Glycoconjugate Journal 18, 589613, 2001


C 2002 Kluwer Academic Publishers. Manufactured in The Netherlands.

REVIEW

Plant lectins:
Occurrence, biochemistry, functions and applications
1
Harold Rudiger

and Hans-J. Gabius2

1

Institut fur
Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universitat,
Am Hubland, Wurzburg,

Germany, 2 Institut fur

Physiologische Chemie, Tierarztliche

Fakultat,
Ludwig-Maximilians-Universitat,
Veterinarstr.

13, 80539 Munchen,

Germany

Growing insights into the many roles of glycoconjugates in biorecognition as ligands for lectins indicates a need to
compare plant and animal lectins. Furthermore, the popularity of plant lectins as laboratory tools for glycan detection and
characterization is an incentive to start this review with a brief introduction to landmarks in the history of lectinology.
Based on carbohydrate recognition by lectins, initially described for concanavalin A in 1936, the chemical nature of the
ABH-blood group system was unraveled, which was a key factor in introducing the term lectin in 1954. How these versatile
probes are produced in plants and how they are swiftly and efficiently purified are outlined, and insights into the diversity
of plant lectin structures are also given. The current status of understanding their functions calls for dividing them into
external activities, such as harmful effects on aggressors, and internal roles, for example in the transport and assembly
of appropriate ligands, or in the targeting of enzymatic activities. As stated above, attention is given to intriguing parallels
in structural/functional aspects of plant and animal lectins as well as to explaining caveats and concerns regarding their
application in crop protection or in tumor therapy by immunomodulation. Integrating the research from these two lectin
superfamilies, the concepts are discussed on the role of information-bearing glycan epitopes and functional consequences
of lectin binding as translation of the sugar code (functional glycomics).
Keywords: affinity chromatography, agglutinin, glycomics, lectin, plant toxin, protein body
Abbreviations: Ara: L-arabinose; ConA: lectin from Canavalia ensiformis; ER: endoplasmic reticulum; Gal: D-galactose;
GalNAc: N-acetyl-D-galactosamine; Glc: D-glucose; GlcNAc: N-acetyl-D-glucosamine; GNA: lectin from Galanthus nivalis
(snowdrop); Fuc: L-fucose; Man: D-mannose; Neu5Ac: N-acetyl-D-neuraminic acid; PHA: lectin(s) from Phaseolus vulgaris (common or French bean); SBA: lectin from Glycine max (soybean); SDS-PAGE: polyacrylamide gel electrophoresis in sodium-dodecyl-sulfate-containing buffer; WGA: lectin from Triticum vulgare (wheat) germ; UEA: lectin from Ulex
europaeus (gorse).

1. Introduction
The ubiquity of glycan chains as integral part of various cell
constituents is reason to envision fundamental functional roles
for these substances. Starting with the monotonous sequence
of cell wall and energy storage polymers chitin, cellulose and
glycogen, structural analysis of the carbohydrate part of glycoconjugates has taught us that monosaccharides as building
blocks for oligomers are better than other compounds (amino
acid or nucleotide) in providing diversity. Calculations of the
To

whom correspondence should be addressed: Harold Rudiger,

Institut fur
Pharmazie und Lebensmittelchemie, Julius-MaximiliansUniversitat,
Am Hubland, 97074 Wurzburg,

Germany. Tel.: +49931-92529; Fax: +49-931-8885462; E-mail: ruediger@mail.uniwuerzburg.de

limits for isomer formation with amino acids and sugars easily
dispel cliches about the role of carbohydrates being limited to
intermediary metabolism. Given 20 letters to form hexamers,
6.4 107 different hexapeptides can be devised. For sugars,
however, 1.44 1015 linear and branched isomers are possible
[1]. Adding a sulfate, phosphate or acyl group, the biochemical
equivalent of Umlaut generation, to a trisaccharide increases
the isomer quantity by one order of magnitude [1]. Since the
oligosaccharides often have only limited conformational flexibility in contrast to peptides, a sugar epitope can present itself
in one or a few distinct structural topologies, adding the third
dimension to structure changes [2,3].
In addition to the unsurpassed structural versatility, two other
arguments further support the concept of the sugar code, i.e.
the elaborate machinery of glycan assembly and modification

590
as well as the spatial accessibility of carbohydrate determinants
especially at the ends of branches [4,5]. If sugars can indeed
be thought of as the hardware for information storage, then decoding by receptors is expected to be commonplace. Directional
hydrogen bonds and stacking/CH- interactions are pivotal factors governing the selectivity and specificity of binding of carbohydrates to proteins [6]. Several classes of proteins share this
ligand capacity, i.e. enzymes acting on carbohydrate substrates
such as glycosyltransferases, glycosidases or sulfotransferases,
sugar-specific immunoglobulins, proteins binding free monoor disaccharides (e.g. transport or chemotaxis proteins), and
lectins, their definition being explained in detail in Section 3
[7]. Using this concept of the sugar code, the widespread occurrence and hitherto mapped diversity of lectins indicate ways in
which this molecular complementarity can be exploited. With
growing insights into lectin structures and functions in plants
and animals, it is now becoming possible to compare different protein classes. In addition, owing to the popularity of plant
lectins as tools in biochemistry, cell biology and medicine, questions arise on their expression, their natural functions and on
simple and inexpensive isolation procedures. Herein we present
an overview of these sugar receptors starting with a glimpse of
the history of plant lectinology followed by a discussion of
lectin occurrence, isolation, biosynthesis, structure, functions
and applications.
2. Historical aspects
The search for the toxic principle in castor beans (Ricinus communis, Euphorbiaceae) prompted one of the most prominent
pharmacologists of his time, Rudolf Kobert (18541918), to ask
his medical student Hermann Stillmark (18601923) to study
this plant. In his thesis of 1888 at the University of Dorpat/Tartu
(Estonia, then one of the Baltic provinces of the Russian Empire), Stillmark described that extracts from castor beans and
four other Euphorbiaceae plants are able to agglutinate blood
cells from different animals, i.e. rabbits, horses, dogs and cats.
He assumed toxicity and agglutinating capability to originate
from the same substance, ricin, which he regarded to be an
enzyme (in those days ferment) [8]. As we now know, castor beans contain a tetrameric protein called Ricinus communis
agglutinin that is able to agglutinate cells but is hardly toxic.
A second, dimeric protein, now called ricin or Ricinus toxin,
is closely related to the agglutinin, and its enzymatic subunit
acts as a highly specific RNA N-glycosidase on 28S rRNA but
is only a weak agglutinin (see Section 8.1 on biological functions). This observation on agglutinating activity resembles that
made by S. Weir Mitchell already in 1860, i.e. the activity of
rattlesnake (Crotalus durissus) venom on pigeons blood [9].
Soon after Stillmarks discovery, ricin and the related toxin
from jequirity beans (Abrus precatorius, Leguminosae) played
a fundamental role as model antigens in the pioneering studies
of Paul Ehrlich [10]. When passing through ricins history and
of note in the era of heightened awareness of biohazard, its

Rudiger and Gabius

extremely potent toxicity even attracted the attention of the
Bulgarian secret service. In 1978, they turned it into a deadly
weapon in the umbrella homicide of the exile-Bulgarian Georgi
Markov who worked for BBC London, prompting infamous
headlines for a lectin [11].
Looking back to the beginning of lectin research, progress
in the field was hampered in the first decades by the crudeness of the fractionation techniques. The first agglutinin to be
isolated was concanavalin A from Jack bean (Canavalia ensiformis) seeds. The prominent American biochemist James B.
Sumner, well-known for demonstrating that an enzyme (urease)
is nothing but a protein (which earned him the Nobel award in
1946), succeeded in purifying also the agglutinating principle
from these seeds by crystallization and called it concanavalin A
(ConA) [12]. He discovered that ConA is able to interact with
red blood cells as well as with starch, glycogen and mucins,
and that this interaction can be prevented by low-molecularweight carbohydrates such as sucrose [13]. This result was the
first clear experimental indication that an agglutinin binds carbohydrates and had significant implications for the study of cell
membrane constituents. Systematic screening of plant extracts
for agglutinating activities led to a breakthrough in haematology and paved the way for coining the term lectin (for a review
of this development, please see [14]).
In fact, Landsteiners discoveries of human isoagglutinins in
1900 in Vienna and the species specificity of plant agglutinins as
well as his comparison of haemagglutinating/haemolyzing activities with natural antibodies led W. C. Boyd to test seeds for
blood group specificity [15]. With extracts of the lima bean
(Phaseolus lunatus limensis, Leguminosae) he found A-type
specificity and proposed the term lectin (from the Latin legere, to choose or to pick out) for these and other antibodylike substances [16]. Drawing on the competitive inhibition of
antibody-antigen reactions by compounds structurally related to
the haptenic group described by Landsteiner and van der Scheer
[17] and the binding of ConA to sugar compounds documented
by Sumner and Howell [13], inhibition of haemagglutination
mediated by eel (Anguilla anguilla) serum and extracts of Lotus tetragonolobus seeds by Fuc and of haemagglutination by
other plant lectins by GalNAc provided first insights into the
chemical nature of the blood group determinants [18]. From this
starting point, the chemical nature of the blood group substances
as oligosaccharides could be delineated. For the reaction of red
blood cells with blood-group-specific lectins, the terminal linked monosaccharides GalNAc (group A), Gal (group B) and
Fuc (group 0(H)) are decisive. It goes without saying that these
studies were further extended thereafter and were crucial for
unravelling the biochemical basis of blood group ABO and
Lewis antigenic specificity [19]. With this focus on haemagglutination it is no surprise that the initial definition of the term
lectin [16] placed special emphasis on just this aspect. As apparent from the example of the homologous lectin subunits of
the Ricinus agglutinin/toxin, however, strict application of the
criterion of haemagglutination would separate related proteins.

Plant lectins
Thus, haemagglutination or precipitation of glycans which depend on at least bivalency are now considered as being only one
example of a broad panel of assays able to detect carbohydrate
binding [5]. Moreover, lectins ought to be distinguished from
other molecules able to clump erythrocytes together.

3. Definition
To qualify as lectin today, a (glyco)protein must meet three
distinct requirements [7,20,21].
3.1. A lectin is a (glyco)protein that binds carbohydrate
By this part of the definition, tannins, certain lipids, cationic
substances and also cognate carbohydrates in carbohydratecarbohydrate interactions that agglutinate cells are excluded.
Since a carbohydrate recognition domain can be linked to other
functional sections in mosaic-like proteins, as encountered especially in animal C-type lectins [21], it is the presence of this
distinct binding activity in a multifunctional protein that justifies to call it a lectin, leaving open the possibility to include the
protein in other categories, too.
3.2. Lectins are separated from immunoglobulins
Originally, lectins were regarded as anti-body-like substances
[16,22]. This term refers to the apparent specificity of binding and was not meant to reflect structural similarity. In fact,
the term lectin was later delimited from immunoglobulins (Ig)
that need an antigenic stimulus to be synthesized. Although
carbohydrate-binding activity is also seen in the family of I-type
animal lectins from the Ig-superfamily [21], all immunoglobulins are thus excluded [7]. Interestingly, lectins are known
that can be induced by an external stimulus distinct from an
antigenic challenge. Plant lectin expression may increase as a
consequence of stress, e.g. virus infection [23], drought [24] or
high salt concentration [25,26].
3.3. Lectins do not biochemically modify the carbohydrates
which they bind
This part of the definition excludes glycosyltransferases, glycosidases and enzymes introducing a substituent like a sulfate
group into the carbohydrate. This addition is necessary, because it is known that certain glycosidases agglutinate cells at
low temperature, if binding to the cell surface carbohydrates
proceeds faster than hydrolysis of glycosidic linkages [27].
Because some plant and also animal lectins can furthermore
harbor enzymatic activities that are independent of the lectins
carbohydrate-binding sites, it is now indispensable to demonstrate already at an early stage of investigation whether activities
can be attributed to the same or more than one center [21,2831].
Lectins are furthermore separated from sensors for free monoor disaccharides acting in chemotaxis or in operon systems and
from transport proteins [7].

591
Having herewith given the criteria for defining a lectin, we
can proceed to survey the occurrence of plant lectins.

4. Occurrence of lectins
As is the case with ricin, Ricinus agglutinin or ConA, the richest source for most lectins are the seeds or, more generally,
the storage organs of plants. These are seeds as in most plants
studied so far, but also roots (Urtica, Phytolacca, Sambucus,
Trichosanthes, Calystegia), tubers or bulbs (Solanum, Galanthus, Scilla, Allium, Crocus, Tulipa, Iris), bark (Sambucus,
Sophora, Robinia, Maackia, Laburnum, Cytisus, Cladrastis,
Hevea, Abies) or leaves (Aloe, Lactuca, Vicia unijuga, Viscum
album) can provide a rich lectin harvest.
Within the cells, lectins are primarily found in protein bodies. They abound in proteins synthesized in the endoplasmic
reticulum (ER) and transported via the Golgi apparatus and
originate by subdividing the vacuole. Viewed from their origin
and their role for protein turnover, protein bodies are related to
lysosomes. The main content of protein bodies are storage proteins (vicilin, legumin and convicilin in Leguminosae, related
proteins in other plants [32]), lectins, hydrolases (glycosidases,
phosphatases) and phytin to store phosphate [33,34]. Proteinbody-like and lectin-containing cell organelles are also present
in non-seed organs such as e.g. bark [35]. In addition to this
major intracellular site lectins have also been found in the cytoplasm [36] and in the intercellular space [37]. The amount
of lectin can vary markedly from one plant species to the other
[38]. To give the reader an idea of lectin quantities found in
seeds, we have compiled data based on purification for various
plants and present them in Table 1 along with the carbohydrate
specificity of each listed lectin. As alternatives to measure lectin
quantities reliably, an immunological assay or tests with neoglycoproteins or neoglycoenzymes [39,40] are routine procedures
of high sensitivity. These techniques verify that purification
yields have reached a satisfying level.

5. Isolation of lectins
Initially, purification of lectins followed the scheme used for
proteins in general without exploiting their special characteristics. The methods used included precipitation by salts, acids
and organic solvents. Of course, the preparations thus obtained
were far from pure, and results obtained with them have to be
interpreted with caution. An exception with celebrity status was
Sumners purification of the abundant ConA by crystallization,
as pointed out above [12]. Progress in this area was achieved
by the introduction of preparative chromatographic methods
with ion exchangers, gel filtration (size exclusion chromatography) media and, above all, affinity adsorbents. Originally developed for isolating enzymes by means of immobilized inhibitors,
the principal strategy was transferred to lectinology, and crosslinked dextrans found versatile application in one-step purification schemes for Glc-binding plant lectins [41]. Agarose, a

592

Rudiger and Gabius

Table 1. Carbohydrate specificity and content of lectin in selected plants (from [38], modified and extended)
Carbohydrate specificity

Plant species

Monosaccharides

Canavalia ensiformis

Man/Glc

Ricinus communis
(Euphorbiaceae)

Gal

Vicia cracca I
Phaseolus vulgaris
Griffonia simplicifolia I

Man/Glc
No binding monosaccharide
known
Gal/GalNAc

Griffonia simplicifolia II

GlcNAc

Glycine max

GalNAc/Gal

Robinia pseudoacacia
Arachis hypogaea

No binding monosaccharide
known
Gal

Sophora japonica

GalNAc

Phaseolus lunatus

GalNAc

Wisteria floribunda/sinensis

GalNAc

Vicia cracca II
Pisum sativum

GalNAc
Man/Glc

Phytolacca americana
(Phytolaccaceae) roots
Dolichos biflorus

GlcNAc

Cytisus scoparius
Euonymus europaeus
(Celastraceae)
Lotus tetragonolobus

GalNAc
No interacting
monosaccharide known
Fuc

Lens culinaris

Man/Glc

Laburnum alpinum

GlcNAc (low affinity)

Triticum vulgare
(Gramineae)

GlcNAc (low affinity)

Caragana arborescens

GalNAc

GalNAc

Glycans
ratio of inhibitory potency compared
with the monosaccharide (italics)

mg lectin/100 g
seeds

GlcNAc2Man6(GlcNAc2Man3)Man4GlcNAc
4200
Gal4GlcNAc2Man6(Gal4GlcNAc2Man3)Man4GlcNAc
50
Unknown
Gal4GlcNAc2Man6(GlcNAc2Man3)(GlcNAc4)Man4GlcNAc
GalNAc3GalNAc3Gal4Gal4Glc
15 (isolectin A-4)
GlcNAc4GlcNAc
3.4
No oligosaccharide known better
than GalNAc
Complex-type N glycan

2100

1400

1400
1200
700
300
300
300

Gal3GalNAc
55
GalNAc6Gal
16
GalNAc3(Fuc2)Gal-R
43
GalNAc6GalNAc
8.8
Unknown
Neu5Ac6Gal4GlcNAc2Man6(Neu5Ac6Gal4GlcNAc2Man3)Man4GlcNAc4(Fuc6)GlcNAcAsn
780
Gal4GlcNAc6Gal
no ratio available
GalNAc3GalNAc3Gal4Gal4Glc
62
Unknown
Gal3(Fuc2)Gal3/4GlcNAc

190

Fuc6GlcNAc
6.5
Neu5Ac6Gal4GlcNAc2Man6(Neu5Ac6Gal4GlcNAc2Man3)Man4GlcNAc4(Fuc6)GlcNAcAsn
6300
GlcNAc4GlcNAc
no ratio available
GlcNAc4GlcNAc4GlcNAc4GlcNAc4GlcNAc
48
GalNAc3GalNAc3Gal4Gal4Glc
9.5

65

170
170
160
150
140

125
110
82
75

60

55
45

35

(Continued on next page)

Plant lectins

593

Table 1. (Continued ).
Carbohydrate specificity

Plant species

Monosaccharides

Vicia faba

Man/Glc

Bauhinia purpurea

GalNAc

Maclura pomifera
(Moraceae)
Ulex europaeus I

GalNAc

Ulex europaeus II

GlcNAc (low affinity)

Fuc

Glycans
ratio of inhibitory potency compared
with the monosaccharide (italics)
Neu5Ac6Gal4GlcNAc2Man6(Neu5Ac6Gal4GlcNAc2Man3)Man4GlcNAc4(Fuc6)GlcNAcAsn
780
Gal3GalNAc
611
Gal3GalNAc
24
Fuc6GlcNAc
4
GlcNAc4GlcNAc4GlcNAc4GlcNAc
2.7 (compared with GlcNAc4GlcNAc)

mg lectin/100 g
seeds
30

28
24
16
9

Unless indicated otherwise, all plants belong to the family of Leguminosae, and the source of the lectins were the seeds.

commercially available natural polysaccharide containing Gal,

was used as an affinity adsorbent for Gal-binding lectins such
as those from Ricinus communis, Bauhinia purpurea, Glycine
max and Wisteria floribunda, and chitin, a polymer of GlcNAc,
for the purification of the lectin II from Griffonia (formerly
Bandeiraea) simplicifolia [42]. The tacit assumption that the
exploited resin has no ligand activity of its own must thus be
reconsidered. When used in gel filtration without haptenic sugar
in the running buffer, a lectin even if its affinity to the matrix is
low will be retarded or even bound, producing erroneous data
on its molecular mass.
Since the range of naturally occurring polysaccharides is restricted and does not cover the broad range of carbohydrate
specificities of all lectins, further development of methods for
immobilizing glycosides, glycopeptides or glycoproteins to any
type of resin followed the use of the unmodified matrix. For this
purpose, cyanogen bromide or divinyl sulfone activation among
various methods have been and are being widely used with high
yields of the product [43]. By selecting suitable glycoproteins
as ligands, it has even become feasible to devise an affinity
matrix presenting a panel of carbohydrate epitopes as potential ligands. Such a matrix can prove its value when screening
extracts for lectin activity in one binding step followed by washing and differential elution. For this purpose, gastric mucin or
ovomucoid were successfully tested as ligands [38]. Since the
reproducibility of purification yields critically depends on the
density of the incorporated ligands, it is essential to determine
the amount that had been bound to a matrix. If the ligand absorbs
UV light as is the case with glycoproteins, the optical density
can readily be measured, background turbidity of the matrix to
be compensated for by running derivative spectra [44].
For eluting a lectin, the obvious method is to exploit the haptenic sugar as competitive inhibitor following the principle of

dissociating antibody-antigen complexes by the soluble hapten

group [17]. As far as monosaccharides are concerned this is generally no problem. Certain lectins, however, bind to oligosaccharides which are not readily accessible or are very expensive.
In such cases lectin elution can be performed by lowering the
pH of the buffer with immediate neutralization of the eluant.
When run as a gradient, it has even been possible to fractionate
isolectin mixtures [45]. Since not all lectins survive exposure
to low pH values, desorption with a borate-containing buffer
can represent a reasonable and affordable alternative. Borate
competes with the lectins carbohydrate-binding sites for immobilized carbohydrate by virtue of its affinity to vicinal hydroxyl groups. Similar to changing the pH value of the buffer,
this procedure also allows to purify isolectins. When the eluant
is applied as a gradient, the five isolectins L4 , L3 E, L2 E2 , LE3
and E4 of the common bean (Phaseolus vulgaris) were separated [46]. Furthermore, subsequent application of Gal and
borate was helpful to resolve from Griffonia simplicifolia seed
extracts the Gal/GalNAc- and GlcNAc-binding species in one
run [38]. After the detection of the lectin by haemagglutination
or neoglycoconjugate binding and its isolation further studies
can then focus on lectin synthesis and processing, structural
aspects and functions.

6. Lectin biosynthesis
6.1. General
The biosynthesis of most plant lectins studied so far proceeds
via the secretory pathway [34]. In brief, the lectin is synthesized
at ribosomes attached to the endoplasmic reticulum (ER), enters the lumen of the ER, and is further transported through the
Golgi apparatus. In contrast to secretory proteins in a narrow

594
sense which move to the cell membrane by vesicular transport
and then leave the cell by exocytosis, lectins (and other proteins as typical storage proteins) end up in the vacuole. During
development of the resting seeds this organelle finally divides
into smaller parts called storage vacuoles or protein bodies. This
routing is disturbed by the presence of monensin, an ionophore.
It intercalates into membranes and allows flow of monovalent
cations including protons, thereby interfering with the intracellular traffic. Monensin effectively impairs the separation of
transport routes to the outer cell membrane and to the vacuolar
membrane, and in turn a lectin will accumulate extracellularly
under these conditions [47].
6.2. Processing
On the way from the site of synthesis to its final destination,
a lectin is subject to a series of covalent modifications that are
common for proteins on this route: the N-terminal signal sequence of about 20 to 30 amino acid responsible for initiating
transmembrane passage is split off cotranslationally as soon as a
sizeable segment of the newly synthesized protein has entered
the lumen of the ER. A further reaction occurring cotranslationally in many lectins is N-glycosylation (O-glycosylation is
found in Solanaceae lectins, please see Section 6.4). Consensus
sequences (Asn-X-Ser/Thr) for N-glycosylation serve as targets for transfer of membrane-bound dolichol-linked oligosaccharides (Glc3 Man9 GlcNAc2 ). They are trimmed in the ER
and can be reglucosylated with one Glc residue as part of the
quality control system to ensure proper folding [48,49]. The
Glc1 Man7-9 GlcNAc2 -binding ER-proteins calnexin and calreticulin prove in this process that lectins can be efficient molecular chaperones. Besides removing N-terminal signals and trimming N-glycans, the protein part can undergo rearrangements
and deletions (please see Sections 6.3.1.3 and 6.3.2). Thus even
lectins that are not decorated by carbohydrate in their mature
state can originate from glycosylated precursors. Modification
reactions continue to take place up to the arrival in the protein
bodies where further proteolysis and in a few cases transpeptidations are carried out.
6.3. Legume lectins
In this respect, legume lectins and their biosynthesis have been
studied thoroughly. This family of lectins sharing extended sequence homology displays an unusual interspecies variability
of sugar target selection (see Table 1). Typically, specificities for
Glc, GlcNAc, Man, but also for Gal, GalNAc, Fuc and complextype oligosaccharides have been detected. Ascribing the capacity to bind sialic acids, a family of monosaccharides not synthesized by plants [50], to certain lectins should be viewed with
caution. So-called sialic-acid-binding plant lectins actually bind
Gal or lactose but do not interact with free Neu5Ac [51]. Their
affinity to Gal or lactose, however, is noticeably enhanced if an
acidic group is in close vicinity. Linking a sialic acid or sulfate
moiety to the Gal unit which occupies the primary binding site

Rudiger and Gabius

provides instructive examples for the supplementary role of the
negatively charged sugar group [52,53]. A notable exception
is wheat germ agglutinin and its binding to both GlcNAc and
Neu5Ac. Evidently, this sialic acid satisfies the stereochemical
requirement determining selectivity for GlcNAc, i.e. presence
of an equatorial N-acetyl group and an adjacent equatorial hydroxyl group [54]. Based on their quaternary structure, legume
lectins are traditionally subdivided into two categories. One
group consists of lectins with identical or nearly identical subunits, while the other category is characterized by different subunit types. A small subgroup of the first category is subject to
the previously mentioned posttranslational transpeptidations.
Though there are borderline cases which question this subdivision, it is maintained in the present article for practical reasons.

6.3.1. Single-chain lectins

6.3.1.1. Phaseolus lectins. A well-studied example of the first
group is given by the lectin fraction from the common or French
bean, Phaseolus vulgaris, its purification and isolectin resolution referred to above [46]. Infamous as toxic ingredients of
insufficiently cooked beans, this lectin fraction can cause maladsorption and irritations in the digestive tract [55]. Another factor contributing to its popularity in research was the abundance
and ease of isolation (ranking it on position four in Table 1)
giving it the status of a role model. Thus, it has been dubbed
phytohaemagglutinin (PHA), a name that strictly speaking
applies to plant lectins in general [56]. PHA consists of two
types of subunits. They are products of tandemly linked genes
with 82% sequence identity on the amino acid level. Being
frequently designated as E- and L-subunits reflects their preferential binding to erythrocytes and leukocytes, respectively.
They are synthesized concomitantly in the ER and assemble
to form tetramers at random, thus giving rise to the known series of the five isolectins mentioned above. As likewise noted,
the release of the N-terminal signal sequence is common to
PHA as for other proteins of the secretory pathway. Even at
the C-terminus, proteolysis may occur but in a comparatively
less precise manner. This leads to the so-called ragged ends
which have been found not only in PHA-E but also in the lectins
from the Leguminosae Erythrina corallodendron, Glycine max,
Arachis hypogaea and Dolichos biflorus and in lectins from
other plant families, e.g. the Gal-binding agglutinin/toxin from
Viscum album. Recombinant lectins, too, have ragged ends but
these can differ from those occurring under natural conditions.
The biological meaning of this type of modification, if not considered to be due to degradative steps after homogenization, is
unclear.
Both PHA subunits contain the characteristic Nglycosylation sequons [48], subunit E at Asn12 , Asn60
and Asn80 , subunit L only at Asn12 and Asn60 . In the mature
proteins, only the first two sites are actually glycosylated. The
glycan at Asn12 belongs to the high-Man type, that at Asn60
to the complex type containing xylose and Fuc in the stem

Plant lectins
region [57]. High-Man type glycans near the N-terminus are
uncommon among animal glycoproteins [51]. To elucidate
whether presence of N-glycans affects further transport,
lectin biosynthesis has been performed in the presence of
tunicamycin, an inhibitor of the first step in N-glycosylation.
The lectins were synthesized in a non-glycosylated form
in the presence of the inhibitor but were processed and
deposited normally [58]. This is in contrast to the conditions
in the Lima bean, Phaseolus lunatus limensis, where, though
belonging to the same genus, the lectin precursor is completely
dependent on its glycans for successful maturation. Inhibition
of N-glycosylation by tunicamycin prevents the lectin from
assembling correctly. Instead, it is retained in the ER and
forms mixed aggregates together with phaseolin, a vicilin-type
storage protein which normally is also glycosylated, and the
chaperone BiP. Notably, processing and transport of another
storage protein, legumin, which is not glycosylated, is not
affected by tunicamycin [59].
6.3.1.2. Glycine max (soybean) lectin (SBA). To illustrate
implications of glycan presence beyond folding and routing,
the delineation of a different function for glycans of the soybean lectin is instructive. The Gal/GalNAc-binding lectin from
Glycine max (soybean agglutinin, SBA) is a glycoprotein with
a high-Man type glycan [60]. Apparently, the glycan moiety is necessary for keeping the subunits together: SBA denatured by guanidinium chloride recombines after dilution.
It fails, however, to do so after removal of the glycan or
in the presence of the competing high-mannose glycopeptide
Man9 GlcNAc2 Asn [61,62]. Using various further methods, the
authors demonstrated conclusively that the non-reducing ends
of the SBA-bound glycans are buried between the subunits
whereas the core regions are exposed [63]. Thus, SBA combines two carbohydrate-binding sites in a single molecule: one
specific for high-Man-type oligosaccharides and directed towards its own core oligosaccharide, the other one specific for
Gal and GalNAc, exposed on the surface and directed towards
external carbohydrates. A report describing the recombinant
expression in Escherichia coli of N-glycan-free but nevertheless active SBA [64] cannot be interpreted unambiguously. As
pointed out by Masaoka et al. [63], the small amount of SBA renatured from the inclusion bodies (0.2 to 0.4%) may have been
stabilized by the inductor isopropyl--D-thiogalactopyranoside
similarly to the known effect of GalNAc on chemically deglycosylated SBA.
6.3.1.3. Concanavalin A (ConA). Concanavalin A (ConA),
the lectin from the Jack bean which had initially been purified
in 1919 by crystallization [12], is one of the most abundant
lectins known (Table 1). Analysis of its synthesis and processing was rendered comparatively unproblematic by this strong
expression. When its amino acid sequence was compared with
that of other legume lectins, a high degree of homology was
found. Surprisingly, alignments to achieve optimal homology

595
arranged the polypeptide chains in such a way that the N- and
C-termini of ConA face certain residues in the middle of the
chains of other lectins and vice versa [65]. The reason for this
seemingly strange phenomenon called circular permutation remained unclear for several years. In 1985, it was found that the
ConA precursor displays the normal sequence, and that the
circular permutation is due to an up to that time unprecedented
posttranslational event in plants, in which the peptide chain is
split at one site and annealed at another one [66]. Though known
from the biosynthesis of bacterial murein, transpeptidations had
never been observed in higher organisms until then. Even up to
now the detection of similar events has remained exceptional
in eukaryotes [67], although they are quite common among
bacteria [68]. Canavalia ensiformis belongs to the Diocleinae
subtribe of the Leguminosae. Presumably, the lectins from other
members of this group (Canavalia maritima, C. gladiata, Dioclea grandiflora, D. lehmanni, D. guianensis, D. floribunda)
with amino acids sequences similar to ConA undergo a similar
processing step [69].
In addition to this rearrangement of peptide stretches, ConA
processing involves an intriguing aspect with respect to glycosylation, as indicated at the end of Section 6.2. Mature ConA is
not glycosylated. Its precursor pro-ConA, however, contains a
high-Man-type glycan but does not bind carbohydrate. The glycan is localized on a segment of 15 amino acid residues which
is lost during maturation [70]. A further peptide segment is concomitantly deleted and the new peptide bond closed, completing a process that takes place in the developing protein bodies
[71]. Presumably, this process (like protein splicing) does not
require significant refolding because the residues participating
are in close proximity already on the level of the precursor.
It is very likely that transpeptidation is an enzymatically catalyzed process. If the cDNA coding for pre-pro-ConA is expressed in Escherichia coli, the product pre-pro-ConA is not
processed at all [72]. This result intimates that not yet known
factors in the plant are responsible for ligation. A more direct
hint to mechanistic details comes from experiments in which
pro-ConA isolated from immature Canavalia seeds was subjected to the action of either an extract of immature cotyledons
or a commercial asparaginyl endopeptidase [73]. In each case,
the product was mature ConA with its full-length chain of 30
kDa together with the so-called fragments of 16.2 and 14.2 kDa
which actually arise from incomplete processing. Asparaginespecific vacuolar processing enzymes are not uncommon in the
plant kingdom [74].
The fact that the biosynthesis of ConA is severely distorted by
tunicamycin underscores that transient presence of the glycan
on the level of the precursor is not without functional implications. In the presence of this inhibitor, most of the unglycosylated pro-ConA is retained in the ER, and the small fraction
that is transported is processed very slowly. In contrast, the
storage protein canavalin which is unglycosylated under normal circumstances is transported in the common way even in the
presence of tunicamycin [71]. The differences in the influence

596
of tunicamycin on processing and routing of PHA, Lima bean
lectin and ConA reveal that it is hardly possible to draw general
conclusions from a limited number of investigations, and that
each case has to be studied separately.
6.3.2. Two-chain lectins
As already alluded to above, legume lectins, in particular
those from the subtribe Vicieae, can be composed of two nonhomologous types of subunit and therefore are called two-chain
lectins, in contrast to the single-chain lectins as PHA, ConA or
SBA. Most lectins from the various Vicia species, from Pisum,
Lens and Lathyrus belong to this group. Following the molecular size order in SDS-PAGE, the smaller one is designated
as -, the larger one as -subunit. As seen for other lectins,
biosynthesis starts with a signal sequence which is not part of
the mature protein, then the - and finally the -subunits follow. This order of sequence has been substantiated in several
cases and is backed up by the occurrence of small amounts
of a /-precursor in pea lectin preparations and by a comparison between the amino acid sequences of two-chain- and
single-chain lectins (for a review see [75]). The final proteolytic
cleavage of the chains occurs in the protein bodies, together with
C-terminal processing which can produce isoforms [76]. The
strict distinction between single-chain and two-chain lectins is
more or less fortuitous. The genus Lathyrus belongs to the Vicieae tribe, and monitored plant species predominantly synthesize two-chain lectins with the exception of Lathyrus nissolia
which produces a single-chain lectin. If the L. nissolia lectin
is incubated with a lectin-free extract of immature L. ochrus
seeds at an acidic pH as it prevails in developing protein bodies, the L. nissolia lectin is split into two dissimilar subunits
as is common among other Vicieae lectins [77]. Apparently,
it is not the lectin per se which determines to which group it
belongs but rather the presence or absence of an appropriate
endopeptidase. Since the -subunits of two-chain lectins bear
asparagine residues at their C-termini, such an enzyme is likely
an asparaginyl endopeptidase [74]. The second position of the
-chains N-terminus is threonine. It thus appears that the endopeptidase cleaves the protein right in the middle of a potential
N-glycosylation site. It is neither known whether this site has
been transiently glycosylated nor whether a glycan if transferred
at this position assists in cleavage. It is interesting to note that
the site in ConA where transpeptidation occurs is Asn118 -Ser119 Thr120 , a potential glycosylation sequon [73]. Taking a look at
those positions which correspond to the cleavage sites of twochain lectins, substitutions to residues other than asparagine
modify this site in most one-chain lectins such as SBA or
PHA, another argument in favor of the assumed endopeptidase
specificity.
6.4. Solanaceae lectins
While biochemical properties and carbohydrate binding of
Solanaceae lectins are well investigated, their biosynthetic route

Rudiger and Gabius

has not been studied in detail. These lectins form a group
by themselves and are not related to legume lectins. All of
them bind GlcNAc and its oligomers. The best studied member
of this group is the lectin from potato tubers. Its biosynthesis starts very early during tuber development, and the lectin
is abundantly present in the epidermis [78]. It is a glycoprotein, but in contrast to the well-studied legume lectins which if
they are glycoproteins bear N-linked glycans, the highly glycosylated (52.3%) potato lectin contains O-glycans linked to
hydroxyproline (Ara) and serine (Gal) residues [79]. This is
a very uncommon structure among plant lectins, whereas it
is quite frequently encountered in typical plant cell wall proteins [80]. The observation that potato lectin was detected, at
least in part, also in the cell wall intimated to count this lectin
to the family of cell wall glycoproteins [81]. More recently,
however, the lectin was reported to reside in the cytoplasm
although in close proximity to the inner wall surface. Upon
wounding the tuber, the synthesis of a new chitin-binding protein is induced which despite being related to the lectin is the
product of a different gene [82]. As a bona fide carbohydratebinding protein, it is justified to call this new protein a lectin,
too. One might note that its expression enhanced by a lesion
is slightly evocative of immune reactions. The upregulation of
lectin expression after virus infection has already been cited
earlier [23].
6.5. Euphorbiaceae lectins
Of the Euphorbiaceae lectins, the toxic ricin is studied most
intensively [83]. Similar to other plant lectins, pre-pro-ricins
sequence starts with a 24-residues N-terminal targeting signal
followed by the toxic A-chain, then a peptide of 12 residues
which does not appear in the mature lectin follows linking the
Gal-binding B-chain with the A-chain [84]. The precursor of
the Ricinus agglutinin has an almost identical sequence [85]
with only a few amino acid exchanges. Nevertheless, these will
account for the already given differences of both proteins in
subunit aggregation as di- or tetramers and in biological activities [83]. Biosynthesis starts at the ER, the precursors are
glycosylated and transported to the protein bodies where they
are cleaved to yield the subunits [86,87]. As documented recently, the presence of the linker peptide is essential for correct
targeting to the vacuole and the protein bodies [88]. Apparently, N-glycosylation is not necessary, because presence of tunicamycin does not impair the transport of the unglycosylated
proteins [89]. This is similar to the events in the processing of
the Phaseolus vulgaris lectins [58]. A pertinent question concerns the way how the plant protects itself against the toxic
principle. Ricin is a potent toxin not only to animals but, albeit
to a lesser extent, also to plants including castor beans if tested in
cell-free systems or if applied exogenously [90]. Since mature
and active ricin is generated exclusively in the protein bodies
and thus does not show up in the cytoplasm, this strict compartmentalization apparently prevents the Ricinus toxin from being
a horror autotoxicus.

Plant lectins
6.6. Lectins from monocot plants
From the monocot plants, special emphasis has been placed
on cereals, in particular on wheat. The lectin from wheat, usually designated as wheat germ agglutinin (WGA), is a rather
small and heat-resistant protein established by hevein domains
(please see Section 7.2). It interacts with GlcNAc (and also
Neu5Ac, see in Section 6.3), preferentially with its 1 4linked oligomers, the constituents of the polysaccharide chitin.
By the way, binding to the natural product chitin offers a clue for
defining natural target molecules (please see Section 8.1.2 on
biological functions). WGA is synthesized in the ER as a glycosylated precursor of 23 kDa, in the presence of tunicamycin
of only 20 kDa. Upon maturation, the glycan is lost together
with a C-terminal segment [91] and subunits of 18 kDa result
which assemble to a 36 kDa dimer. Structural aspects of this
lectin will be continued to be discussed in the Section 7.2 on
lectin structure. The lectin is present in the embryo where it
is deposited in storage vacuoles preferentially at the periphery
[92], whereas typical storage organs as the endosperm, scutellum and aleuron layer are devoid of it. On germination, de
novo synthesis starts in the roots where it is localized at the tip.
The synthesis of WGA, as already mentioned in Section 3, is
enhanced under stress conditions as drought [24] or osmotic
strain [25,26]. Rice (Oryza sativa), a further important cereal,
contains a similar lectin. While still in the ER, it displays a
Mr of 23 kDa in SDS-PAGE but is then processed to first
yield an 18 kDa chain which finally is split into 8 kDa and 10
kDa subunits forming the mature lectin [93]. In rice, a chitinbinding endosperm-specific lectin which is similar to but not
identical with the embryo-specific lectin has also been found
[94,95].
In the search for alternatives to chemical pesticides in pest
control, the insecticidal action of the snowdrop (Galanthus nivalis) bulb lectin GNA [96] has prompted numerous studies on
its properties, biosynthesis and application. At the monosaccharide level, this lectin binds Man but in contrast to Man-binding
legume lectins such as ConA it does not accommodate Glc
into its carbohydrate-binding site. Binding to microvilli in the
tomato moth larvae is instrumental for the transport into cells of
the gut and malphigian tubules [97]. The mature lectin consists
of 13 kDa subunits which combine to tetramers [98]. Its biosynthesis was followed in detail in developing ovaries. Comparable
to many other lectins, the pre-pro-lectins sequence starts with
a signal stretch of 23 residues and contains a C-terminal extension of 29 residues. Expectedly, biosynthetic processing takes
place in the ER and the final deposition site are the storage
vacuoles [99].

597
has been gathered that there are exceptions to this rule, as already indicated above. Nearly two decades ago, two different
yet related lectins from the Leguminosa Dolichos biflorus were
detectable at different sites. Whereas the classical seed lectin
is deposited in the protein bodies, the second lectin which is
found in stem and leaves resides at the inner periphery of the
cells in loose association with the cell wall [37], a characteristic
similar to that of the potato agglutinin [81]. More recently, a
lectin was isolated from rhizomes of the Convolvulacea hedge
bindweed (Calystegia sepium) that binds to Man [100]. When
its cDNA sequence was determined, it turned out that the mature
lectins protein sequence corresponds to the open reading frame
of the cDNA and that it is neither preceded by a signal sequence
nor undergoes proteolytic processing [101]. The lack of a signal sequence suggested a localization different from most other
lectins. Therefore, the intracellular localization was probed in
Calystega rhizomes by immunofluorescence. Indeed, the vacuoles which constitute most of the cell volume are entirely free
of lectin whereas the thin cytoplasmic layer between the vacuole and cell membrane/wall is stained by the antibody [36].
Deduced from its amino acid sequence, the Calystegia lectin is
related to jacalin, a lectin from the Moracea Artocarpus integrifolia. This plant is taxonomically distinct from Calystegia.
Jacalin, however, binds Gal rather than Man and is processed
and deposited in the normal way, i.e. in protein bodies [36].
Thus, jacalin and the Calystegia lectin, though possessing homologous amino acid sequences, display different carbohydrate
specificities and are localized in different intracellular compartments, a phenomenon that is also known from C-type lectins
of animals binding to Man/GlcNAc or Gal [21]. Further monitoring of lectin properties disclosed that the Calystegia lectin
belongs to an extended family. Lectins with similar carbohydrate specificities and similar jacalin-related amino acid sequences have been found in several plants that are taxonomically far apart such as those in the Asteracea Jerusalem artichoke (Helianthus tuberosus) [102] and in the Musacea banana
(Musa acuminata) [103]. The occurrence of a similar lectin in
salt-stressed rice plants is of particular interest due to possible economic implications [104,105]. Regarding the situation
in animals, it is notable that intrafamily diversity can lead to
expression of various family members in the same cell and
to a complex network of functional additivity/synergism or
divergence that is beginning to be analyzed, for example for
galectins [106,107]. Interestingly, this animal lectin family has
a folding profile also seen in leguminous agglutinins (please
see Section 7.1 and Figure 1).

7. Lectin structures
6.7. Uncommon locations
Up to this point, the take-home message apparently emerges
that most plant lectins travel intracellularly along the secretory
pathway after synthesis in the ER with final destination in protein bodies or storage vacuoles. Growing evidence, however,

So far, sequences have primarily been considered to infer evolutionary relationships and to reflect processing routes. Access
to sequences is also an essential step on the way to solve folding
patterns of plant lectins and their modes how to accommodate
carbohydrate ligands in their binding pockets.

598

Rudiger and Gabius

Figure 1. Appearance of a common folding pattern in plant and animal lectins. Illustration of the versatile jelly-roll motif with its
antiparallel -strand arrangement in a pentraxin (1SAC) with its bound Ca2+ -ions and in a legume lectin (1AXY). While sharing the
general folding pattern the binding sites for the ligand in the pentraxin (SAP), plant lectin (Erythrina corallodendron) and galectin
(CG-16, from [116]) are at different places, shown by arrows (kindly provided by A. Romero, Madrid).

7.1. Legume lectins

As other fields of lectin research, structural investigations began
with the lectin from the Jack bean Canavalia ensiformis, and
the method used was x-ray crystallography [108]. ConA turned
out to be mainly built up of -sheets that are connected by loops
whereas -helical elements are almost absent. In the following
years, this folding pattern was likewise seen for various other
legume lectins by x-ray crystallography, circular dichroism and
calculation of hydropathic profiles from amino acid sequences.
Over the years, however, x-ray crystallography proved to be the
method of choice to map plant lectin structure. Despite their diversity in carbohydrate-binding specificity, the folding patterns
of subunits of legume lectins share secondary and tertiary structures to the extent that they are superimposable [109113]. They
consist of a flat six-stranded back sheet, a curved seven-stranded
front sheet and a smaller five-stranded sheet (S-sheet) that holds
the two larger sheets together. The structures resemble flattened
bell-shaped domes containing a shallow pocket at their apex
which forms the carbohydrate-binding site. The bottom of the
pocket contains binding sites for bivalent metal ions. If demetallized ConA requires to be reconstituted, binding of manganese
has to precede that of calcium ions. The presence of these ions
is essential for correct folding and internal arrangements of the
carbohydrate-binding site to reduce entropic expenses by restricting mobility for the orchestrated ligand contact. The metal

ions do not participate directly in the interaction with the ligand as is the case in C-type animal lectins. In addition to
the metal-ion- and carbohydrate-binding sites, many legume
lectins harbor binding sites for hydrophobic molecules [75].
When the overall folding pattern of legume lectins is run through
computer searches to pick up similarities, the jelly-roll motif
is seen (or predictable) for galectins and serum pentraxins as
shown in Figure 1, also for some bacterial glycohydrolases
(e.g. Bacillus 1,3-1,4--glucanase) and for the intracellular
lectin ERGIC-53 (endoplasmic reticulum-Golgi-intermediatecompartment protein at 53 kDa) and calculated for VIP-36
(vesicular integral membrane protein at 36 kDa), with ligandand also metal-ion-binding sites (if present) at variable positions
[21,114,115].
As a special feature of ConA, binding of Ca2+ induces an isomerization of the non-proline Ala207 -Asp208 peptide bond from
the trans to the cis conformation allowing Asp208 to flip into
its carbohydrate-binding position [116]. Though not studied
as detailed as ConA, the subunits of lectins from the Leguminosae Vicia faba, Pisum sativum, Lathyrus ochrus, Erythrina
corallodendron and Griffonia simplicifolia (lectin IV) adopt an
identical structural fold with only minor changes. The question arose as to whether this structure will invariably lead to
carbohydrate binding or whether changes in the actual binding
domain can be tolerated. In the course of extensive structural

Plant lectins

599

work it was discovered that non-lectin proteins occurring in

Phaseolus species, namely arcelin and the -amylase inhibitor,
resemble the Phaseolus lectin in their primary, secondary and
tertiary structures [117,118]. Thus, legume lectins apparently
belong to a larger protein family that includes also proteins that
lack contact sites to bind carbohydrate, a situation similarly
seen for C-type animal lectins and related proteins with C-type
lectin-like domains without carbohydrate-binding capacity [5].
On the other hand, lectins from other plant families though sharing with legume lectins their ability to bind carbohydrate are
definitely not structurally related to them [110,111].
In contrast to similarities of folding patterns of the subunits,
remarkable diversity is observed in the mode of association of
subunits. Table 2 gives a brief overview of known dimeric structures. Within the class of legume lectins, fully active species can
be either dimers or tetramers resulting from dimer association,
Table 3 giving an insight into different ways of dimer assembly
governed by protein-protein interactions. Among plant lectins,
the diversity of the legume lectins in carbohydrate specificities

is unique. As structural studies show, conserved amino acid

residues are responsible for the affinity to a given monosaccharide whereas the specificity is determined by variable lengths
of loops [109]. When oligosaccharides or glycosides with hydrophobic aglyconic moieties are bound, additional residues in
the vicinity of the primary carbohydrate-binding site participate
to accommodate the ligands noncarbohydrate tail. Concerning
lectins from non-legume plants, general features for comparison have also been elucidated and will be outlined in the next
sections.
7.2. Lectins from other plants
WGA, the wheat germ agglutinin, has already been introduced.
It belongs to the superfamily of chitin-binding proteins including not only lectins but also certain enzymes (chitinases).
Hevein from the rubber tree (Hevea brasiliensis) with its sequence of 43 amino acids is the prototype of this protein
class [119]. WGA is composed of two 18 kDa subunits each

Table 2. Association of legume lectin subunits

Type

Description

Occurrence

Canonical

Side by side association of the back sheets which

leads to a continuous arrangement of twelve
-strands.
Back sheets are packed face to face with the
-strands running perpendicularly to each other.
The interface between both subunits consists mainly
of the side chain of the upper -strands of the
back sheets.
The subunits associate with their flat back sheets
facing each other while the side chains intercalate.
The C-terminal peptide of one subunit is positioned
in the central cavity. This peptide is not present in
the other subunit.

ConA, lectins from Pisum sativum, Vicia faba, Lathyrus

ochrus (-amylase inhibitor from Phaseolus vulgaris)

GS IV type
EcorL type

DB58 type

Lectin IV from Griffonia simplicifolia

Lectins from Erythrina corallodendron and Psophocarpus
tetragonolobus
Leaf and stem lectin from Dolichos biflorus

Table 3. Assembly of legume lectin dimers to tetramers

Type

Description

Occurrence

ConA type

Two canonical dimers are packed against each other

with the central part of their continuous twelve
strand -sheets turned around by 90 .
An association of two GS-IV dimers where a subunit
of the first dimer combines with a subunit of the
second dimer in a canonical way.
The tetramer can be regarded either as a dimer of two
canonical dimers or alternatively as a dimer of two
DB58 dimers, depending on which subunits are
combined to form dimers.
Interaction of the monomers by comparatively small
buried interfaces provided mainly by nonpolar
residues.

ConA

PNA type

PHA-L type

GS I-B4 type

Lectin from Arachis hypogaea

Lectins L4 from Phaseolus vulgaris, B4 from Vicia

villosa, II from Ulex europaeus, from Dolichos
biflorus and Glycine max
Lectin I-B4 (demetallized) from Griffonia simplicifolia
[115]

600
consisting of four independently folded and helically assembled hevein-like domains held together by four disulfide bridges
lending stability to the molecule. Its four carbohydrate-binding
sites for GlcNAc (or Neu5Ac) are located at the interface between the subunits [54]. Besides crystallographic analyses, the
hevein domains and their binding of sugar have been studied
in solution by NMR spectroscopy using proton-proton distance
constraints for hevein, pseudohevein and the B domain of WGA
and explaining the multivalent chitin binding of these defense
proteins in structural terms [120, 121 and references therein].
Positioning of the disulfide bridges and key aromatic residues,
which are crucial for ligand contact in plant hevein domains
[122] and shown in Figure 2, as well as superimposable folds
give reason to attribute the origin of chitin-binding lectins in
invertebrates to convergent evolution [123]. As noted above in
the section on biosynthesis, another target, i.e. Man, can be implicated in defense by a lectin leading to the discussion of the
structure of GNA.
Several Man-binding lectins from monocot species (Amaryllidacae, Liliaceae, Alliaceae, Orchidaceae, Araceae) have been
studied. Their main representative is the snowdrop lectin GNA

Figure 2. Depiction of the positioning of amino acid residues

in a binding site of a plant lectin. Illustration of the structure of
the hevein-(GlcNAc)2 complex as obtained by molecular dynamics simulations, kindly provided by M. Frank, H.-C. Siebert and
C.-W. von der Lieth (Heidelberg/Munich). The contact between
the ligand and key aromatic residues is readily visible.

Rudiger and Gabius

(Galanthus nivalis), whose insecticidal action has been referred
to above. GNA (50 kDa) is a tetramer constituted by four 13
kDa subunits. Each subunit consists of three subdomains forming a twelve-stranded -barrel structure. Each subdomain is
formed by a bundle of four antiparallel strands of -sheets connected by loops. The subunits associate to form a crownshaped
tetramer. The twelve Man-binding sites are provided by clefts
on the surface of each subdomain [124].
An instructive example for the adaptability of a folding pattern is given by the -prism fold. Jacalin from Jackfruit (Artocarpus integrifolia, Moraceae) is a lectin that binds preferentially to the oncofetal TF-antigen Gal 3GalNAc. The
subunits consist of an unusually small (24 residues) -chain and
a larger (133 residues) -chain which are derived from a precursor by posttranslational processing. Together both chains form
the subunits which consist of three bundles of four -sheets
strands arranged into the -prism structure [125]. Subunits associate to dimers and tetramers. The carbohydrate-binding site
involves the N-terminus (Gly) of the -chain which is generated by processing [110,125]. In contrast to most other lectins,
jacalin has a rather broad specificity interacting also with Man
and Glc. A study making use of of surface plasmon resonance,
x-ray crystallography and molecular modeling revealed that the
unusually broad specificity can be attributed to a carbohydratebinding cleft that is much more extended than in comparable
lectins [126]. A similar architecture has been found for the lectin
from the osage orange (Maclura pomifera), another Moracea
[127]. Interestingly, this folding pattern is shared by lectins
binding other sugars (e.g. Man) from other plants, for example the Asteracea Helianthus tuberosus [128]. As already noted
above, a basic fold can be adapted to various targets by sitespecific sequence alterations.
Amaranthus caudatus and other Amaranthaceae species contain lectins that similarly to jacalin blind the TF antigen Gal
3GalNAc. The lectins subunits of 34 kDa combine to homodimers of 64.5 kDa. Their subunits are oval in shape and composed of an N-terminal and a C-terminal domain connected
by a short helical segment. Each domain forms a -trefoil
fold established by six strands of antiparallel -sheets which
form a -barrel. Both subunits assemble to dimers in a headto-tail fashion, and the carbohydrate-binding sites are formed
by two shallow invaginations at the interface between the subunits [110]. For ricin, subunit assembly by a disulfide bridge
brings the Gal-binding B-chain and the toxic A-chain together
[83]. The lectin parts structurelike that of hevein-domaincontaining lectinsis another example of the origin of domains
by gene duplication with two homologous domains forming
the lectin, each containing three homologous 40-residue folding units which pack around a pseudo-threefold axis [129].
The outlined way how the plant protects itself from the toxic
action of ricin (please see Section 6.5 on lectin biosynthesis)
and the cooperation of the two subunits to exert the potent
toxic activity render it likely to assume that ricin can be considered as protection against animal predators. This comment

Plant lectins
opens the discussion on the functional significance of plant
lectins.
8. Biological functions
A central question which has often been asked but up to now
not yet been answered definitively is that on the biological function(s) of plant lectins. Not surprisingly, reviews have dealt with
this issue with increasing level of experimental input over the
last two decades [110,130134]. Similar to the situation in animals [21,135], there are evidently diverse families of lectins
and intrafamily divergence adapted to distinct roles, to be elucidated case by case. Even for lectins with homologous sequences
as those from the Leguminosae (or the galectins in animals), a
common function cannot be readily ascribed to them because
individual parameters such as carbohydrate specificities, location and time of appearance differ. Therefore, every description
of a function requires clear-cut evidence which cannot be substituted by analogy considerations. The main lines of current
ideas in this area are divided into two groups: one in which a
lectin is assumed to bind ligands from foreign sources such
as animal, fungal or bacterial aggressors or symbionts, and
another one in which a lectin interacts with ligands from the
plant.
8.1. External activities
It is reasonable to regard toxic lectins such as those from Ricinus
communis and from Phaseolus vulgaris as protectants against
animal predators. Ricin toxicity is brought about by an elaborate
transport mechanism akin to that of bacterial AB5 toxins: the
lectin subunit binds to the cell surface and makes the molecule
penetrate the cell membrane. The imported protein then moves
on a retrograde migration route along the exocytic pathway
of eukaryotic cells and finally arrives at the ribosomes. Here it
turns on its deadly enzymatic activity by splitting off an exposed
and unpaired adenine at position 4324 from the 28S rRNA (rat)
which is essential for binding of elongation factors and thus for
protein synthesis in general [136]. This mechanism of action is
shared by several other plant proteins, generally combined by
the designation ribosome-inactivating proteins class II (RIP
II). Whether other presumed enzymatic activities, specifically
nucleolytic capacity, might contribute to the final consequence,
will have to be clarified with preparations that are guaranteed
free of contaminations [137]. The mechanism by which ricin
acts on cells fully justifies Stillmarks original statement of ricin
as being a highly toxic enzyme (=ferment), referred to in Section 2 on historical aspects. Agglutination of red blood cells by
Ricinus extracts, however, is brought about by its accompanying
agglutinin.
The following description is intended to accentuate lectin
interactions with insects. Due to their interaction with the digestive tract, the lectins from Phaseolus vulgaris (PHA) are
toxic for mammals in general [55]. The same does not hold
true for insects. Initially, it was assumed that also the cowpea

601
weevil (Callosobruchus maculatus) is harmed by feeding a
PHA-containing diet but later it was found that sufficiently purified PHA is actually nontoxic to this insect [138], and that the
active compound of Phaseolus vulgaris beans that is toxic to the
cowpea weevil is the -amylase inhibitor [139]. On the other
hand, PHA, in particular the isolectin E4 , is apparently toxic
to the potato leafhopper (Empoaca fabae) [140]. As shown by
light and electron microscopy, PHA-E4 binds to the midgut
epithelial cells and leads to severe disorganization and finally
to occlusion of the lumen [141]. Whereas PHA is not active
against the cowpea weevil, the wheat germ lectin (WGA) is
[138]. Also, the GlcNAc-specific lectin II from Griffonia simplicifolia (GSA-II) inhibits growth of the cowpea weevil [142]
by virtue of its carbohydrate-binding site [143]. WGA, though
also binding GlcNAc, does not harm the potato leafhopper except at high doses [144]. Anti-nutritive effects of WGA in humans are not known. With 450 mg lectin per kg present in
wheat germ (Table 1) the natural concentration is more than
an order of magnitude lower than the effective dosage for insects. Designed to mimic a diet under these conditions it is
disconcerting to read that rats fed on a fully-balanced semisynthetic diet with 93 g lactalbumin/kg and 7 g WGA/kg for 10
days showed reduced utilization of dietary proteins and growth
[145]. These selected and representative examples demonstrate
that interactions between plant lectins and insects are quite specific and can neither be predicted nor generalized. They also intimate that genetic manipulation to obtain transgenic crop plants
rich in insecticidal lectin may not be without consequence for
humans.
8.1.1. Protection from insects; transgenic plants
During the last decade, feeding trials to assess the insecticidal action of lectins as potential biological pesticide have focused on the Man-binding lectin GNA, as already mentioned.
This lectin has a rather broad insecticidal activity. After initial experiments in which various insects were fed with artificial diets supplemented with GNA, transgenic plants containing the GNA gene were increasingly used. Transgenic plants
include potato [96,146], rice [147149] and wheat [150]. Insects affected are aphids [96,150,151], moths [146,152] and
plant hoppers [147,148]. In this context it is essential to underscore that the implications of lectin presence in an ecosystem is not confined to the desired effect in crop protection. In
a tritrophic study in which ladybirds fed on aphids that had
been sucking on transgenic GNA-containing potato plants, adverse effects on the development of the coleopterous predators were observed [151]. In related studies where the moth
Lacanobia oleracea reared on GNA-potatoes was exposed to
the wasp Eulophus pennicornis, no significant [152] or even a
beneficial effect [153] on the wasp progeny occurred, demonstrating that long-term effects in complex systems will have
to be evaluated very carefully and patiently prior to rushing to claims for safe and efficient application. A similar
reasoning will be presented in Section 9.2 concerning the

602
immunomodulatory effects of lectins and claims for clinical
benefit.
8.1.2. Protection from fungi
With the insecticidal action of plant lectins the spectrum of external functions is certainly not yet completely covered [154].
Binding to cell wall constituents in fungi can interfere with their
growth. Fungal cell walls contain chitin, the 1 4 linked
polymer of GlcNAc. As already suggested in Section 6.6 when
discussing sugar binding by WGA, it is, therefore, likely that a
fungicidal action is exerted by GlcNAc-binding lectins. In fact,
this is the case for several lectins in vitro. In order to unequivocally demonstrate the fungicidal action of a lectin, however, it
is important to avoid the use of lectins that are contaminated by
other fungicidal proteins such as for example chitinases [132].
8.1.3. Symbiosis with bacteria
Plant lectins are not only assumed to be part of the defense
system. Regarding bacteria and animal lectins, this aspect prevails with serum and surfactant collectins and also ficolins as
molecular guards against infection [21,155]. In the case of plant
lectins, however, their interaction with cell surface compounds
is also considered to initiate new and desired contacts. Several
plants, in particular Leguminosae, are known for their ability
to establish a symbiosis with soil bacteria of the genus Rhizobium and related genera which are able to fix atmospheric
nitrogen, rendering plants independent of supply of external
nitrogen fertilizer. This symbiosis is species specific: a given
Rhizobium strain will nodulate only a single legume species or
at best a limited number. It is thus suggestive, together with
the widespread and abundant occurrence of lectins in the seeds
of Leguminosae, that lectins might participate in establishing
the symbiosis between plant roots and rhizobial bacteria. Initial
studies appeared to support this hypothesis but were questioned
by further scrutiny [130]. These experiments were performed
with seed lectins. Naturally, they will not contact soil bacteria
in vivo.
Recent studies focused on root lectins, some of them being
similar to seed lectins. Indeed, it could be shown that by transferring the pea lectin gene to white clover roots, lectin expression enables them to host also pea-specific bacteria [156], and
that this effect requires an intact sugar-binding site of the lectin
[157]. This topic has been dealt with extensively in several reviews [133,158160]. A new aspect emerged with the detection
of the nodulation (Nod) factors, i.e. lipochitooligosaccharides
that are produced by the bacteria as a response to plant-derived
stimuli (flavonoids). That a lectin is part of the communication
network involved in nodulation is substantiated by the trnsformation of clover roots with the pea lectin gene [156,157]. This
manipulation broadens the specificity of the roots to various
Nod factors enabling them to respond with enhanced cortical
cell growth [161]. Presumably, this is an indirect effect since the
pea lectin, being specific for -linked Man or Glc residues will

Rudiger and Gabius

not bind to -linked GlcNAc residues which form the backbone
of Nod factors [162]. Work is currently in progress to identify
the receptors of Nod factors which by definition are lectins.
From roots of alfalfa (Medicago sativa) plants, proteins were
isolated by affinity chromatography on immobilized Nod factor
analogues (GlcNAc or its trimer) which interact with authentic
Nod factors in vitro [163]. From horse gram (Dolichos biflorus)
roots, a lectin known for many years and originally described to
bind GalNAc similar to the seed lectin of the same plant [164]
was recently found to weakly interact with GlcNAc, better with
chitooligomers and best with authentic Nod factors [29]. Since
the phosphatase activity, a further novel property of this lectin,
was enhanced in the presence of Nod factors, this protein might
function as a signal transducer [29]. With this functional connection, evocative of the signal-triggering properties of animal
lectins [165], the separation between external and internal functions becomes blurred. Interestingly, genome analysis of Arabidopsis thaliana has uncovered a further trait of animal lectins
in a plant gene, i.e. association of a legume-lectin-like extracellular domain with a receptor-like serine/threonine kinase motif
(Ath.lecRK-al-a4) [166].
8.2. Internal activities
8.2.1. Interaction with storage proteins
This leads our considerations to a further set of hypotheses.
According to them lectins act within the plant. A rather passive
function as storage proteins is self-evident, because seed lectins
are generally degraded during germination. This proposal, however, does not yet satisfactorily answer the question why they
possess binding sites for carbohydrates and other substances.
Clues to address this issue could be gained by characterizing
the nature of lectin ligands, in the case of animal lectins for
example matrix glycoproteins to aid cell attachment [21,167].
When pea or lentil seed extracts were passed over a column
with an affinity adsorbent containing (pea, lentil) lectin, two
fractions of lectin-binding material were obtained, one which
is bound by ionic interaction and the other one which binds via
the lectins carbohydrate-binding site. Electrophoretic analysis
revealed that the lectin-binding material belongs to the storage
proteins, the first fraction being a mixture of legumin and nonglycosylated vicilin, the second one consisting of glycosylated
vicilin [168171], a result corroborated independently [172].
Differences in affinity of the lectin-binding fractions towards
the lectin as compared to the bulk storage proteins are also reflected by physicochemical (calorimetry [173], nephelometry
[170]) and immunochemical [174,175] properties. How these
disparities translate into differences in routing or packaging is
unclear at the moment.
8.2.2. Interaction with enzymes
The interaction of lectins with storage proteins is clearly only
one aspect of the interactions of lectins within the protein
body. In addition to storage proteins, also hydrolytic enzymes

Plant lectins
(glycosidases [169,170] and phosphatases [176]) are bound by
the lectin in a carbohydrate-, ion strength- or pH-dependent
manner. A striking example is the -mannosidase from
Canavalia ensiformis seeds which despite being a glycoprotein reacts with the lectin from the same plant, ConA, not via
its carbohydrate moiety but by ionic interaction most effectively
at pH 5 [177,178]. When isolated protein body membranes are
brought into contact with lectin, pH- or carbohydrate-dependent
interactions between them are observed [179,180]. Calcium
ions which stabilize the conformation of the protein (please see
Section 7.1) are released when lowering the pH. This is probably
the reason why the interaction of Ca2+ -dependent lectins with
binding partners is abolished at low pH values [116]. Moreover,
pea protein body membranes contain a protein that is crossreactive with the lectin [181]. Its presence may result from
residual firm lectin binding to the membrane, a phenomenon
also demonstrated by in vitro studies [182], or it may represent
a modified form of the lectin.
The dual binding ability of lectins to storage proteins and
to protein body membranes suggests that lectins might form
a reversible glue between protein and membrane, an idea that
is supported by the time course of lectin and storage protein
biosynthesis during seed maturation [183]. Such interactions
could be modulated by small alterations of the ionic or pH
environment within the protein bodies. In consequence, participation of lectins in organizing the protein body content has
been suggested, a concept also discussed for Erythrina indica
[184] and for rice (Oryza sativa) seeds [95,185] as well as
for soybean (Glycine max) leaves [186]. This mechanism to
bridge an effector to a target might also be at work for a lectin
with myrosinase-binding capacity in seeds of Brassica napus,
possibly ensuring that products of glucosinolate hydrolysis are
made available in the vicinity of an aggressor traced by the
lectin [187]. Mutatis mutandis, the principle to take advantage
of bi- or oligofunctional lectins is also illustrated by the concept
that comitin, a 24 kDa actin-binding protein, might link actin
cytoskeleton to Man-presenting Golgi vesicles [188].
Lectins may not only bind enzymes but also modify their
activities. More than a decade ago it was observed that wheat
germ and potato lectins are able to activate endogenous phosphatases from the respective plant [189,190]. More recently,
the lectin from a mushroom (Pleurotus ostreatus) turned out to
activate a phosphatase from the same source in a carbohydratedependent manner [30,191]. In addition, this lectin is closely
associated with an -galactosidase activity [30]. At present, it
is not possible to provide a straightforward molecular explanation for these phenomena. It may be instructive to note reports on unexpected activities of proteins originally described
as lectins. The B-chain of ricin is reported to display a lipolytic
activity which is asserted to contribute to ricins toxicity [192]
under the precondition that the preparation employed was completely pure [137]. As mentioned above, a root lectin from the
legume Dolichos biflorus recently shown to bind Nod factors exhibits a nucleotide phosphohydrolase (apyrase) activity which is

603
enhanced in the presence of haptenic carbohydrates [29]. Actually, the modulation of properties of a second independent
site by the lectin domain is similarly operative in animal lectinology, for example in the case of the 67 kDa elastin-/lamininbinding protein or CBP70 interacting with galectin-3 [21,193].
Further association of a lectin-like domain with a hydrolytic
activity (-galactosidase, endo-1,4-glucanase) is seen in two
enzymes of ripening strawberries [31]. Certain bacterial lectins,
too, were shown to modify enzymatic activities [194] or to harbor enzymatic activities of their own [195197]. It is attractive
to envision a cooperation of the two active sites, the lectins
domain guiding the enzymes activity to a site of action (see
above), as likewise suggested for sperms proteolytic acrosin
by its Fuc-binding site homing in on zona pellucida glycans
[21]. Cellulases and xylanases of aerobic microorganisms with
non-catalytic cellulose-binding domains optimizing enzymesubstrate proximity add to the list of modular proteins harboring
enzyme and lectin activity [198].
8.2.3. Knock-out experiments
Most studies aiming at defining the biological function of lectins
have been performed with isolated proteins in vitro. Recently,
genetic engineering was introduced to addressing this question
by knocking out their expression. Transgenic alfalfa (Medicago
sativa) plants were constructed with engineered antisense genes
of the (putative) lectin genes MsLEC1 and MsLEC2 [199]. As
KO animals for lectins have provided clues for in vivo functions
[21], this approach is valuable for delineating answers to the
same question in plants. In antisense plants, embryogenesis was
severely impaired and both vegetative and reproductive development was disturbed whereas development and growth were
normal in controls (vector control, sense-transgenic plants)
[199]. This result points to an involvement of the targeted lectins
already at early stages, a challenge for further investigations in
this field. They will enhance our knowledge in terms of a broader
panel of species studied and with regard to the individual steps
that bring about the observed phenomena both outside and inside the plant. In what respect the mitogenic activity of plant
lectins, initially detected by Nowell for PHA and resting lymphocytes [200], might play a role in development in situ will
have to be clarified. Regarding plant lectins as tools, this example together with the blood-group-specific binding discussed
above attests the potential of lectins in medical applications.
9. Applications of plant lectins
In addition to the increasingly sophisticated description of the
occurrence and structural characteristics of plant lectins, this aspect has been and continues to be a driving force for the growth
of literature in glycosciences [201]. Numerous questions in basic and medical sciences have been and are being addressed
by exploiting plant lectins (Table 4). Laboratory manuals illustrate that lectin-involving methods have well matured and

604
Table 4. Common applications of plant lectins as tools in basic
and medical sciences
Biochemistry
Detection of defined carbohydrate epitopes of glycoconjugates
in blots or on thin-layer chromatography plates
Purification of lectin-reactive glycoconjugates by affinity
chromatography
Glycan characterization by serial lectin affinity chromatography
Glycome analysis (glycomics)
Quantification of lectin-reactive glycoconjugates in
enzyme-linked lectin-binding assays (ELLA)
Quantification of activities of
glycosyltransferases/glycosidases by lectin-based detection
of products of enzymatic reaction
Cell biology
Characterization of cell surface presentation of
glycoconjugates and their preceding intracellular assembly
and routing in normal and genetically engineered cells
Analysis of mechanisms involved in correct glycosylation by
lectin-resistant cell variants
Fractionation of cell populations
Modulation of proliferation and activation status of cells
Model substratum for study of cell aggregation and adhesion
Medicine
Detection of disease-related alterations of glycan synthesis
Blood group typing and definition of secretor status
Quantification of aberrations of cell surface glycan
presentation, e.g. in malignancy
Cell marker for diagnostic purposes incl. infectious agents
(viruses, bacteria, fungi, parasites)

reached the status of routine handling [202,203]. Together with

carbohydrate-specific monoclonal antibodies lectins afford the
possibility to profile glycan populations. Mindful of the conclusion that oligosaccharides are ideal for generating compact
units with explicit informational properties [204], complexity in the pattern of code units (the glycome) with cell type
selectivity and modulation of their expression in response to
changes of the status of differentiation or onset of disease (e.g.
malignant transformation) can be predicted. In this sense, plant
lectins have made themselves known to assess a defined aspect
of activity of the complex machinery for glycan assembly and
modification, starting with synthesis of nucleotide sugars and
their transport into the Golgi lumen [4,48].
9.1. Profiling of glycosylation
Indeed, the plethora of studies in this research area confirms
the expectation that glycan synthesis fulfils the requirements of
a flexible process. Figuring prominently, it is definitely capable to generate a wide variety of sugar determinants. Sorting
through disease states as an example, multiple alterations of
N- and O-glycan structures were disclosed worthy of further
tests whether they can eventually be referred to as markers with
clinical relevance [205207]. Analogous lessons were drawn

Rudiger and Gabius

from the study of development in various systems [208]. As
compiled in Table 4, the structural changes cannot only be detected but also quantified and characterized on the molecular
level by plant lectins. The development of serial lectin affinity
chromatography had a major impact on glycan isolation and
gained access to salient sequence information [209,210]. The
topological route how these lectin-reactive epitopes are transported following their synthesis and then presented on the cell
surface as well as their lateral movements in the membrane
are readily followed by lectin cytochemistry using fluorescent
or biotinylated lectins or lectin-coated colloidal gold granules
[211,212]. Examples for the ample return after investing efforts into basic research on plant lectins are given in the next
paragraphs.
In diagnostic pathology, especially the possibilities to stain
vascular endothelia and tumors by the Fuc-specific lectin
UEA-I and to distinguish pathogens, for example fungi such
as Aspergillus fumigatus, Candida albicans or Rhizosporus
oryzae, underscore the usefulness of these sugar-specific probes
(Table 4) [213217]. However, care is to be exercised and
methodological aspects closely scrutinized before clinically
predictive power of lectin binding is claimed, as thoughtfully
discussed by Walker in a special case [218]. Regarding limits of
plant lectin application in histochemistry, distinct epitopes with
functional relevance in mammalian cell adhesion such as sulfated Lewisx [193,219] cannot be monitored, because no plant
source for a lectin of this specificity is known. In other words,
plant lectins should not be relied upon to completely cover
the wide variety of sugar code words in mammalian glycoconjugates. Consequently, to close this gap tissue lectins have
been introduced to classical lectin histochemistry [220,221].
The endogenous lectins together with carrier-immobilized carbohydrate ligands and plant lectins establish the complete panel
of tools in current glycohistochemistry [135,222,223]. Owing
to the immunogenicity of plant lectins it is similarly reasonable to include endogenous lectins (bioaddressins) in work on
targeted drug delivery [224].
9.2. Ligand cross-linking and mediator release
In cell biology, toxic lectins (ricin) are potent tools to select resistant cell variants on the way to pinpoint genetic defects of the
glycosylation machinery (Table 4) [225]. As model substances
to induce cell aggregation and adhesion, their cell-binding capacity enables to assay synthetic inhibitors such as glycodendrimers and to dissect signaling cascades relevant for these
processes [226,227], the same aim as can be attained with mitogenic lectins in the study of molecular mechanisms of proliferative control and mediator release [228]. In such instances, the
cross-linking capacity of lectins which form ordered networks
with ligands is vital to accomplish initiation of the signaling
cascade [165,229]. By activating immune cells, inducing apoptosis or modulating their cytokine secretion, the respective plant
lectins effectively function as biological response modifiers. An
example is given by the Gal-binding lectin from mistletoe, an

Plant lectins
abundant agglutinin related to ricin/Ricinus communis agglutinin with similar capacity to induce secretion of proinflammatory cytokines from mononuclear cells at non-toxic quantities
[230]. Unquestionably, immunomodulatory activity should not
be assumed to translate automatically into clinical benefit [230].
We have already alerted the reader to an analogous concern not
to rush to lectin application prematurely in the context of noting
the lack of knowledge concerning ecological long-term effects
in lectin-dependent crop protection. In fact, the spectrum of
lectin-reactive cells responding to the mitogenic stimulus can
include tumor cells, and activation of immune cells may render growth-promoting factors available to let tumor cells thrive
more rapidly and/or aggressively than without immunomodulation [231]. In fact, intratumoral macrophages reactive with the
lectin could release proinflammatory cytokine in a paracrine
manner (Figure 3). When the mistletoe lectin, a component of
proprietary extracts from alternative/complementary medicine,
was tested at its immunomodulatory dosis in tumor models,
this treatment modality indeed led to tumor growth stimulation
in vitro with certain lines and a part of the tested histotypic
cultures, in vivo in mice after transplantation and in rats after
chemical carcinogenesis [232235]. In a recent report on a trial
with melanoma patients receiving a proprietary mistletoe exR
tract (Iscador

) it was concluded that for patients with lymph
node metastases, treatment with Iscador may accelerate and
alter the course of the disease; a significant increase in brain
metastases and a significant decrease in overall survival rates
were observed for this very high-risk group [236]. Although
it is not clear whether the clinically examined preparation contained the immunomodulatory dose of the lectin, these data seriously question the practice to exclude treatment modalities of

605
alternative/complementary medicine from rigorous safety tests
[237]. They also underline the principally double-edged character of lectin-induced immunomodulation in tumor biology.
9.3. Nuclear transport and perspectives
An intriguing example for plant lectin application in cell biology and glycan analysis comes from the detection of OGlcNAcylation of nucleocytoplasmic proteins by WGA [238].
From the panel of plant lectins, only Gramineae proteins detect
this special type of O-glycosylation separate from mucin-type
O-glycans [48,54,239,240]. Their availability enables not only
monitoring of dynamic changes on the extent of this modification but also blocking studies to infer a role of this sugar
e.g. in nuclear transport. This application is a further example
epitomizing the very handy target specificity of lectins when
dealing with complex-glycan-containing samples (Table 4). It
is therefore a good advice to keep an updated list of lectin
specificities on the desk [241] to select the proper tool when
neededand further additions to the list of plant lectins with
new properties are certainly always welcome to match an upcoming problem with a proper tool. The abundance of lectins in
plants (Table 1), the availability of efficient purification methods
(see Section 5 on isolation of lectins) and their stability (probably connected to the outlined external functions in defense and
conferring eminent resistance to detergents and aprotic solvents
to them [242,243]) render it likely that the literature on lectin
applications will continue to grow. Conceptually, the equivalent
of genome and protein analysis, termed glycomics or glycome
profiling [5,244] (Table 4), will gear up the output of pertinent
publications on the way to correlate cellular glycan (code word)
display with functions ( functional glycomics).
10. Conclusions

Figure 3. Illustration of localization of lectin-reactive cell surface glycoconjugates using a biotinylated probe. Localization
of intratumoral macrophages reactive with the immunomodulatory mistletoe lectin (arrows) in a section of an axillary lymph
node metastasis from a mammary carcinoma. Lectin binding
can elicit in situ cytokine release with its inherent ambivalence,
as described in the text.

Plant lectins are established laboratory tools with applications

for example in glycan profiling in cyto- and histochemistry and
as elicitors of cellular activities such as mitosis. These properties in signaling have contributed to delineation of mechanisms
of signal transduction from initial cell binding and ligand crosslinking to the final response. In this respect, they can mimic
properties of endogenous lectins. These insights pave the way
for growing appreciation of the range of activities of proteincarbohydrate recognition in situ and help to familiarize with this
concept. Equally important, the analysis of plant lectin structures teaches intriguing lessons also relevant for the strategic
design of animal lectins. As discussed, the spatial integration of
enzymatically active and lectin sites in modular proteins provides a means to target the catalytic center to distinct places
via the lectin activity. Considering structure, the occurrence of
the jelly-roll-like folding pattern in plant and animal lectins affords a notable role model to define distinct ways how a binding
site for carbohydrate ligands can be positioned into the same
basic structure. Moving from the analysis of the carbohydratebinding sites architecture to the level of the complete protein,

606
it next appears possible that occupancy of this site can influence the functionality at other parts of a mosaic-like molecule.
Finally, the effectiveness of plant lectins to interact with animal
cells can now rightfully be viewed in an ecological context, e.g.
as protection of the plant against predators.
Acknowledgments
We gratefully acknowledge the contributions and enthusiasm of
our coworkers and collaborators in the course of the studies in
our laboratories. We also want to express our sincere gratitude to
Dr. J.H. Wu (Taipei, Taiwan) for promptly and precisely sending published data to be included in Table 1, to Drs. M. Frank
and C.-W. von der Lieth (Heidelberg, Germany), Dr. A. Romero
(Madrid, Spain) and Dr. H.-C. Siebert (Munich, Germany) for
kindly providing ilustrations, and to Dr. B.B. Namirha (Munich,
Germany) for competently polishing the text. A sincere apology
is directed to colleagues whose original work could not be fully
given reference to due to the very strict space limitations further
enforced during the review process. The financial support from
the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Dr.-M.-Scheel-Stiftung fur Krebsforschung,
the VW-Stiftung and the Wilhelm-Sander-Stiftung as well as
DAAD programs and NATO fellowships for travel grants are
also gratefully acknowledged.
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Received 24 October 2001; revised 29 July 2002;

accepted 30 July 2002