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Food Reviews International, 25:1–16, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 8755-9129 print / 1525-6103 online
DOI: 10.1080/87559120802458115
Biological Properties and Characterization of Lectin
from Red Kidney Bean (Phaseolus Vulgaris)
1525-6103
8755-9129
LFRI
Food
Reviews International
International, Vol. 25, No. 1, October 2008: pp. 1–34
Red Kidney
Zhang
et al. Bean Lectin
JIANSHEN ZHANG1, XIANQUAN SHI1, JOHN SHI2,
SANJA ILIC3, SOPHIA JUN XUE2, AND YUKIO KAKUDA3
1
Department of Bioengineering and Environmental Science, Changsha University, 5
Changsha, Hunan, China
2
Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph,
Ontario, Canada
3
Department of Food Science, University of Guelph, Ontario, Canada
Red kidney beans (Phaseolus vulgaris) contain significant amounts of lectins which 10
have both beneficial and detrimental biological properties. Lectins are carbohydratebinding glycoproteins that can react specifically with human blood cells, preferentially
agglutinate malignant cells, and undergo mitogenic stimulation of lymphocytes. Some
lectins are resistant to heat and proteolytic enzymes and can enter the circulatory system intact. Phytohaemagglutinin (PHA)—a lectin isolated from the red kidney bean— 15
consists of four subunits with a molecular weight of 125 kDa. This bioactive compound
has been partially purified by affinity chromatography using Affi-gel Blue. PHA has
been shown to inhibit the viral enzymes, immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), and a- and b-glucosidases. This paper will review the chemical
properties, biological activity, distribution, isolation, and heath benefits of red kidney 20
bean lectin.
Keywords Lectins, biological properties, bioactivity analysis, antinutritional factors,
red kidney beans
Introduction
Lectins are carbohydrate-binding glycoproteins of non-immune origin capable of specific 25
recognition of and reversible binding to carbohydrates, without altering their covalent
structure.(1) Lectins are essential and ubiquitous plant constituents. Over 70 different seed
lectins have been identified in various legumes. Among other possible functions, they are
responsible for innate immunity and defence mechanisms in plants and interactions with
symbionts. Although the amount of lectins in edible plants vary, the daily ingestion of lec- 30
tins by both humans and animals is significant. Since lectins are often resistant to heat and
proteolytic enzymes, including those of intestinal microflora, the effects of consumption
of these proteins deserves special attention.(1,2)
In the last two decades, there has been increased interest in the potential health benefits
of bioactive proteins from plants, including lectins from legumes because of the extensive 35
studies which showed that lectins exhibit antiproliferative, antitumor, immunomodulatory,
antifungal, antiviral, and HIV-1 reverse transcriptase inhibitory activities.(3,4,5) Lectins are
glycoproteins that selectively and reversibly bind carbohydrates. This property gives them
Address correspondence to John Shi, Guelph Food Research Center, Agriculture and Agri-Food
Canada, Guelph, Ontario N1G 5C9, Canada. E-mail: [email protected]
1
2
Zhang et al.
a diversity of biological functions, many of which are still unknown. Even for lectins with
homologous amino acid sequences as those from legumes, a common function cannot
be ascribed to them because individual parameters such as carbohydrate specificity and
other influencing factors differ. This is the reason why every description of a proposed
biological function requires experimental evidences and cannot be assumed by analogy
considerations.(2)
In addition to increasingly sophisticated descriptions of the occurrence and structural
characteristics of lectins, their potential to enhance health has been a driving force for the
expanding interest in lectinology.(6) Kidney bean lectin—PHA—is one of the most thoroughly studied lectins due to its abundance and availability. Additional knowledge of its
structure, binding, specificity, and isolations methods is needed in order to understand
potential applications of this compound. Possible benefits from the reported inhibitory
effect of kidney bean lectin on HIV-1 reverse transcriptase was an incentive to review
published literature describing this property of lectins.
Affinity chromatography has been widely used to purify lectins from beans due to its
high specific selectivity.(7,8) The assay of hemagglutinating activity, assay of antiproliferative
activity, and assays of antifungal and HIV-1 reverse transcriptase inhibitory activities are normally used to analyse the bioactivity of lectins.(3,5) Circular dichroism (CD) spectroscopy,
molecular mass determination by Sodium dodecyl sulfate-polyacrylamide gel electrophresis
(SDS-PAGE), and gel filtration, as well as the N-terminal amino acid sequence determination, are commonly used for bioactivity analysis and characterization of lectins.(8,9)
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60
65
Lectins in Edible Plants
Lectins are present in numerous edible plants. It has been long known that they occur in
legumes that are a major food source for both humans and animals. Amounts of lectin in
legumes vary significantly (Table 1). The content of total protein in kidney bean seeds is
reported to be between 17–23%, of which 2.4–5% is PHA.(2, 10, 11) Soybeans (Glycine
max) contain 34% protein, of which 0.8% is lectin.(2, 11) Lima bean (Phaseolus lunatus)
lectin makes up about 0.8% of the total protein content (21%), and the amount of garden
peas (Pisum sativum) lectin is around 0.6% of the total protein (24–25%).(2, 11)
Besides legumes, lectins are found in other plants that serve as food or feed, many of
which are consumed raw, like fruits and vegetables. These food items include ingredients,
spices, and dry cereals. Table 2 provides a list of edible plants in addition to legumes that
have been demonstrated to exhibit agglutinating activity towards erythrocytes from various species.
The human population is constantly exposed to dietary sources of lectins. Although
lectin containing foods are frequently consumed cooked or otherwise processed, these
treatments may not always inactivate the lectins, although some lectins are heat labile. For
example, lectins have been detected in roasted peanuts(12) and slow cooking of beans without boiling does not always eliminate activity, as was observed with kidney beans cooked
for 11 h at 82°C, or 5 h at 91°C. However, some lectins can be removed from foods by different technological processes. Adeparusi(13) reported that the lectin in Phaseolus lunatus
L was completely destroyed by soaking, autoclaving, and toasting processes. Autoclaving
lima beans for 20 min was found to eliminate all anti-nutrients, except tannins. The uses of
conventional or microwave ovens are not effective methods for inactivating lectin in the
bean. Moreover, the use of an autoclave or boiling water is needed in order to cause irreversible lectin denaturation. For instance, Hemagglutinating activity was eliminated
within 5 min of heat treatment at 92°C on newly developed cultivars.(14)
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75
80
85
90
3
PNA
SJL
Arachis hypogaea (peanut)
Sophora japonica
(Japanese pagoda tree)
Phaseolus lunatus (Lima bean)
Pisum sativum (pea)
LBA
PSL
SBA
Glycine max (soya bean)
Ricin
PHA
Euphorbiaceae
Phaseolus vulgaris
GSL-I
RCA
Ricinus communis (Castor bean)
Griffonia simplicifolia I
ConA
Canavalia ensiformis (Jack Bean)
Plant species
Name/
abbreviation
GlcNAcb2Mana6(GlcNAcb2Mana3)
-Manb4GlcNAc4200
Galb4GlcNAcb2Mana6
(Galb4-GlcNAcb2Mana3)Manb4GlcNAc50
GalNAc
Man/Glc
GalNAca3(Fuca2)Galb-R43
Neu5Aca6Galb4GlcNAcb2Mana6(Neu5Aca6Galb4GlcNAcb2Mana3)Manb4GlcNAcb4(Fuca6)GlcNAcbAsn780
No binding
Galb4GlcNAcb2Mana6(GlcNAcb2-Mana3)
monosaccharide
(GlcNAcb4)Manb4GlcNAc
known
Gal/GalNAc
GalNAca3GalNAcb3Gala4Galb4Glc15
(isolectin A-4)
GalNAc/Gal
No oligosaccharide known better
than GalNAc
Gal/GalNAc
Galb3GalNAc 55
Gal/GalNAc
GalNAcb6Gal 16
Gal/GalNAc*
Man/Glc
Monosaccharides
Olygosaccharides ratio of inhibitory potency
compared with the monosaccharide
Carbohydrate binding specificity
Table 1
Plant lectins: names, carbohydrate specificity, are contents(4, 5)
(Continued)
170
140
190
170
300
700
1200
1400
2100
Content Mg lectin/
100g seeds
4
Bauhinia purpurea
Maclura pomifera Osage
orange (Moraceae)
Triticum vulgare (wheat)
(Gramineae)
Vicia faba (fava bean)
Phytolacca americana
(pokeweed) (Phytolaccaceae)
Dolichos biflorus (horse gram)
Lotus tetragonolobus
(asparagus pea)
Lens culinaris (lentil)
Plant Species
BPA
MPA
Favin
WGA
LCL
GalNAc
GalNAc
GlcNAc
(low affinity)
Man/Glc
Man/Glc
Neu5Aca6Galb4GlcNAcb2Mana6(Neu5Aca6Galb4GlcNAcb2Mana3)Manb4GlcNAcb4(Fuca6)GlcNAcbAsn6300
GlcNAcb4GlcNAcb4GlcNAcb4GlcNAcb4GlcNAc48
Neu5Aca6Galb4GlcNAcb2Mana6(Neu5Aca6Galb4GlcNAcb2Mana3)Manb4GlcNAcb4(Fuca6)GlcNAcbAsn780
Galb3GalNAc 611
Galb3GalNAc 24
GalNAca3GalNAca3Galb4Galb4Glc62
Fuca6GlcNAc 6.5
DBL (DB58 root) GalNAc
LTA
Fuc
Olygosaccharides ratio of inhibitory potency
compared with the monosaccharide
Galb4GlcNAcb6Gal no ratio available
Monosaccharides
GlcNAc
PWM root
Name/
abbreviation
Carbohydrate binding specificity
Table 1
(Continued)
28
24
30
45
60
110
65
125
Content Mg lectin/
100g seeds
Red Kidney Bean Lectin
5
Table 2
Edible plants (besides legumes) that exhibit lectin activity(4)
Scientific name
Common name
Scientific name
Common name
Cereals
Avena sativa
Hordeum vulgare
Oryza sativum
Oats
Barley
Rice
Secale cereale
Triticum vulgare
Zea mays
Rye
Wheat
Corn
Vegetables
Apium graveolens
Asparagus officinalis
Beta vulgaris
Caspicum annum
Chicorium inbitus
Cucubrita peppo
Cucubrita sativum
Celery
Asparagus
Beet
Sweet pepper
Chicory
Zucchini
Cucumber
Ipomea batatas
Lepidium sativum
Lycopersicon esculentum
Medicago sativum
Petrosecinum hortense
Rheum rhapontium
Solanum tuberosum
Sweet potato
Cargen cress
Tomato
Alfalfa
Parsley
Rhubarb
Potato
Fruits
Curica papaya
Citrus aurantum
Citrus medica
Citrullus vulgaris
Cuccumis melo
cantalupensis
Fragaria vesca
Malus species
Papaya
Orange/lemon
Grapefruit
Watermelon
Cantaloupe
Musa paradisiac
Prunus Americana
Prunus avium bigarreaus
Punica granatum
Ribes rubrum
Banana
Plum
Cherry
Pomegranate
Currant
Strawberry
Apple
Rubrus idaeus
Rubrus fructicosus
Raspberry
Blackberry
Spices
Alliuum sativum
Labiacae organum
Myristica fragrans
Garlic
Marjoram
Nutmeg
Menta piperita
Pimenta officinalis
Peppermint
Allspice
Other
Agarus bisporus
Carum carvi
Coccus nucifera
Coffea Arabica
Corylus avelania
Mushroom
Caraway seeds
Coconut
Coffee
Hazelnut
Halianthus annus
Juglans regia
Phaseolus mungum
Sesamum indicum
Theobremo cacao
Sunflower seeds
Walnut
Mung bean sprouts
Sesame seeds
Cocoa
Chemical Properties of Lectins
Lectins are proteins or glycoproteins of non-immune origin which specifically bind (or
cross-link) carbohydrates.(15) Ligand binding, which is the first step in the reaction 95
between lectins and carbohydrates, is similar for both lectins and carbohydrate-reactive
enzymes (Fig. 1). In contrast to the actions of enzymes, formation of the lectin-sugar
complex is not followed by splitting of chemical bonds in the covalent structure of the
carbohydrate ligands.(16) Lectins exhibit many interesting biological properties, such as specificity for human blood types, preferential agglutination of malignant cells, and mitogenic 100
6
Zhang et al.
Figure 1. Structure of Phytohaemagglutinin (PHA) (Protein Data Bank identifier 1FAT).
stimulation of(17) lymphocytes. As a result, they are being utilized extensively as macromolecular carbohydrate-specific reagents for probing the structure, organization, and
function of cell-surface glycoconjugates, and the changes they undergo during cell growth
and malignant transformations.
Lectins are essential and ubiquitous plant constituents. Among other functions, they
are responsible for innate immunity and defense mechanisms in plants. As many foods are
of plant origin, the daily ingestion of lectins by both humans and animals is significant. In
an ad hoc survey, 53 edible plants were shown to contain lectins and approximately 30%
of fresh and processed foods regularly consumed by humans had significant hemagglutinating activity.(6) Since lectins are often resistant to heat and proteolytic enzymes, including
those of intestinal microflora, the effects of consumption of these proteins deserve special
attention.
Based on their quaternary structure, legume lectins are traditionally subdivided into
two categories. One group consists of lectins with identical or nearly identical subunits,
such as Concanavalin A, while the other category is characterized by different subunit
types. Although legume lectins share an extended sequence homolog, they display an
unusual interspecies variability of sugar specificity (Table 1). Typically, specificities for
Glc, GlcNAc, Man, but also for Gal, GalNAc, Fuc, and complex types oligosaccharides
have been detected. Phytohaemagglutinin (PHA), a lectin isolated from the red kidney
bean (Phaseolus vulgaris), belongs to this group. It consists of four subunits. There are
two different types of subunits. One appears to be involved primarily in red cell agglutination and has been designated the “E” subunit (for erythroagglutinin). The other type is
involved in lymphocyte agglutination and mitogenic activity and has been termed the “L”
subunit (for leucoagglutinin). These subunits are synthesized alongside in the endoplasmatic reticulum and then randomly combined to produce five isolectins that are assigned
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120
125
Red Kidney Bean Lectin
7
the structures L4, L3E1, L2E2, L1E3, and E4. All of these isolectins are glycoproteins of
approximately 125 kDa.(18) However, these isolectins have similar physicochemical properties and structural homology, but differ in their relative biological activities, resulting in
the biological and biochemical properties of PHA showing inconsistencies. The different
biological actions of the isolectins may reflect different bioactivity of lectins in the beans.
For example, PHA-L and PHA-E possesses different agglutination activity. E4 isolectin
has higher agglutination activity, and agglutination activity decreased through L1E3, L2E2,
and L3E1, but L4 isolectin does not show the activity.(11) Therefore, the PHA isolectins
have unique value for revealing lectin action. Moreover, the isolated pure isolectins are
useful as cell surface probes to study how these structural differences confer different biological activities and how valence affects cell-lectin interactions and mitogenic activity.
Both PHA subunits contain the characteristic N-glycosylation sequence(19) 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 highmannose type, while glycan at Asn60 belongs to the complex type containing xylose and
fucose.(20)
Lectins bind carbohydrates reversibly and non-covalently, detecting subtle differences in their complex structure.(21) According to their carbohydrate specificity, they can
broadly be divided into those that bind monosaccharides, as well as oligosaccharides and
those that recognize oligosaccharides exclusively. However, since lectins with the same
specificity may show considerably different sugar-binding preferences, more recent classification depends on the source.(19)
Their carbohydrate specificity is the basis for many of the biochemical functions of
lectins. This property, in addition to their abundance in plants, gives them a significant
popularity in research. Names, carbohydrate specificity, and contents of lectins from various edible plants are given in Table 1.
In addition to the sugar binding properties of the legume lectins, a number of legume
lectins can also bind adenine.(20) High affinity binding sites for adenine and its derivatives
have been found for PHA-E, Dolichos biflorus seed lectin (DBL), D. biflorus stem and
leaves lectin (DB58), soybean agglutinin, and Phaseolus lunatus lectin (LBL).(22, 23)
All legume lectins possess two bound metal ions (one calcium ion and one transition
metal ion, mainly Mn2+) per monomer, in the vicinity of the sugar binding site. The presence of these two bound metal ions is vital for the sugar binding capabilities of the legume
lectins.(24)
Isolation of lectin from kidney bean seeds starts with an extraction step in water or
buffer, followed by salt precipitation. Figure 2 shows a general isolation procedure for
lectins.(17) Further purification of lectin by affinity chromatography is based on the ability
of kidney bean lectin to specifically bind sugars.
In affinity chromatography, the lectin binds specifically to an immobilized ligand
while the remaining molecules in the mixture flow through the column. The bound lectin
is then eluted from the column under conditions, which disrupts its interaction with the
ligand.(25) Purification of lectins by affinity chromatography is similar in principle to that
of other biopolymers with specific combining sites. Lectins do not biochemically modify
the carbohydrates which they bind. As a result, lectin can be displaced from the affinity
columns by the sugars for which they are specific. Knowledge of the specificity of lectin
allows the use of suitable purification procedures.(25)
The biospecific adsorbents for Phaseolus lectins are matrix-bound glycoproteins and
glycopeptides. The isolation of this lectin has been successfully performed using Affi-gel
Blue (Bio Rad).(26–28) A summary of purification with Affi-gel Blue is shown in Table 4.
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140
145
150
155
160
165
170
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Zhang et al.
Dry red kidney bean seed
Soaking
Grinding
Grinding
Swollen seeds
Red kidney bean powder
Delapidation
Homogenization
by blending
Lipid free material
Extraction with buffer TRIS pH = 7.2
Centrifugation 25 min 10,000 rmp
Crude extract
Precipitation with
ammonium sulphate 40%
Supernatant 40%
Precipitate 40%
Grinding
Precipitate 40%
Grinding
Pre-purified
Precipitation with ammonium sulphate 80%
Supernatant 80%
Pre-purified
AFFINITY
CHROMATOGRAPHY
PURIFIED LECTIN
Figure 2. Scheme for lectin purification (modified from Ref.(4)
Table 3
Biological activities of plant lectins(4)
Lectin activity
Cell agglutination
Inhibition of nuclear transport in vivo
Immunosuppressive effects in vivo
Inhibition of growth and killing of tumour cells
Insulin like effects on fat cells
Mediation of lysis of antigenically unrelated cells
by cytotoxic T-cells
Mitogenic stimulation of lymphocytes
Preservation of primitive progenitor cells
Protection of human T-cells against HIV infection
Examples of lectins involved
Most plant lectins
WGA
Con A
Ricin, Viscum album agglutinin
WGA
Can A, CSL-I
PHA, Con A, PMW, artocarpin
FRILc, PvFRIL
NPL, BMA, GNA, cyanovirin-N
Red Kidney Bean Lectin
9
Table 4
Summary of purification of lectin from red kidney bean seeds(6)
Fraction
Q1
Protein* (mg)
Crude extract
Adsorbed on Affi-gel blue gel
Adsorbed on CM-sepharose
10,580
347
55
*Protein obtained from 200 g of red kidney bean seeds.
Biological Activities of Lectins
All cells come in sugar coating, which consists of carbohydrate chains, membrane glycoproteins and glycolipids, or polysaccharides. These carbohydrates are potential sites of
attachment for lectins. Attachment may induce variety of changes in the cell, such as cell
agglutination, mitogenic stimulation, cytotoxicity and insulin-like effects. Table 3 shows
biological activities of some plant lectins.(21)
Agglutination is the most easily detectable manifestation of the interaction of a lectin
with a cell. For agglutination to occur, a lectin molecule must form multiple cross-bridges
between opposing cells. Agglutination is a process affected by properties of the lectin,
such as number of sugar binding sites and molecular size, as well as cell-surface properties, such as accessibility of receptor sites or membrane fluidity. It is also affected by
external conditions such as temperature, cell concentration, etc.(29)
Another property of some lectins is mitogenic stimulation of lymphocytes, which are
normally not dividing. It is not clear why some lectins are mitogenic since the structures to
which mitogenic lectins bind are not necessarily the same, and not all lectins with similar
binding specificity are mitogenic. It is likely that binding to the cell surface alone is not sufficient to cause mitosis but that other interactions on the cell surface are equally important.
Most mitogenic lectins, like Concanavalin A and PHA, stimulate only the thymus dependant lymphocytes (T-cells). Unlike antigens that stimulate only 0.1% or less of the total
number of lymphocytes, mitogenic lectins stimulate as much as 80% of the susceptible
cells.(25)
Lectins are proteins that have extraordinary resistance to proteolytic enzymes found
in the digestive tract. The stability of plant lectins in the stomach has been investigated
using Con A, PHA and WGA. When fed to rats, between 50 and 90% of these lectins
were recovered after 1 hour from the stomach tissue.(29) In a few experiments with
humans that ate lectin containing food, such as tomatos(24) and red kidney beans, the lectins withstood the acidity and proteolytic enzymes of the intestinal tract and a significant amount reached the circulatory system with unimpaired hemagglutinating and
immunological activity. Lectins are also resistant to breakdown by most bacteria found
in the intestinal microflora.(29)
Upon reaching the intestinal tract, the bulk of the lectin may bind to carbohydrates on
the epithelial cells.(30) Binding is most pronounced in the small intestine, but it takes place
throughout the entire digestive system. A major consequence is damage to the absorption
of nutrients across the intestinal wall due to changes in intestinal permeability. Food lectins interfere with the normal process of food absorption by interacting with brush border
hydrolases that play a role in the digestion of proteins and carbohydrates.(17)
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190
195
200
205
210
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Zhang et al.
Health Benefits of Lectins
Kidney Bean Nutritional Benefits
Health benefits of beans are manifold. Their low glycemic index (class III) helps maintain
healthy blood glucose levels and healthy weight levels. Beans are rich in folate that plays
a role in reduction of birth defects and maintain healthy vascular heart condition. The 215
abundance of iron and other vitamins and minerals, such as B1, B3, and panthotenic acid,
potassium, copper, and phosphorus, in beans can promote overall health.
Inhibitory Effect of Lectin on HIV-1 Reverse Transcriptase
Balizarini et al.(31) noted that the mannose specific lectins from Cymbidium hybrid
(pink orchid), Epipactis helleborine (helleborine orchid) and Listeria ovata and the
N-acetylglucosamine specific Urtica diodica (Stinging Nettle) lectin potently and selectively inhibited HIV-1 and HIV-2 in MT4-cells.* Protection of human T-cells against HIV
infection was noted for a number of lectins (NLP, BMA, GNA, and cyanovirin-N).(32, 33)
An inhibitory effect of some plant lectins on human immunodeficiency virus type 1
reverse transcriptase (HIV1-RT) has been reported.(29, 34–38) In several studies they have
isolated lectin from kidney bean seeds and demonstrated that Phaseolus vulgaris lectin
was able to directly inhibit HIV-1 reverse transcriptase, an enzyme crucial for HIV replication,(22, 23, 34) as well as β-glucosidase, which has a role in HIV-1 envelope protein gp
120 processing.(20) A dose-response curve for inhibitory effects of PHA on HIV-1 reverse
transcriptase is given in Table 5.
Human immunodeficiency virus reverse transcriptase (HIV-RT) is one of the enzymes
essential for HIV-1 replication cycle. This important role of RT in HIV replication has made
the enzyme a primary target for antiretroviral chemotherapy. Figure 3 shows HIV replication
in lymphocytes, indicating where reverse transcriptase inhibitors act.(31)
Reverse transcriptase is a multifunctional enzyme utilized by HIV and other retroviruses to convert their single stranded viral RNA into a single stranded DNA, and to subsequently construct a complementary strand of proviral DNA, forming a double helix
capable of integration into host cell chromosomes (polymerase function) where it is integrated into the host cellular DNA by the activity of viral integrase.(30, 32, 33, 37–39) HIV RT
then degrades the RNA template (RNase H function).(26) Because of the importance of RT
to HIV replication reverse transcriptase, HIV-1 RT inhibitors can reduce the viral load of
HIV-1 infected individuals. This makes the inhibitors potential therapeutic agents in the
battle against HIV. Functional HIV1-RT is a heterodimer containing subunits of 66 kDa
(p66) and 51 kDa (p51). The first unit contains two domains, the N-terminal polymerase
Table 5
Inhibitory effects of Phaseolus vulgaris lectin on HIV-1 reverse
transcriptase: dose response study(6, 15)
Phaseolus vulgaris
lectin
PHA pure from sigma
Red kidney bean lectin
% Inhibition
5 mg/ml
0.5 mg/ml
0.05 mg/ml
EC
95.4 ± 7.8
80.2 ± 3.5
21.6 ± 1.8
—
0.6 ± 0.1
—
2.19
—
220
225
230
235
240
Red Kidney Bean Lectin
11
Figure 3. Replication of HIV in CD4 lymphocytes, lifecycle of HIV replication in CD4 lymphocytes, indicating where different antiretroviral inhibitors act.(24)
domain (440 residues) and the C-terminal RNase H domain (120 residues). Portions of 245
both the p51 and the polymerase domain of p66 can be described as a “right hand” that
contains three subdomains: fingers, palm, and thumb. The connection subdomain
connects the hand of the polymerase domain and the RNase H domain in p66, which
provides the ribonuclease activity of HIV-RT. Although p51 contains a connection
subdomain, it lacks an RNase domain. The connection subdomains and the palm subdo- 250
mains contain three-stranded α-sheets with α-helices on one side. The thumb subdomains comprise three α-helices.(34)
12
Zhang et al.
The majority of the template/primer-RT interactions are thought to occur between the
sugar-phosphate backbone of the DNA/RNA and p66.(34) The two α-helices of the thumb,
in combination with the fingers, serve as a clamp, holding the nucleic acid in place over 255
the palm that contains the polymerase active site. In the functional heterodimer, the p51
subunit has been modeled to bind the anticodon stem and loop of tRNA at the start of
reverse transcription. The thumb of p66 partially embeds itself in the minor groove of the
DNA and the thumb of p51, plus the connection domains of p66 and p51 form the floor of
the binding cleft. The active site of p66 contains three catalytic residues in the palm 260
subdomain that may bind metal ions (Asp185, Asp186, and Asp110). This active triad is positioned close to the 3’ - OH terminus of the DNA primer.(32)
HIV-RT Inhibitors. Because of the importance of RT to HIV replication, reverse transcriptase inhibitors can decrease the viral load of HIV-1 infected individuals. This makes 265
them potential therapeutic agents in the battle against HIV. Reverse transcriptase inhibitors can be classified into two major groups, depending upon their structure: nucleoside
analogs and non-nucleoside RT inhibitors (NNRTIs).(29) Nucleoside inhibitors like AZT,
ddI, and ddC are dideoxy compounds lacking a3’ oxygen, causing DNA chain termination
when they are incorporated into a growing DNA strand.(34) NNRTIs act in different ways 270
to block HIV-RT polymerase activity. They have been shown to bind in a pocket, ~10 Å
away from the polymerase active site. The internal surface of this pocket is predominantly
hydrophobic, constructed primarily from leucine, valine, tryptophan, and tyrosine residues. Although chemically diverse compounds, the NNRTIs bind in a common mode.(32)
Assay for Reverse Transcriptase Inhibitory Activity. Reverse transcriptase ELISA assay
is used for detection and quantification of enzyme activity and in vitro screening for RT
inhibitors. Non-radioactive enzyme immunoassay from Roche Applied Science gave satisfactory results in the studies of the inhibitory effects of plant lectins on HIV-1 RT.(16, 17,
24, 26)
The method takes advantage of the ability of reverse transcriptase to synthesize
DNA, starting from the template/primer hybrid poly (A) × oligo (dT)15. Digoxigenin- and
biotin-labeled nucleotides in an optimized ratio are incorporated into the same DNA molecule, which is newly synthesized by the RT. The amount of synthesized DNA is quantitated as a parameter for RT activity following a sandwich ELISA protocol. In the first
step, biotin-labeled DNA binds to the surface of microtiter plate (MTP) modules that have
been precoated with streptavidin. In the next step, an antibody to digoxigenin, conjugated
to horseradish peroxidase (anti-DIG-POD), binds to the digoxigenin-label of the bound
DNA. In the final step, the chemiluminescent peroxidase substrate luminol/4-iodophenol
is added. Peroxidase in the presence of hydrogen peroxide (H2O2) catalyzes the oxidation
of luminol, resulting in reaction products in an electronically excited state. The excited
state molecules emit light during decay to the ground state, a process which is strongly
enhanced by the radical transmitter 4-iodophenol. The emitted light is quantified using a
microtiter plate chemiluminescence reader (96-well format) and is directly correlated to
the level of RT activity in the sample.
275
Inhibitory Effect of Lectin on Tumor Growth
295
Rudiger and Gabius(2) reported that certain types of malignant cells are more readily
agglutinated by lectins then the corresponding normal cells. Preferential agglutination of
cancer cells by wheat germ agglutinin (WGA) was reported by Pusztai.(10, 41) Similar
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285
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Red Kidney Bean Lectin
13
discoveries followed for other lectins, which resulted in these compounds gaining an
important role in cancer research.
Numerous studies have been conducted comparing the extent and pattern of lectin
binding to normal and malignant cells. Such investigations have contributed to the knowledge
of the structural changes in cell surface saccharides that accompany malignant transformation,
as well as those that characterize the process of metastasis.
Phytohaemagglutinin from kidney bean has been recently reported to inhibit growth
of lymphoid tumor in vitro and in vivo.(30) This study has shown that PHA can inhibit
incorporation of 3H-thymidin in a variety of lymphoid tumors, including cells from mouse
and human origin of both T-cell and B-cell tumor lines. The inhibitory effect was enhanced
when PHA was used in conjunction with the cell cycle directed drug 5-fluorouracil (5-FU).
PHA not only altered lymphoid cell growth in vitro, but it has shown to be able to impede
(and in some cases eradicate) B-cell tumor in vivo when used alone or in combination with
5-FU, showing no toxic side effects in the animals.(30)
Results of another study proved that growth of an established murine non-Hodgkin
lymphoma tumor is limited by switching to a phytohaemagglutinin containing diet.(40,41)
The growth of a non-Hodgkin lymphoma in mice was markedly diminished by including
PHA from raw kidney bean in the diet. The tumor was first allowed to develop for five
days before switching the animals to a diet containing PHA at different concentrations.
This switch of diets proved to be effective in slowing down growth of the lymphoma
tumor. The reduced rate of tumor growth occurred in a dose-dependent manner.(40)
Anticancer activities of other lectins from edible plants have been reported (Fig. 3).
The antitumoral and immunostimulating properties of rViscumin (recombinant mistletoe
lectin) has been investigated in two mouse tumor models.(42) An overall prolonged
survival time after treatment with rViscumin, and a reduction in the number of tumor
colonies after administration of certain rViscumin doses indicated that rViscumin had antineoplastic properties and might therefore be a promising candidate in cancer therapy.(42)
Viscum album agglutinin I- has also shown to induce enhanced proliferation and apoptosis
of murine thymocytes in vivo.(43)
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Q2
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325
Conclusion
A low glycemic index (class III) helps maintain healthy blood glucose levels and healthy
weight levels. Beans are rich in folate that plays a role in reduction of birth defects and
maintain healthy vascular heart condition. Their abundance in iron and other vitamins and
minerals, such as niacin, panthotenic acid, potassium, copper, and phosphorus, helps promote overall health. Kidney beans are high on the list of those nutriments rich in fiber and
which positively affect colon health. A recent study confirms that eating high fiber foods
such as kidney beans helps prevent heart disease.
It is known that lectins can cause antinutritional and allergic effects as they react with
the surface epithelium of the digestive tract. However, studies on lectin’s effect in the gut
have revealed that oral administration of low doses can have many beneficial effects on
digestive efficiency, the immune system, and the body’s endocrine system, with beneficial
consequences for general metabolism. With this wide range of potential applications,
lectins may be a practical and natural means of improving both the natural value and the
safety of a diet.
From the data currently available it is reasonable to expect PHA to have inhibitory
activity towards HIV-1 reverse transcriptase. This property may have potential for the use of
kidney beans as a food based treatment to ameliorate the effects of HIV infection.
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Zhang et al.
The content of total protein in kidney bean seeds is reported to be between 17–23%, of
which 2.4–5% is lectin. In comparison with other edible plants, kidney beans are rich in lectin, which makes them very attractive for additional research, including clinical trials. Therapeutic effects, nutritional benefits, and toxic consequences, should continue to be examined.
Acknowledgments
350
The authors gratefully acknowledge the contribution of the Guelph Food Research Center, Agriculture and Agri-Food Canada (AAFC Journal Series No. S386); and China’s NSFC Supports
(#30571414 and 30640015) to J. Zhang.
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