# Purification and Characterization of a Lectin fromPhaseolus vulgaris cv. (Anasazi Beans)

Authors: Arishya Sharma; Tzi Bun Ng; Jack Ho Wong; Peng Lin
Journal: Journal of Biomedicine and Biotechnology
 (2009)

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## Abstract

A lectin has been isolated from seeds of thePhaseolus vulgaris cv. “Anasazi beans” using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to other Phaseolus lectins. The hemagglutinating activity of the lectin was stable within the pH range of 1–14 and the temperature range of 0–80∘C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with an IC50 of 1.3 μM, and inhibited the activity of HIV-1 reverse transcriptase with an IC50 of 7.6 μM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.


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## Body

## 1. Introduction

Lectins are defined as proteins/glycoproteins
possessing at least one non-catalytic domain which binds reversibly to a
specific mono- or oligosaccharide [1]. Over the last few decades, lectins have
become a topic of interest to a large number of researchers owing to their
potentially exploitable biological properties including antitumor [2, 3],
immunomodulatory and anti-insect [4], antifungal [5], antibacterial [6], anti-HIV
[2, 5, 7], and mitogenic [8] activities. Because of their sugar binding
properties, lectins have been extensively studied and used as molecular tools for
the study of carbohydrate architecture and dynamics on the cell surface, and
have been exploited for such practical applications as distinguishing between
normal and malignant cells [9, 10], purification of glycoconjugates [11], and
coating of drugs to enhance their gastrointestinal tract absorption [12, 13].
Further, specific amino acid residues are essential for maintaining the carbohydrate
binding and hemagglutinating activities of lectins [14–16].
Identification of these amino acid residues is a prerequisite for investigating
the structure-function relationships of lectins. Chemical modification with
group-specific modifying agents provides a general approach for identification
of the amino acid residues present in the functional or active site of proteins,
including lectins [14, 16]. Hence, elucidation of biological activities of
lectins and amino acid residues essential to these activities is a meaningful undertaking. Although lectins are found ubiquitously
in plant species, they have variable structures and specific activities according
to the plants they originate from [9, 10]. Thus, purification and
characterization of lectins from a variety of plant species interests
researchers in the field of glycobiology. The more is known about the lectins,
the wider the applications of this type of proteins that can be achieved. This
study reports the purification and some properties of a new lectin isolated
from seeds of the Anasazi cultivar of Phaseolus vulgaris. To date, the isolation of a lectin
from the Anasazi bean and examining it for various potentially exploitable
biological activities such as mitogenic, antitumor, immunomodulatory, and HIV-1
reverse transcriptase inhibitory activities have not been attempted. In this
study, a lectin was isolated from Anasazi beans. It was assayed for the various
aforementioned activities. In order to further characterize the lectin, a
chemical modification study was undertaken to determine the involvement of
different amino acid residues in its hemagglutinating activity.


## 2. Materials and Methods

### 2.1. Materials

Anasazi beans, a product of Western
Family Foods Inc., Portland, Ore, USA, were purchased from a local supermarket.
Two hundred grams of Anasazi beans were soaked in 700 mL distilled water at
room temperature for 4 hours to soften the beans. Then they were blended, after
addition of 700 mL of distilled water in a Waring blender into a slurry.
Subsequently, the mixture was centrifuged at 14000 rpm for 30 minutes at 4°C.
Tris-HCl buffer (1 M)
at pH 7.3 was added to the supernatant collected after the centrifugation until
the final Tris concentration reached 10 mM. The supernatant was loaded on an
Affi-gel blue gel (Bio-Rad, Calif, USA) column (5×18 cm) which had been
equilibrated with the same buffer. After the unbound proteins had been eluted,
bound proteins with hemagglutinating activity were eluted with 1 M NaCl in 10 mM Tris-HCl buffer (pH
7.3), dialyzed, lyophilized, and then subjected to ion exchange chromatography
by fast protein liquid chromatography (FPLC) using an AKTA purifier (GE Healthcare,
Hong Kong, China) on a 1 mL Mono S column (GE Healthcare, Hong Kong) in 20 mM
Tris-HCl buffer (pH 7.3). The adsorbed fraction was further purified by
FPLC-gel filtration on a Superdex 200 column (GE Healthcare) in 20 mM Tris-HCl
buffer (pH 7.3).


### 2.2. Molecular Mass Determination by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and by FPLC-Gel Filtration

Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was carried out in
accordance with the procedure of Laemmli and Favre [17], using a 12% resolving
gel and a 5% stacking gel. At the end of electrophoresis, the gel was stained
with Coomassie brilliant blue. After destaining, the electrophoretic mobilities
of the marker proteins and the purified lectin were determined. The molecular
mass of the lectin was estimated from the standard curve plotting
electrophoretic mobility against molecular mass. FPLC-gel filtration was
carried out using a Superdex 200 HR 10/30 column (GE Healthcare) that had been
calibrated with molecular-mass standards (GE Healthcare) to estimate the
molecular mass of the purified lectin.


### 2.3. Analysis of N-Terminal Amino Acid Sequence

Amino acid sequence analysis was carried out using a Hewlett Packard
(HP) G1000A Edman degradation unit and an HP 1000 HPLC system.


### 2.4. Assay of Hemagglutinating Activity

In the assay for lectin (hemagglutinating) activity, a serial twofold dilution of
the lectin solution in microtiter U-plates (50μL) was mixed with 50 μL of a 2%
suspension of rabbit red blood cells in phosphate-buffered saline (pH 7.2) at
20°C. The results were read after about 1 hour, when the blank had fully
sedimented. The hemagglutination titer, defined as the reciprocal of the
highest dilution exhibiting hemagglutination, is reckoned as one
hemagglutination unit. Specific activity is the number of hemagglutination
units per mg protein [18].


### 2.5. Inhibition of Lectin-Induced Hemagglutination by Carbohydrates

The hemagglutinating inhibition tests
to investigate inhibition of lectin-induced hemagglutination by various
carbohydrates were performed in a manner analogous to the hemagglutination
test. A serial twofold dilution of each sugar samples was prepared in
phosphate-buffered saline. All of the dilutions were mixed with an equal volume
(25μL) of a solution of the lectin with 16 hemagglutination units. The mixture
was allowed to stand for 30 minutes at room temperature and then mixed with 50 μL
of a 2% rabbit erythrocyte suspension. The minimum concentration of the sugar
in the final reaction mixture which completely inhibited 16 hemagglutination
units of the lectin preparation was calculated [18].


### 2.6. Effect of Temperature on Lectin-Induced Hemagglutination

The effect of temperature on hemagglutinating
activity of the lectin was examined as previously described [19]. A solution of
the lectin with 16 hemagglutination units was incubated at various temperatures
for 30 minutes: 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C,
90°C, and 100°C. The tubes were then put on ice, and assay of
hemagglutinating activity was then carried out.


### 2.7. Effect of pH on Lectin-Induced Hemagglutination

The pH stability of the lectin was determined by incubation
of the lectin (1 mg/mL) in buffers of different pH values ranging from pH 1.0–14.0. for 60 minutes.
The pH of the lectin solution was adjusted to 7.0 by the addition of 0.1 N HCl
or 0.1 N NaOH before hemagglutination activity was determined.


### 2.8. Assay of Antiproliferative Activity on Tumor Cell Lines

Breast cancer MCF-7 cells or hepatoma HepG2
cells were suspended in RPMI medium and adjusted to a cell density of5×104 cells/mL. A 100 μL aliquot of this cell suspension was seeded to a well of a
96-well plate, followed by incubation for 24 hours. Different concentrations of
the lectin in 100 μL complete RPMI medium were then added to the wells followed
by an incubation for 24 hours. After 24 hours, 50 μL of a 5 mg/mL solution of
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) in
phosphate buffered saline was spiked into each well, and the plates were
incubated for 2 hours. The plates were then centrifuged at 2500 rpm for 5 minutes.
The supernatant was carefully removed, and 150 μL of dimethyl sulfoxide was
added in each well to dissolve the MTT (formazan) at the bottom of the wells. The absorbance at 590 nm was then measured with a microplate reader within 10
minuets.


### 2.9. Assay of HIV Reverse Transcriptase Inhibitory Activity

The assay of Anasazi bean lectin for ability to inhibit HIV-1 reverse
transcriptase was carried out by using an enzyme-linked immunosorbent assay kit
from Boehringer Mannheim (Germany) as described by Collins et al. [20]. The
assay takes advantage of the ability of reverse transcriptase to synthesize
DNA, starting from the template/primer hybrid poly(a)·oligo(dT) 15. Instead of radio-labeled
nucleotides, digoxigenin and biotin-labeled nucleotides in an optimized ratio
are incorporated into one and the same DNA molecule, which is freshly
synthesized by the reverse transcriptase (RT). The detection and quantification
of synthesized DNA as a parameter for RT activity follows a sandwich enzyme-linked
immunosorbent assay protocol. Biotin-labeled DNA binds to the surface of
microtiter plate modules that have been precoated with streptavidin. In the
next step, an antibody to digoxigenin, conjugated to peroxidase (anti-DIG-POD),
binds to the digoxigenin-labeled DNA. In the final step, the peroxidase
substrate is added. The peroxidase enzyme catalyzes the cleavage of the
substrate, producing a colored reaction product. The absorbance of the samples
at 405 nm can be read using a microtiter plate (ELISA) reader and is directly
proportional to the level of RT activity. A fixed amount (4–6 ng) of
recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of
the purified protein was calculated as percent inhibition as compared to a
control without the lectin [20].


### 2.10. Assay of Nitric Oxide Production by Murine Peritoneal Macrophages

The assay was conducted as described
by Wong and Ng, 2006 [21]. Macrophages were collected from the peritoneal
cavity of mice after an intraperitoneal injection of a 3% thioglycolate
solution. The cells were washed and resuspended in RPMI medium containing 10%
heat-inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL
streptomycin. Cells (2×105 cells/well) were seeded in a 96-well culture
plate for 1 hour, before incubation with the Anasazi bean lectin for 24 hours.
The amount of nitric oxide in the culture medium was
determined by a colorimetric method. In the assay, a 100 μL aliquot of
cell-free culture medium from each culture well was allowed to react with 50 μL
of Griess reagent (1% sulfanilamide in 5%  H3PO4-0.1%
naphthalene-ethylenediamine dihydrochloride) for 10 minutes before the
absorbance was read at 540 nm using a microplate reader. Lipopolysaccharide was
used as a positive control in this assay. Dexamethasone (5 μM) was used as
inducible nitric oxide synthase (iNOS) inhibitor in this assay.


### 2.11. Assay of Antifungal Activity

The antifungal activity of Anasazi bean lectin towardBotrytis cinerea,
Mycosphaerella arachidicola, and Fusarium oxysporum was examined
using 90×15 mm Petri plates that contained 10 mL of potato dextrose agar, in
view of the report that some lectins displayed antifungal activity [4]. After
the mycelial colony had developed, sterile blank paper disks (0.625 cm in
diameter) were placed at a distance of 0.5 cm away from the rim of the mycelial
colony. An aliquot of a solution of Anasazi bean lectin was added to a disk.
The plates were kept at 25°C for 72 hours until mycelial growth had enveloped
disks containing the control and crescents of inhibition had formed around
disks containing samples with antifungal activity.


### 2.12. Assay of Mitogenic Activity

Four C57BL/6 mice (20–25 g) were killed
by cervical dislocation, and the spleens were aseptically removed. Spleen cells
were isolated by pressing the tissue through a sterilized 100-mesh stainless steel
sieve and resuspended to5×106 cells/mL in RPMI 1640 culture medium
supplemented with 10% fetal bovine serum, 100 units penicillin/mL, and 100 μg
streptomycin/mL. The cells (7×105 cells/100 μL/well) were seeded
into a 96-well culture plate, and serial dilutions of a solution of Anasazi bean
lectin in 100 μL medium were added. After incubation of the cells at 37°C in a
humidified atmosphere of 5% CO2 for 24 hours, 10 μL methyl [H3]thymidine
(0.25 μCi, GE Healthcare) was added, and the cells were incubated for further 6
hours under the same conditions. The cells were then harvested with an
automated cell harvester onto a glass fiber filter and the radioactivity was
measured with a Beckman model LS 6000SC scintillation counter. All reported
values are means of triplicate samples [22].


### 2.13. Effect of Chemical Modification of Amino Acid Residues on Hemagglutinating Activity

For serine modification, the lectin
(100μg) in 0.1 mL of 50 mM Tris-HCl buffer (pH 7.4) was incubated with 5 mM
phenylmethylsulfonyl fluoride (PMSF) at 27°C for 1 hour [23]. Aliquots were
removed at 15 minutes intervals. Excess reagent was removed by ultrafiltration,
followed by determination of residual hemagglutinating activity.
Lectin incubated without PMSF served as a control.Reduction of the thiol groups of Anasazi bean
lectin was carried out by incubating the lectin (100μg) in 0.1 mL of 50 mM phosphate
buffer (pH 8.0) with 0.1 mM 5, 5′-dithiobis-(2-nitrobenzoic acid (DTNB) at 27°C for 1 hour. Aliquots were removed at different time intervals. Excess reagent
was removed by ultrafiltration, followed by determination of residual
hemagglutinating activity. Lectin incubated in the absence of DTNB served as a
control [24].For lysine modification, 0.5 mg of NaBH4 was added to the lectin (5 mg) in 2 mL of 0.2 M sodium borate buffer (pH 9.0)
at 4°C, followed by six aliquots (5 μL each) of 3.5% formaldehyde at 10 minutes
intervals. Excess reagent was removed by ultrafiltration, followed by
determination of residual hemagglutinating activity. Lectin incubated in the
absence of sodium borohydride (NaBH4) served as a control [25].Modification of tryptophan residues was carried out
according to the method of Spande and Witkop [26]. The lectin was dissolved in
NaOAc buffer (0.1 M, pH 5.0) to 1 mg/mL. The modification was carried out at 20°C. N-bromosuccinimide (NBS) (10 μL, 10 mM) was added every 5 minutes. The
number of tryptophan residues was calculated as described by Spande and Witkop
[26]. The samples were then desalted by ultrafiltration to remove excess
reagent, and then assay of hemagglutinating activity was carried out. Lectin
incubated in the absence of NBS served as a control.Acetylation of the tyrosine side chain phenoxy
groups of the lectin was done at room temperature by incubating 300μg lectin
in 1.0 mL of 25 mM phosphate buffer (pH 7.5) containing 60-fold molar excess
(0.1–10 mM) of
N-acetylimidazole. Excess reagent was removed by ultrafiltration,
followed by determination of residual hemagglutinating activity. Lectin incubated
in the absence of N-acetylimidazole served as a control [27].


## 2.1. Materials

Anasazi beans, a product of Western
Family Foods Inc., Portland, Ore, USA, were purchased from a local supermarket.
Two hundred grams of Anasazi beans were soaked in 700 mL distilled water at
room temperature for 4 hours to soften the beans. Then they were blended, after
addition of 700 mL of distilled water in a Waring blender into a slurry.
Subsequently, the mixture was centrifuged at 14000 rpm for 30 minutes at 4°C.
Tris-HCl buffer (1 M)
at pH 7.3 was added to the supernatant collected after the centrifugation until
the final Tris concentration reached 10 mM. The supernatant was loaded on an
Affi-gel blue gel (Bio-Rad, Calif, USA) column (5×18 cm) which had been
equilibrated with the same buffer. After the unbound proteins had been eluted,
bound proteins with hemagglutinating activity were eluted with 1 M NaCl in 10 mM Tris-HCl buffer (pH
7.3), dialyzed, lyophilized, and then subjected to ion exchange chromatography
by fast protein liquid chromatography (FPLC) using an AKTA purifier (GE Healthcare,
Hong Kong, China) on a 1 mL Mono S column (GE Healthcare, Hong Kong) in 20 mM
Tris-HCl buffer (pH 7.3). The adsorbed fraction was further purified by
FPLC-gel filtration on a Superdex 200 column (GE Healthcare) in 20 mM Tris-HCl
buffer (pH 7.3).


## 2.2. Molecular Mass Determination by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and by FPLC-Gel Filtration

Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was carried out in
accordance with the procedure of Laemmli and Favre [17], using a 12% resolving
gel and a 5% stacking gel. At the end of electrophoresis, the gel was stained
with Coomassie brilliant blue. After destaining, the electrophoretic mobilities
of the marker proteins and the purified lectin were determined. The molecular
mass of the lectin was estimated from the standard curve plotting
electrophoretic mobility against molecular mass. FPLC-gel filtration was
carried out using a Superdex 200 HR 10/30 column (GE Healthcare) that had been
calibrated with molecular-mass standards (GE Healthcare) to estimate the
molecular mass of the purified lectin.


## 2.3. Analysis of N-Terminal Amino Acid Sequence

Amino acid sequence analysis was carried out using a Hewlett Packard
(HP) G1000A Edman degradation unit and an HP 1000 HPLC system.


## 2.4. Assay of Hemagglutinating Activity

In the assay for lectin (hemagglutinating) activity, a serial twofold dilution of
the lectin solution in microtiter U-plates (50μL) was mixed with 50 μL of a 2%
suspension of rabbit red blood cells in phosphate-buffered saline (pH 7.2) at
20°C. The results were read after about 1 hour, when the blank had fully
sedimented. The hemagglutination titer, defined as the reciprocal of the
highest dilution exhibiting hemagglutination, is reckoned as one
hemagglutination unit. Specific activity is the number of hemagglutination
units per mg protein [18].


## 2.5. Inhibition of Lectin-Induced Hemagglutination by Carbohydrates

The hemagglutinating inhibition tests
to investigate inhibition of lectin-induced hemagglutination by various
carbohydrates were performed in a manner analogous to the hemagglutination
test. A serial twofold dilution of each sugar samples was prepared in
phosphate-buffered saline. All of the dilutions were mixed with an equal volume
(25μL) of a solution of the lectin with 16 hemagglutination units. The mixture
was allowed to stand for 30 minutes at room temperature and then mixed with 50 μL
of a 2% rabbit erythrocyte suspension. The minimum concentration of the sugar
in the final reaction mixture which completely inhibited 16 hemagglutination
units of the lectin preparation was calculated [18].


## 2.6. Effect of Temperature on Lectin-Induced Hemagglutination

The effect of temperature on hemagglutinating
activity of the lectin was examined as previously described [19]. A solution of
the lectin with 16 hemagglutination units was incubated at various temperatures
for 30 minutes: 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C,
90°C, and 100°C. The tubes were then put on ice, and assay of
hemagglutinating activity was then carried out.


## 2.7. Effect of pH on Lectin-Induced Hemagglutination

The pH stability of the lectin was determined by incubation
of the lectin (1 mg/mL) in buffers of different pH values ranging from pH 1.0–14.0. for 60 minutes.
The pH of the lectin solution was adjusted to 7.0 by the addition of 0.1 N HCl
or 0.1 N NaOH before hemagglutination activity was determined.


## 2.8. Assay of Antiproliferative Activity on Tumor Cell Lines

Breast cancer MCF-7 cells or hepatoma HepG2
cells were suspended in RPMI medium and adjusted to a cell density of5×104 cells/mL. A 100 μL aliquot of this cell suspension was seeded to a well of a
96-well plate, followed by incubation for 24 hours. Different concentrations of
the lectin in 100 μL complete RPMI medium were then added to the wells followed
by an incubation for 24 hours. After 24 hours, 50 μL of a 5 mg/mL solution of
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) in
phosphate buffered saline was spiked into each well, and the plates were
incubated for 2 hours. The plates were then centrifuged at 2500 rpm for 5 minutes.
The supernatant was carefully removed, and 150 μL of dimethyl sulfoxide was
added in each well to dissolve the MTT (formazan) at the bottom of the wells. The absorbance at 590 nm was then measured with a microplate reader within 10
minuets.


## 2.9. Assay of HIV Reverse Transcriptase Inhibitory Activity

The assay of Anasazi bean lectin for ability to inhibit HIV-1 reverse
transcriptase was carried out by using an enzyme-linked immunosorbent assay kit
from Boehringer Mannheim (Germany) as described by Collins et al. [20]. The
assay takes advantage of the ability of reverse transcriptase to synthesize
DNA, starting from the template/primer hybrid poly(a)·oligo(dT) 15. Instead of radio-labeled
nucleotides, digoxigenin and biotin-labeled nucleotides in an optimized ratio
are incorporated into one and the same DNA molecule, which is freshly
synthesized by the reverse transcriptase (RT). The detection and quantification
of synthesized DNA as a parameter for RT activity follows a sandwich enzyme-linked
immunosorbent assay protocol. Biotin-labeled DNA binds to the surface of
microtiter plate modules that have been precoated with streptavidin. In the
next step, an antibody to digoxigenin, conjugated to peroxidase (anti-DIG-POD),
binds to the digoxigenin-labeled DNA. In the final step, the peroxidase
substrate is added. The peroxidase enzyme catalyzes the cleavage of the
substrate, producing a colored reaction product. The absorbance of the samples
at 405 nm can be read using a microtiter plate (ELISA) reader and is directly
proportional to the level of RT activity. A fixed amount (4–6 ng) of
recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of
the purified protein was calculated as percent inhibition as compared to a
control without the lectin [20].


## 2.10. Assay of Nitric Oxide Production by Murine Peritoneal Macrophages

The assay was conducted as described
by Wong and Ng, 2006 [21]. Macrophages were collected from the peritoneal
cavity of mice after an intraperitoneal injection of a 3% thioglycolate
solution. The cells were washed and resuspended in RPMI medium containing 10%
heat-inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL
streptomycin. Cells (2×105 cells/well) were seeded in a 96-well culture
plate for 1 hour, before incubation with the Anasazi bean lectin for 24 hours.
The amount of nitric oxide in the culture medium was
determined by a colorimetric method. In the assay, a 100 μL aliquot of
cell-free culture medium from each culture well was allowed to react with 50 μL
of Griess reagent (1% sulfanilamide in 5%  H3PO4-0.1%
naphthalene-ethylenediamine dihydrochloride) for 10 minutes before the
absorbance was read at 540 nm using a microplate reader. Lipopolysaccharide was
used as a positive control in this assay. Dexamethasone (5 μM) was used as
inducible nitric oxide synthase (iNOS) inhibitor in this assay.


## 2.11. Assay of Antifungal Activity

The antifungal activity of Anasazi bean lectin towardBotrytis cinerea,
Mycosphaerella arachidicola, and Fusarium oxysporum was examined
using 90×15 mm Petri plates that contained 10 mL of potato dextrose agar, in
view of the report that some lectins displayed antifungal activity [4]. After
the mycelial colony had developed, sterile blank paper disks (0.625 cm in
diameter) were placed at a distance of 0.5 cm away from the rim of the mycelial
colony. An aliquot of a solution of Anasazi bean lectin was added to a disk.
The plates were kept at 25°C for 72 hours until mycelial growth had enveloped
disks containing the control and crescents of inhibition had formed around
disks containing samples with antifungal activity.


## 2.12. Assay of Mitogenic Activity

Four C57BL/6 mice (20–25 g) were killed
by cervical dislocation, and the spleens were aseptically removed. Spleen cells
were isolated by pressing the tissue through a sterilized 100-mesh stainless steel
sieve and resuspended to5×106 cells/mL in RPMI 1640 culture medium
supplemented with 10% fetal bovine serum, 100 units penicillin/mL, and 100 μg
streptomycin/mL. The cells (7×105 cells/100 μL/well) were seeded
into a 96-well culture plate, and serial dilutions of a solution of Anasazi bean
lectin in 100 μL medium were added. After incubation of the cells at 37°C in a
humidified atmosphere of 5% CO2 for 24 hours, 10 μL methyl [H3]thymidine
(0.25 μCi, GE Healthcare) was added, and the cells were incubated for further 6
hours under the same conditions. The cells were then harvested with an
automated cell harvester onto a glass fiber filter and the radioactivity was
measured with a Beckman model LS 6000SC scintillation counter. All reported
values are means of triplicate samples [22].


## 2.13. Effect of Chemical Modification of Amino Acid Residues on Hemagglutinating Activity

For serine modification, the lectin
(100μg) in 0.1 mL of 50 mM Tris-HCl buffer (pH 7.4) was incubated with 5 mM
phenylmethylsulfonyl fluoride (PMSF) at 27°C for 1 hour [23]. Aliquots were
removed at 15 minutes intervals. Excess reagent was removed by ultrafiltration,
followed by determination of residual hemagglutinating activity.
Lectin incubated without PMSF served as a control.Reduction of the thiol groups of Anasazi bean
lectin was carried out by incubating the lectin (100μg) in 0.1 mL of 50 mM phosphate
buffer (pH 8.0) with 0.1 mM 5, 5′-dithiobis-(2-nitrobenzoic acid (DTNB) at 27°C for 1 hour. Aliquots were removed at different time intervals. Excess reagent
was removed by ultrafiltration, followed by determination of residual
hemagglutinating activity. Lectin incubated in the absence of DTNB served as a
control [24].For lysine modification, 0.5 mg of NaBH4 was added to the lectin (5 mg) in 2 mL of 0.2 M sodium borate buffer (pH 9.0)
at 4°C, followed by six aliquots (5 μL each) of 3.5% formaldehyde at 10 minutes
intervals. Excess reagent was removed by ultrafiltration, followed by
determination of residual hemagglutinating activity. Lectin incubated in the
absence of sodium borohydride (NaBH4) served as a control [25].Modification of tryptophan residues was carried out
according to the method of Spande and Witkop [26]. The lectin was dissolved in
NaOAc buffer (0.1 M, pH 5.0) to 1 mg/mL. The modification was carried out at 20°C. N-bromosuccinimide (NBS) (10 μL, 10 mM) was added every 5 minutes. The
number of tryptophan residues was calculated as described by Spande and Witkop
[26]. The samples were then desalted by ultrafiltration to remove excess
reagent, and then assay of hemagglutinating activity was carried out. Lectin
incubated in the absence of NBS served as a control.Acetylation of the tyrosine side chain phenoxy
groups of the lectin was done at room temperature by incubating 300μg lectin
in 1.0 mL of 25 mM phosphate buffer (pH 7.5) containing 60-fold molar excess
(0.1–10 mM) of
N-acetylimidazole. Excess reagent was removed by ultrafiltration,
followed by determination of residual hemagglutinating activity. Lectin incubated
in the absence of N-acetylimidazole served as a control [27].


## 3. Results

Purification of the Anasazi bean seed lectin
involved initial extraction in 10 mM Tris-HCl buffer (pH 7.3) and three-step
chromatography including affinity chromatography on Affi-gel blue gel, ion-exchange
chromatography on Mono S, and gel filteration on Superdex 200. Fractionation of
the crude seed extract using Affi-gel blue gel revealed the presence of a
slightly larger unadsorbed fraction designated as B1, and a slightly smaller
adsorbed fraction designated as B2 (Figure1(a)). The latter fraction, in which
hemagglutinating activity was concentrated, was subsequently fractionated on
Mono S into a large unadsorbed fraction and a small adsorbed fraction (data not
shown). The adsorbed fraction with hemagglutinating activity was resolved into a
large peak (SU1) and a small peak (SU2) by FPLC-gel filtration on Superdex 200.
Hemagglutinating activity was located in SU1 (Figure 1(b)). The purified lectin,
represented by SU1, appeared as a single band with a molecular mass of 30 kDa
in SDS-PAGE (Figure 2)
and a 60 kDa absorbance peak in gel filtration on Superdex 200 (Figure 1(b)). The
protein yield and specific hemagglutinating activity of the product at each
stage of the purification scheme are shown in Table 1. Anasazi bean lectin
demonstrated remarkable N-terminal sequence similarity to those of Phaseolus lectins (Table 2). The hemagglutinating activity of purified Anasazi bean
lectin could not be inhibited by any of the simple sugars tested at 1.56–100 mM, α-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine,
D(+)-galactosamine, (+)-lactose, D(+)-melibiose, L(+)-mannose, D(+)-mannose,
D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose, galacturonic acid, and N-acetyllactosamine,
and also not by the glycoproteins heparin, thyroglobulin, lactoferrin, human
chorionic genadotropin, and ovalbumin. The hemagglutinating activity was
completely stable between 0°C and 80°C. Considerable loss in activity
occurred at 90°C. Some activity was discernible at 100°C (Figure 3). The
lectin exhibited remarkable stability over the entire range of pH 1–14 (data not
shown). The Anasazi bean lectin inhibited HIV-1 reverse transcriptase with an
IC50 of 7.6 μM
(Figure 4). The antiproliferative activity of Anasazi
bean lectin toward Hep G2 and MCF-7 cells is shown in Figure 5. The lectin
displayed only slight inhibition of proliferation toward HepG2 cells with an IC50 beyond 8 μM while its IC50 toward MCF-7 cells was 1.3 μM. The lectin
stimulated the mitogenic response of mouse splenocytes with an optimal response
at 1.04 μM
(Figure 6). However, the hemagglutinin did not affect nitric oxide
production by mouse macrophages (Figure 7). Anasazi bean lectin exhibited no
effect on mycelial growth in the fungal species examined, that is, Botrytis
cinerea, Mycosphaerella arachidicola, and Fusarium oxysporum (data
not shown). The effects of various types of chemical modifications on
hemagglutinating activity of the purified lectin are summarized in Table 3.
DTNB, reductive methylation and N-acetylimidazole treatments did not produce
any alterations in the hemagglutinating activity of Anasazi bean lectin, suggesting
that cysteine, lysine and tyrosine residues, respectively, did not play any
important role in its hemagglutinating activity. However, 67% loss of
hemagglutinating activity after NBS treatment was noted, whereas no change in
the control was detected. These results strongly suggest a considerable
involvement of tryptophan residues in hemagglutinating activity, and stability
of the lectin. PMSF treatment resulted in 43% loss in hemagglutination activity
suggesting partial
involvement of serine in the lectin activity. Spectrophotometric monitoring
revealed that the controlled addition of 10 μL of 10 mM NBS under
mildly acidic conditions (pH 5.0) led to a progressive decrease in absorption
at 280 nm, indicative of oxidation of tryptophan residues. A concomitant drop in lectin activity was clearly seen upon
modification of tryptophan residues (Figure 8).Table 1
Yields and specific hemagglutinating activities of chromatographic fractions obtained
at different stages of purification of Anasazi bean lectin.
Specific activity (titer/mg)Total activity (titer)Yield (mg)Fold purificationCrude Extract27081920030331.00Affi-gel blue gel fraction B213217372805584.89Mono S adsorbed fraction407678643219215.09Superdex fraction SU1 (purified lectin)42671146882615.80Table 2
Comparison of N-terminal amino acid sequence of Anasazi bean lectin with
those of otherPhaseolus lectins.
Sample nameSequence% IdentityPhaseolus vulgaris (cultivar Anasazi bean)1ANQIYFNFQRFNETNLILQR 20100Phaseolus vulgaris (cultivar Pinto bean)1ASETSFSFQRFVETNLILQR 2070Phaseolus vulgaris (cultivar Haricot bean)1ASESYFNFQRFEETN1573.3Phaseolus vulgaris (cultivar Red Kidney bean)1ASETSFSFERFNETNLILQR 2070Differences are underlined: 1A refers to A being the 1st residue in the lectin; R20 refers to R being the 20th residue in the lectin; N15 refers
to N being the 15th residue in the lectin.Table 3
Effect of chemical modification on hemagglutinating activity of Anasazi bean lectin.
TreatmentModified group/amino acid% Hemagglutinating activity remainingPhenylmethylsulfonyl fluoride (PMSF)Serine575,5′-Dithiobis-(2-nitrobenzoic acid (DTNB)Thiol group100Reductive methylationLysine100N-Bromosuccinimide (NBS)Tryptophan33N-AcetylimidazoleTyrosine100(a) Fractionation of the crude extract of Anasazi
beans on an Affi-gel blue gel column equilibrated with the binding buffer (10 mM Tris-HCl, pH 7.3). The
column was washed initially with the binding buffer to remove B1 and then
eluted with 1000 mM NaCl in 10 mM Tris-HCl buffer, (pH
7.3) to desorb B2. (b) Superdex 200 column chromatography.
Buffer 20 mM Tris-HCl buffer, (pH
7.3), flow rate: 0.5 mL/min, fraction size: 1.0 mL. Only the major peak (SU1)
exhibited hemagglutinating activity. mAU = milli-absorbance units.
(a)(b)Figure 2
SDS-PAGE of Anasazi bean
lectin. Lane 1: Anasazi bean lectin. Lane 2: molecular mass standards. From top
downward, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin
(43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), andα-lactalbumin (14 kDa).Figure 3
Effect of temperature on hemagglutinating
activity of Anasazi bean lectin.Figure 4
HIV-1 reverse transcriptase inhibitory
activity of Anasazi bean lectin (data represent means ±SD,n=3).
IC50= 7.6 μM. (Data represent means ±SD, n=3.)Figure 5
Inhibitory effect of Anasazi bean lectin
on proliferation of cancer cell lines. Cell proliferation was determined by MTT
assay (Data represent means ±SD,n=3.)Figure 6
Mitogenic effect of Anasazi bean lectin
and Con A toward mouse splenocytes. (Data represent means ±SD,n=3.)Figure 7
Effects of lipopolysaccharide (LPS), Anasazi
bean lectin, and dexamethasone on nitric oxide production by mouse peritoneal
macrophages. (Data represent means ±SD,n=3.)Figure 8
Effect of the modification of tryptophan
residues on the hemagglutinating activity of Anasazi bean lectin. (Data represent means ±SD,n=3.)


## 4. Discussion

In this study, a plant lectin has been purified by
three-step chromatography from seeds of the Anasazi bean. Similar to pinto bean
lectin [28], Anasazi bean lectin is adsorbed on Affi-gel blue gel. However,
unlike the former lectin, the latter is adsorbed on Mono S as well. The homogeneity
of the Anasazi bean lectin preparation was evidenced by the presence of a
single band in SDS-PAGE. The results of SDS-PAGE and gel filtration
chromatography together revealed that the lectin exists as a dimer of two
subunits. The molecular mass and dimeric nature of Anasazi bean lectin are
similar to those of pinto bean lectin and most of the other Phaseolus lectins
[2, 28]. On the other hand, it differs from a tetrameric 115–120 kDa lectin
from tepary bean (Phaseolus acutifolius)  [29], Phaseolus acutifolius var. latifolius
lectin from which consists of four subunits of 21 kDa molecular mass [30], and
a tetrameric 94 kDa immunosuppressive lectin isolated from seeds of Phaseolus
vulgaris cv Cacahuate [31]. Lectins from some cultivars of Phaseolus vulgaris are oligomeric [32], whereas
Anasazi bean lectin is dimeric. The subunit molecular weight of the oligomeric
lectins is similar to that of Anasazi bean lectin. Isolectins are absent in Anasazi beans but
present in some cultivars of P. vulgaris such as red kidney bean [32–34]. The isolectins differ from one another by the
number of erythrocyte-reactive (E) subunits and lymphotcyte-reactive (L)
subunits that they possess. There are
five such isolectins: L4, L3E1, L2E2, L1E3, and E4. Although there is striking homology between Anasazi
bean lectin and other Phaseolus lectins in N-terminal sequence, Anasazi bean
lectin exhibits absence of simple sugar specificity. Simple sugars, the
N-acetyl sugar N-acetyllactosamine and the glycoproteins heparin thyroglobulin,
lactoferrin, human chorionic gonadotropin, and ovalbumin are not able to
inhibit the hemagglutinating activity of Anasazi bean. In this aspect, Anasazi bean lectin is
dissimilar from arcelin-1, a lectin-like protein from wild varieties of kidney
bean which interacts with complex glycans [35], and also from lectins from many
other P. vulgaris cultivars such as the galactose specific pinto bean
lectin [28]. Similar to pinto bean lectin [28], Anasazi bean lectin is fairly
thermostable because its hemagglutinating activity is stable at temperatures up
to 80°C, and is reduced
only at 90°C. Interestingly,
some activity remained even after heating at 100°C for 30 minutes. Haricot bean and ground bean
lectins, however, have been shown to lose activity beyond 40°C in a temperature-dependent manner [19, 36]. The
lectin shows remarkable pH stability, its activity being unaffected throughout
the entire range of pH from 1 to 14. This is in contrast to lectin from Parkia
javanica beans which is stable in pH 7–10 [37]. Like
many lectins such as mushroom lectins, haricot bean lectin, and others [2, 3, 31], Anasazi bean lectin exhibits an antiproliferative activity toward tumor
cell lines. Interestingly, the Anasazi bean lectin exhibits only slight
antiproliferative activity toward Hep G2 cells while it has been shown to have
a significant antiproliferative effect against MCF-7 cells. Only a very small
number of lectins have been shown to have antifungal activity [2]. Like most other
lectins, Anasazi bean lectin does not display antifungal activity. Anasazi bean
lectin inhibits HIV-1 reverse transcriptase with an IC50 below those
of some anti-HIV natural products [38]. The IC50 is almost twice
that of pinto bean lectin [28]. However, it is almost one-tenth of that of ground
bean lectin [19]. The mechanism of inhibition probably involves protein-protein interaction.
It is known that HIV-1 protease inhibits HIV-1 reverse transcriptase with a
similar mechanism [39]. Antifungal proteins [40–42], ribosome
inactivating proteins [43–45], and lectins [2, 5, 7, 21, 46] of plant origin
have been shown to inhibit HIV-1 reverse transcriptase. The ability of Anasazi bean
lectin to inhibit HIV-1 reverse transcriptase is interesting in view of the
inhibition of DNA polymerase alpha activity by lectins including Concanavalin A and
ricin [47, 48] and DNA polymerase beta activity by red kidney bean agglutinin [47].
Induction of mitogenicity is a common feature elicited by most lectins of Phaseolus genus, such as pinto bean lectin, haricot bean agglutinin [32], and PHA-L4,
a lectin from kidney beans [8]. However, GNL-2, a lectin from great northern
beans [49], is devoid of this activity. Other lectins may also have mitogenic
activity [50]. Anasazi bean lectin is capable of eliciting a mitogenic response from
mouse splenocytes. The magnitude of the maximal response is about one-fourth of
that of Con A; the lectin concentration needed to bring about the maximal
response is 64 times less than that of Con A. Nitric oxide (NO) is known to
play an important role in immunomodulation. Proinflammatory cytokines, for
example, tumor necrosis factor-α (TNF-α) and bacterial products, for example,
lipopolysaccharide (LPS) are well established NO stimulators. The steroid,
dexamethasone, is an inhibitor of NO production. These agents up- or
downregulate the activity of the inducible form of nitric oxide synthase [51].
NO is also involved in cytotoxity against tumor cells [52], and it is possible
that NO is implicated in tumor lysis induced by lectins with an antitumoral
effect [53–55]. Previously,
emperor banana lectin [21] has been reported to induce nitric oxide production
by mouse macrophages. Anasazi bean lectin, in the present report, is not
capable of augmenting nitric oxide production by mouse macrophages. Chemical
modification studies were carried out to investigate the role of specific amino
acids in the hemagglutinating activity of Anasazi bean lectin. The results
disclose that tryptophan and serine are important to the hemagglutinating activity,
the contribution of tryptophan being more important. Previous studies have
reported that lysine, tyrosine, and tryptophan (e.g., in Dolichos lab-lab bean
[16]) or tryptophan alone are indespensable for the hemagglutinating activity
of some legume lectins [56]. Specific amino acids may be involved in either
direct interaction with the sugar or may have a role in maintaining
conformation of the sugar binding pocket, and hence contribute to the
hemagglutinating activity of lectins [14]. The application of three successive
chromatographic steps, namely, affinity chromatography on Affi-gel blue gel,
ion exchange chromatography on Mono S, and gel filtration on Superdex 200, is
effective in isolating Anasazi bean lectin, with an approximately 15-fold
purification. It is known that the sugar snap produces an antifungal protein,
miraculin [57], whereas the garden pea produces a ribosome inactivating
protein, pisavin, instead [58], although they are two cultivars of the same
species, Pisum sativum. Thus different cultivars of the same species may
produce different proteins. Anasazi bean lectin differs from pinto bean lectin,
its counterpart from another cultivar of the same species, in the
presence/absence of some features, such as sugar specificity, antiproliferative
activity, HIV-1 reverse transcriptase inhibitory activity, although the two
beans are two different cultivars of the same species (Phaseolus vulgaris).
The present study on Anasazi bean lectin has revealed that tryptophan and
serine residues are involved in the activity of the lectin. The involvement of
other amino acid residues and groups in maintaining the conformation of the
lectin could be further investigated by circular dichroism analysis [14]. The
interesting features of Anasazi bean lectin comprise the following: absence of
simple sugar specificity which is dissimilar to phytohemagglutinins from many
other P. vulgaris cultivars, antiproliferative activity against tumor
cells, potent HIV-1 reverse transcriptase inhibitory activity, mitogenic activity
in mouse splenocytes, and remarkable pH stability and thermostability.



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*Source: 929568-2009-03-25.xml*