cold room at 2°C and centrifuged (8,000g for 10 min). The resulting supernatant was brought at 1 M ammonium sulfate, centrifuged (8,000g for 10 min), and filtered through filter paper (3MM, Whatman, Beverly, MA). The cleared extract was loaded onto a column (5 x 5 cm; approximately 100-mL bed volume) of Gal-Sepharose 4B equilibrated with 1 M ammonium sulfate. Binding of the lectin was monitored by regular checking of the agglutination activity of the eluate. After loading the extract, the column was washed with 1 M ammonium sulfate until the A280 fell below 0.01 and the bound lectin eluted with 500 mL of 20 mM Tris-HCl (pH 10). The pH of the affinity-purified lectin fraction was adjusted to 7.5 with 1 N acetic acid, solid NaCl was added to a final concentration of 0.2 M, and the solution was centrifuged (8,000g for 10 min). The supernatant was loaded onto a column (20 x 2.6 cm; approximately 100-mL bed volume) of Gal-Sepharose 4B equilibrated with 20 mM Tris-HCl (pH 7.8) containing 0.2 M NaCl. After extensive washing with the same Tris buffer, the bound lectin was eluted with 500 mL of 0.1 M Gal in 0.2 M NaCl. The affinity-purified lectin fraction was brought at 1 M ammonium sulfate and loaded onto a column (10 x 2.6 cm; approximately 50-mL bed volume) of phenyl-Sepharose equilibrated with 1 M ammonium sulfate. After loading the lectin solution, the column was washed with 500 mL of 1 M ammonium sulfate (to remove any traces of Gal), and the lectin was eluted with 75 mL of 20 mM Tris-HCl (pH 10). The lectin solution was dialyzed against water and lyophilized. Using this procedure, approximately 500 mg of Gleheda was obtained from 5 kg of leaves.
Tissue samples were extracted with mortar and pestle in 4 volumes (v/w) of 20 mM unbuffered 1,3-diaminopropane. The homogenates were transferred to Eppendorf tubes and centrifuged (12,000g for 10 min).
Treatment of Excised Leaves with Phytohormones
Wounding and Insect Herbivory
Senescence
Agglutination assays were carried out in small glass tubes or in the wells of 96 U-welled microtiter plates in a final volume of 50 µL containing 40 µL of a 1% (v/v) suspension of trypsin-treated rabbit erythrocytes and 10 µL of extracts or lectin solutions. Agglutination was monitored visually after 1 h of incubation at room temperature. To quantify the lectin activity, crude extracts were serially diluted with 2-fold increments, and the dilution endpoint was determined. The absolute lectin content of the extracts was calculated by comparison with the agglutination activity of a dilution series of a lectin solution with known concentration. To check a large number of leaf samples for the presence of Gleheda, a simplified detection method was used that is based on an agglutination assay on microscope glass slides. A piece of leaf tissue was squeezed between two glass slides and to the resulting sap a small droplet (50–100 µL) of a 2% (v/v) suspension of trypsin-treated rabbit erythrocytes in 1 M ammonium sulfate was added. The sample was gently shaken, and the agglutination was visually inspected after 10 min of incubation at room temperature.
Proteins were separated by SDS-PAGE using 15% (w/v) acrylamide gels, as
described by Laemmli (1970
Proteins were separated by SDS-PAGE and electroblotted on an Immobilon P membrane (Millipore, Bedford, MA). Before immunodetection, the free binding sites on the membrane were blocked with 5% (w/v) bovine serum albumin (BSA) in 10 mM Tris, 150 mM NaCl, and 0.1% (v/v) Triton X-100, pH 7.6 (TSB), for 1 h at room temperature. After washing the membrane with TSB for 5 min, the membrane was consecutively treated with primary antibody (overnight incubation at room temperature), goat-anti-rabbit antibody (1 h incubation at room temperature), and peroxidase-anti-peroxidase-complex (1 h incubation at room temperature). After every treatment, the membrane was washed three times with TSB for 5 min. Before the immunodetection, the membrane was washed for 5 min with 0.1 M Tris-HCl (pH 7.6). The peroxidase reaction was carried out in a fresh solution of 0.1 M Tris-HCl (pH 7.6) containing 0.7 mM 3,3'-diaminobenzidine tetrahydrochloride and 0.01% (v/v) H2O2. The reaction was stopped by washing the membrane in distilled water.
Polyclonal antibodies were raised against Gleheda in a male New Zealand
white rabbit. The animal was injected subcutaneously with 1 mg of purified
Gleheda dissolved in phosphate-buffered saline (PBS; 1.5 mM
KH2PO4, 10 mM Na2HPO4,
3 mM KCl, and 140 mM NaCl, pH 7.4) and emulsified in 1
mL of Freund's complete adjuvant. Four booster injections with 1 mg of Gleheda
in 1 mL of PBS were given with 10-d intervals. Ten days after the final
injection, blood was collected from an ear marginal vein. After clotting, the
crude serum was prepared by centrifugation. Because western-blot analysis of
crude extracts from ground ivy demonstrated that the crude antiserum reacted
not only with the lectin polypeptides but also with several other proteins,
the antiserum was further purified. Affinity chromatography of the crude
antiserum on immobilized Gleheda did not markedly improve the specificity of
the antiserum. Even after an additional affinity chromatography on immobilized
Robinia pseudoacacia lectin (which removes most of antibodies that
are directed against N-glycans;
Desmyter et al., 2001
Small pieces of leaves were fixed with 4% (w/v) paraformaldehyde/0.1% (v/v)
Triton X-100 in PBS, embedded in polyethylene glycol, and cut as described
(Hause et al., 1996 For electron microscopy, leaf material was fixed with 3% (w/v) paraformaldehyde/0.2% (v/v) glutaraldehyde in PBS and dehydrated in a graded ethanol series. Ethanol of specimens was substituted by LR White (Polysciences, Warrington, PA). Immunolabeling of ultrathin sections was carried out with purified primary antibodies against Gleheda (diluted 1:500 in PBS containing 1% [w/v] acetylated BSA and 0.1% [v/v] Tween 20) and a goat anti-rabbit IgG conjugated with 10 nm of colloidal gold (Sigma-Aldrich, St. Louis). After immunolabeling, sections were post-stained with uranyl acetate and lead citrate. Sections were visualized with a Zeiss TEM 900 electron microscope.
The agglutination activity of Gleheda toward normal and polyagglutinable
human erythrocytes was estimated by determining the minimal concentration
required to agglutinate the different cells. The polyagglutinable cells used
were recovered from liquid nitrogen storage, except Tk, which was prepared by
endo-
Purified Gleheda was tested for insecticidal activity against larvae of the
Colorado potato beetle (Leptinotarsa decemlineata Say; [Coleoptera;
Chrysomelidae]). Newly moulted (0–12 h) last (fourth)-instar larvae were
selected from a continuous culture that was maintained under standard
conditions (23°C ± 2°C; 65% ± 5% relative humidity;
photoperiod, 16-h light and 8-h dark) and provided fresh potato (Solanum
tuberosum cv Bintje) foliage ad libitum
(Smagghe and Degheele, 1994
Gleheda was evaluated for cytotoxic/cytostatic activity against murine
leukemia L1210, murine mammary carcinoma FM3A, human T-lymphocyte Molt 4/clone
8, and human CD4-positive T lymphocytes (CEM cells). Cells were seeded in
96-well microtiter plates at approximately 50,000 to 75,000 cells per 200
µL well in RPMI-1640 cell culture medium in the presence of serial
dilutions of Gleheda, the highest concentration tested being 400 µg
mL–1. After 2 d (L1210 and FM3A) or 3 d (Molt
4/clone 8 and CEM) incubation at 37°C, cell numbers were determined. Data
are expressed as 50% inhibitory concentration or compound concentration
required to inhibit cell proliferation by 50%. The methodology for testing the
antiviral activity of Gleheda toward human immunodeficiency virus has been
described previously (Balzarini et al.,
1991
In vitro microtiter plate assays (according to
Broekaert et al., 1990
We thank Prof. J. Balzarini and L. Van Berckelaer (Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium) for testing the cytotoxicity and antiviral activity of Gleheda. Received March 19, 2003; returned for revision March 26, 2003; accepted March 30, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.023853.
1 This work was supported in part by the Fund for Scientific
Research-Flanders (Belgium; grant no. G.0113.01). W.W. received scholarships
from the Education Ministry of China, the Flemish Community, the Catholic
University of Leuven, and Ghent University to finish her PhD research. * Corresponding author; e-mail ElsJM.VanDamme{at}rug.ac.be; fax 32–9–264–6219.
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ABSTRACT
RESULTS
-mercaptoethanol.
-linked to Ser or Thr (i.e.
Tn antigen), the blood group specificity of Gleheda toward normal and
polyagglutinable human red blood cells was studied in some detail. As shown in
