INTRODUCTION
The high toxicity of castor beans (sjemenke ricinusa) was recognized during the last century when the extract was found to agglutinate a suspension of erythrocytes of different animal species. Since then, lectins were studied and extracted from plants, including fungus and lichens, as well as in animals.
Lectins are glycoproteins of 60,000-100,000 MW that are
known for their ability to agglutinate
(clump) erythrocytes in vitro. There are over 400,000
estimated binding sites for kidney bean (grah)
agglutinin on the surface of each erythrocytes. Lectins
are found in most types of beans, including
soybeans. Reduced growth, diarrhea, and interference
with nutrient absorption are caused by thisclass of toxicants. Different
lectins have different levels of toxicity, though not all lectins are toxic,
though no all are toxins. The bright scarlet seeds of precatory bean Abrus
precatorius contain
the highly toxic glycoprotein, abrin. Less toxic lectins
can be fatal if ingested in high amounts.
Some of such lectins is concanavalin A from Concanavalia
ensiformis (jack bean). Others may
exhibit no hemagglutinating activity as in the case of
ricin from castor bean and yet it is one of the
most toxic substances.
The terms phytohemagglutinins, phytagglutinins, and lectins
are used interchangeably.
Lectins-containing plants have been found in many botanical
groups including mono- and
dicotyledons, molds and lichens, but most frequently
they have been detected in Leguminoseae
and Euphorbiaceae. They may exist in various tissues
of the same plant and have different cellular localizations and molecular
properties.
Lectins interaction with certain carbohydrate is very
specific. This interaction is as specific as the
enzyme-substrate, or antigen-antibody interactions. Lectins
may bind with free sugar or with
sugar residues of polysaccharides, glycoproteins, or
glycolipids which can be free or bound (as in
cell membranes). The term lectin refers to the specificity
of the reaction (legere = to choose).
One of the major interest in this class of glycoproteins
is the therapeutic use against HIV-1.
Jacalin, a plant lectin, is found to completely block
human immunodeficiency virus type 1 in vitro
infection of lymphoid cells. This activity of the jacalin
is attributed to its ability to specifically
induce the proliferation of CD4+ T lymphocytes in human.
LECTINS IN FOODS
The ability to agglutinate human erythrocytes or representatives
of human indigenous microflora
was detected in 29 of 88 food items. Many foods contained
substantial amounts of agglutinating
activity, and lectins extracts could be diluted several
folds and still produce agglutination. Great
variation was observed in agglutination activity in the
same food item purchased from different
stores or from the same store on different days. Sometimes
a food that possessed substantial
activity on one day was found to have little or even
no activity on other day.
A survey of the fresh and processed foods foundlectins in about 30% of the food stuffs tested, including such common foods as salad, fruits, spices, dry cereals, and roasted nuts. However, dry heat may not completely destroy lectin activity. Hemagglutinating activity is found in the processed wheatgerm, peanuts, and dry cereals. Several lectins are resistant to proteolytic digestion e.g., wheatgerm agglutinin, tomato, lectin, and navy bean lectin.
FUNCTIONS OF LECTINS
Not much is known about the functions of lectins in the
organism they are formed. There is
evidence that lectins may be involve in the recognition
between cells or cells and various
carbohydrate- containing molecules. This suggests that
they may be involved in the regulating
physiological functions. They seem to play an important
role in the defence mechanisms of plants
against the attack of microorganisms, pests, and insects.
Fungal infection or wounding of the
plant seems to increase lectins. In legumes, the role
of lectins in the recognition of nitrogen-fixing
bacteria Rhizobium genus, which have sugar-containing
substances, has received a special
attention.
Binding Nitrogen-Fixing Bacteria to legume Roots: among
the possible functions of plant lectins is
their participation in binding nitrogen-fixing bacteria
to legume roots. In this reaction bacteria of
the genus Rhizobium adhere to the surface of differentiated
roots cells and are then internalized
into the root hair to form nitrogen-fixing nodules. The
symbiosis is specific in that certain species
of Rhizobium can only associate with a particular species
of legume. Such observations were made
from the studies that were done on soybean Rhizobium
japonicum and the clover Rhizobium
trifolii. Other functions of lectins in plants may include:
Enzymes (but unknown substrate)
Storage of proteins
Defense mechanism
Cell wall extension
Mitogenic stimulation
Transport of carbohydrates
Packaging and/or mobilization
of storage materials
LECTINS STRUCTURE
One major property of lectins is their specific saccharide-
binding sites. Some lectins are
composed of subunits with different binding sites. These
include the lectin from the red kidney
bean, Phaseolus vulgaris. It is composed of two different
subunits combined into five different
forms of noncovalently bound tetramers. Since subunits
have very different specificities for cell
surface receptors, each combination is considered to
have a different function. The specificity of
the binding sites of the lectins suggest that there are
endogenous saccharide receptors in the tissues
from which they are derived or on other cells or glycoconjugates
with which the lectin is
specialized to interact.
Metal Binding Sites
Biological activity of the lectins may be attributed to
the metal ions which are the essential part of
the native structure of most leguminous lectins. The
most studies and fully sequenced lectin is
concanavalin A. The metal binding sites of the concanavalin
A are situated in the amino terminal
part of the polypeptide chain. In this lectin, each subunit
has aspartic 10 and 19, asparagine 14,
histidine 24, serine 34, glutamic acid 8, and tyrosine
12 that are involved in the binding to one
calcium and one magnesium ion.
Lectins of soybean, peas, faba bean, lentils, and sainfoin
have amino acids that are involved in
metal binding, which are conserved. The exception is
of the tyrosine residue at position 12 of
concanavalin A which is replaced with by phenylalanine
in the other legume lectins.
Hydrophobic Sites
The stability of the native structure of most lectins
is thought to be caused by the hydrophobic
interactions. Such hydrophobic sites, forming cavities
in the lectins structure, may play an
important biological role. The hydrophobic binding sites
of auxins, or cytokinin and adenine, for
instance, by concanavalin A may enhance the functions
of lectins on the plant life cycle.
Glycosylation Sites
Despite the generalization that most lectins are considered
glycoproteins, concanavalin A, lentils
lectin, and wheatgerm agglutinin contain no covalently
attached carbohydrates. However,
non-glycoprotein lectins are believed to be synthesized
as glycosylated precursors. This is
supported by the following observation:
1.1) pro-concanavalin A is an inactive glycoprotein from
which the glycosidic side chain is
removed during post-translational
processing.
2.2) Non-glycoprotein wheatgerm agglutinin molecules
are produced by removing a carboxyl
terminal glycopeptide from the
glycosylated precursor during post-translational processing.
All glycoprotein lectins contain a peptide sequence: asparagine-
X-threonine/serine, which is
characteristic of glycosylation sites. These sequences
are different in the non-glycoprotein lectins.
Also, peptide sequences, which in one glycoprotein lectin
contain the glycosidic side-chains, are
not necessarily conserved in another glycoprotein lectin.
This may suggest that the biological
activity of the lectins may not be determined by carbohydrate
part of their structure.
Carbohydrate Binding Sites
Lectins differ markedly in their sugar-binding specificity. A sequence participating in carbohydrate-binding site of concanavalin A, for instance, are poorly conserved in other lectins.
Three Dimensional Structure
The three dimensional structures of lectins can be used to show the structural similarities and homologies of legume lectins. When the secondary structure of Vicieae lectins, for instance, is compared with those of concanavalin A, an identical or homologous B-turn structures are found in these lectins. Hydropathic profiles of two chains and single-chain lectins are superimposed, and concanavalin A has three domains that appear in some two-chain lectins of Vicieae.
The two-chain and single-chain legume lectins exhibit
high homology in the primary sequences
and three-dimensional structures. Therefore evolution
seems to have imposed only slight
modification in the genetic coding for these lectins.
This fact suggests the possibility of using
lectins as an acceptable phylogenetic markers.
LECTINS IN PLANT TISSUES
Lectins in soybean plant are different from those in seeds.
Dolichos bifluorus's lectins were studies
in both the leaves and seeds. Lectins found to be produced
with low level and constant for the
period between 2-8 weeks after germination, then increased
several folds in the next ten weeks. At
this time extracts of stems and leaves contain several
nanograms of lectin- like material per
microgram of nitrogen, whereas mature seeds have about
1000 ng lectin per microgram. It is
notable that in developing seeds the amount of lectin
rises very abruptly. It is undetectable during
the first 26 days after flouring but reaching a maximal
level by day 28. Lectins from the stems and
leaves don not agglutinate erythrocytes that could be
agglutinated by seeds lectins. Stems and
leaves lectin is a dimer of a molecular weight of 68,000,
in contrast to the tetrameric seed lectin
with a molecular weight of 110,000.
TOXICITY OF LECTINS
Pathological lesions occur in animals injected with kidney
beans extracts. Various tissues suffer
from parenchymatous, fatty degeneration, and edema. In
the liver local necrosis and fatty changes
can be observed. Hemorrhages are observed in the stomach,
the intestinal wall, and other organs.
Distentions of capillary vessels may present in the Kidney
and myocard with numerous thrombi.
Morphological changes in rats fed navy beans include:
increased weight of kidney and heart,
pancreatic acinar atrophy, and fatty metamorphosis of
the liver. Such changes may be attributed
to the low availability of essential amino acids and
low food intake of the animals consuming the
raw bean diet. For example, rats fed raw kidney beans
develop multiple histological lesions. Also,
lectins from red kidney beans are found to induce small
intestinal epithelial growth, crypt cell
hyperplasia and DNA synthesis. Small amounts of isolated
black bean agglutinin show low food
absorption and nitrogen retention in rate.
The absorption of glucose from a ligated intestinal loop
in anesthetized rats, previously fed a bean
diet or given the black bean agglutinin by stomach tube,
was much decreased. Raw kidney beans
were found to interfere with vitamin E utilization in
chicks. The hypoglycemia observed in rats fed
a bean diet may indicate a reduced intestinal absorption
of glucose.
Diet rich in raw soybean has a goitrogenic effect. This
is indicated by the fact that fecal loss of
thyroxine from the gut is higher in animals fed raw soybeans
than in the controls. Raw soybean
meals reduces fat and fatty acids absorption (not soybean
trypsin inhibitor) in young chicks. Such
meals also depresses the utilization of vitamin D in
turkey. These effects are not found when the
meals include heated soybean.
Ricin, abrin, crotin, and related toxins, produce similar
macroscopic and microscopic pathological
lesions. The intensive inflammation with destruction
of epithelial cells, edema, hyperemia, and
hemorrhages in the lymphatic tissues are very common.
Several signs of toxicity may include:
fatty degeneration and necrosis in the liver, degenerative
lesions of the myocard, and extension
and presence of blood clots of capillaries of all organs.
At the site of lectin application, local
hemorrhages are frequently observed.
In vitro, plant lectins effect lymphocyte mitogenesis,
aggregate immunoglobulin induce histamine
release from basophils and mast cells. When raw ground
garden beans are supplemented with
essential nutrients diet, weight lose and death to rats
may occur within 1-2 weeks, due to the
toxicity of the lectins in the beans. Raw navy beans
have been found toxic for Japanese quail but
not toxic for germ-feed birds. However, several cases
of human intoxication were reported due to
ingestion of raw or partially cooked beans.
Effect on the Gastrointestinal Tract
When given orally to experimental animals, lectins interact
with the mucosa of the gastrointestinal
tract causing acute gastrointestinal symptoms, failure
to thrive and even death. When
administered parenterally, they can alter host resistance
to infection or to tumor challenge. They
even can be highly allergenic under certain conditions.
In vitro and in vivo tests show that
intestinal cell damage are caused by bean lectins. In
this regard, intestinal invertase is strongly
inhibited by bean lectin as well as the absorption of
vitamin B. One of the possible explanation of
the toxicity of the lectins, which are resistance to
gastric and intestinal digestion, is the binding to
the cell lining of the intestinal walls, causing lesions
and interference with nutrients absorption.
Effects on Cell Membrane
The reaction between the agglutinin and the cell membrane
is believed to result in an alteration of
the cell function thus producing the toxic effect. Only
those cells bearing the specific receptor
groups for the respective lectin would be effected. For
instance, significant changes in membrane
properties are induced by binding of lectins to liver
cells of diabetic rats. The changes may
influence such cellular properties as aggregation and
deformability of erythrocytes, permeability,
electrical resistance, and binding properties of mitogen,
hormone, and lipoprotein receptors. The
binding of concanavalin A and ricin, for instance, can
be reduced by 20-25% as a result of a
membrane reduction of glycoprotein carbohydrates content.
Ricin Toxicity
After intravenous injection of ricin, both the intestinal
tissue and the intestinal juice of rabbits
became highly toxic, indicating its concentrating in
this tissue and its secretion into the intestinal
lumen, but it can not be found in urine. Ricin may be
appear in the milk of lactating guinea pigs
which had been injected after the birth of the litters
(suckling young became markedly resistance
against subsequent injections of the toxin).
Magnesium blood level decreases after injection of ricin
in cats. Quantitative analysis of plasma,
liver, and urine, of rats acutely poisoned with ricin,
along with the observation of the reduced
respiratory quotient of the liver, can be used to conclude
that: the toxic action of ricin may be
explained by an interference with some metabolic process
in the liver, possibly the Krebs cycle.
Also, a rise of the blood values of urea, glucose, bilirubin,
transaminases, and lactic
dehydrogenase in rats fed ricin could be observed. However,
detection of albumin and hematuria
could lead to the conclusion that a hepatonephritis with
hepatic cytolysis may be an early
manifestation of ricin intoxication. A lag period between
the injection of a lethal amount of ricin
and death is not less than 12 hours. Ricin is many times
more toxic when injected than when given
orally.
DETOXIFICATION
The destruction of ricin toxicity by heat has long been
recognized (1889 by Stillmark). The
presence of more than one toxic principle which differ
in heat resistance in castor beans must be
taken into account when working with products derived
from these seeds.
Although the castor bean allergen is more resistance to
boiling than ricin, it can be inactivated by
autoclaving. For its safe use as fertilizer and for animal
feeding, detoxification of castor pomace is
essential. Steam heating significantly reduces the toxicity
of pomace to become harmless for
sheep, rabbits, and rats, when the beans is about 10%
of their diet. When used before heating
castor bean, calcium hydroxide may aid in achieving complete
destruction of the ricin and the
allergen.
However, animals can be immunized successfully when injected
with heated ricin solution in order
to resist the toxicity of castor bean cake. Mice can
also be protected against the fatal action of
ricin if injected with blood serum from immunized goats
(when given not later than 6 hours after
the ricin injection).
Autoclaving is proved very helpful in enhancing the nutritive
value of legumes, an effect that is
probably related to the destruction of toxic hemagglutinins
and other growth inhibiting factors.
For complete elimination of the toxicity of kidney bean
and field bean, preliminary soaking prior
to autoclaving is required. In addition, autoclaving
for 5 minutes is sufficient to eliminate the
toxicity of finely ground navy bean meal.
However, dry heating has been found to be less effective.
Thirty minutes of dry heating had little
effect on hemagglutinating activity of certain varieties
of V. vulgaris, and activity was still
detectable after 18 hours of heating. Whereas, heating
of the soaked beans or autoclaving was
fully effective.
Moreover, formaldehyde has been recognized to reduce the
agglutinating and toxic actions of
ricin and agglutinating activity of bean lectin, but
phenol was inactive in this respect. Potato lectin
was more rapidly destroyed by phenol than by formaldehyde.
Adsorption of lectins on erythrocytes or stroma that are
brought into solution is observed after
heating to 56 degree C. The agglutinating and the toxic
activity from a bean lectin solution
disappear when it is treated with stroma. For both ricin
and kidney bean agglutinin, no
agglutination of erythrocytes occurs at low pH, and adsorbed
lectins are observed to dissociate
from the erythrocytes.
LECTINS AND CANCER
lectins present on the surface of tumor cells are targeted
for therapeutic purposes. It has been
found that treatment with anti- lectin antibodies can
suppress growth of tumor cells in agarose,
and inhibit lung colonization in vivo. Lectins have the
potential use in cancer treatment strategies
due to the fact that lectins present on the surface of
tumor cells are capable of binding exogenous
carbohydrate-containing molecules and internalize them
by endocytosis.
For example, wheat germ lectin (WGA) is found to induce
lectin- dependent macrophage-mediated
cytotoxicity against human bladder cancer (T-24) cells.
Alveolar macrophage (AM) are
phagocytes, mainly present in the pulmonary alveoli,
are important in the antitumor defense
mechanism of the lung because they can bind to the target
cell- but are unable to induce cytolysis.
However, studies have revealed that human AM tumorcidal
activity can be induce by wheat germ
lectins. Another finding is that the sensitivities of
six human tumor cell lines depend on the
number of receptor sites exist on the surface of WGA.
Although the effector mechanism is still
unknown, the binding of AM with tumor cells initiated
by WGA may increase sensitivity to the
cytotoxicity mediated by human AM.
In addition, WGA is found to enhance the cell killing
ability of murine peritoneal macrphages. In
vivo studies show that WGA has an inhibitory effect on
the growth of murine tumors. The
tumoricidal activity of human blood monocytes can be
induced by the WGA. As a result, the
monocytes are able to become cytotoxic to four different
human tumor cell lines: T-24 bladder
carcinoma, A-375 melanoma, ACHN renal carcinoma, and
U373MG glioblastoma. Murine
systems also show similar response. However, concanavalin
A, PHA, PWM and SBA are unable to
produce tumoricidal monocytes.
Tareq Al-Ati taa1@cornell.edu
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