A lectin (termed NNTL) was purified from the extracts of Nymphaea nouchali tuber followed by anion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on HiTrap Phenyl HP and by repeated anion-exchange chromatography on HiTrap Q FF column. The molecular mass of the purified lectin was 27.0 ± 1.0 kDa, as estimated by SDS/PAGE both in the presence and in the absence of 2-mercaptoethanol. NNTL was an o-nitrophenyl β-D-galactopyranoside sugar-specific lectin that agglutinated rat, chicken and different groups of human blood cells and exhibited high agglutination activity over the pH range 5–9 and temperatures of 30–60°C. The N-terminal sequence of NNTL did not show sequence similarity with any other lectin and the amino acid analysis revealed that NNTL was rich in leucine, methionine and glycine residues. NNTL was a glycoprotein containing 8% neutral sugar and showed toxicity against brine shrimp nauplii with an LC50 value of 120 ± 29 μg/ml and exerted strong agglutination activity against four pathogenic bacteria (Bacillus subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei). In addition, antiproliferative activity of this lectin against EAC (Ehrlich ascites carcinoma) cells showed 56% and 76% inhibition in vivo in mice at 1.5 and 3 mg·kg−1·day−1 respectively. NNTL was a divalent ion-dependent glycoprotein, which lost its activity markedly in the presence of denaturants. Furthermore, measurement of fluorescence spectra in the presence and absence of urea and CaCl2 indicated the requirement of Ca2+ for the stability of NNTL.

INTRODUCTION

Lectins are a group of proteins found in all types of living organisms, either in soluble or in membrane-bound form that recognize specific carbohydrate structures and thereby agglutinate cells by binding to cell-surface glycoproteins and glycoconjugates [1]. In general, they are structurally complex molecules with one or more carbohydrate recognition domains [2]. Many lectins have been isolated and characterized from plant and animal sources. Plant lectins have different defensive roles such as insecticidal, anti-fungal, anti-microbial as well as being toxic to birds and mammals [3]. Based on structural and evolutionary development, most of these plant lectins have been classified as legume lectins, chitin-binding proteins, type 2 ribosome-inactivating proteins, monocotyledon mannose-binding lectins, amaranthins, Cucurbitaceae phloem lectins and jacalin-related lectins [4]. Each family has its own characteristic carbohydrate recognition domain. The rich sources of lectins in plants are particularly their organs such as seeds, tubers, bulbs, rhizomes, bark, etc.

In general, plant tubers are rich in starch and indeed they are often considered solely as a source of carbohydrate for diets and industrial uses. However, they do contain protein that varies in amount from approx. 1 to 10% (dry weight). Plant tubers are known to possess defence-related proteins such as chitinase and lectins [1] that are responsible for various defence-related functions. Lectins were purified from tubers of Arisaema jacquemontii [5], Arisaema helleborifolium [6] and Arum maculatum [7] with anti-insect activity. Chitinase, which exists in tubers of the yam Dioscorea japonica [8,9], acts as fungicides and insecticides. Recently, lectins have also been purified from Dioscorea batatas [10], Helianthus tuberosus L. [11] and Solanum tuberosum L. tubers [1214]. In the recent years, scientists have become interested in the plant lectins due to their potential application as anti-tumour agents [1517].

Water Lily, locally known as ‘Shapla’ in Bangladesh, is an aquatic plant of the genus Nymphaea. Water lily has large, disc-like, floating leaves and showy flowers. There are approx. 50 species growing in different countries. In Bangladesh, two commonly available species are Nymphaea pubescens (blue variety) and Nymphaea nouchali (white variety). Both species grow abundantly as a mixed population in almost all shallow natural water bodies, but the latter is more frequent and popular in Bangladesh and has been designated as the country's national flower. It is seen in abundance mainly in the monsoon season. The peduncle is a popular vegetable to villagers but the tuberous rhizomes are also eaten.

Although different proteins were purified from various tubers and other parts of plant, in the present paper, we are reporting isolation and characterization of a Ca2+-dependent o-nitrophenyl β-D-galactopyranoside-specific lectin from white water lily (N. nouchali) tuber.

MATERIALS AND METHODS

Materials

DEAE-cellulose was procured from Wako Chemical (Japan) and HiTrap Phenyl HP and HiTrap Q FF were procured from Healthcare Bio-Sciences. All other chemicals and reagents were of the highest grades commercially available. The tubers of N. nouchali were collected from local market and stored at 4°C.

Purification of proteins

N. nouchali tuber was homogenized in 10 mM Tris/HCl buffer (pH 8.2) containing 0.15 M NaCl (5 ml of buffer for 1 g of tuber). The homogenate was centrifuged at 24000 g/min for 20 min and the supernatant was dialysed against distilled water and then against 10 mM Tris/HCl buffer, pH 8.2. After dialysis, crude sample was centrifuged at 16000 rev./min for 15 min and the supernatant was collected and subjected to anion-exchange chromatography on a DEAE-cellulose column (1.5 cm × 12 cm), previously equilibrated with the same buffer. The elution was performed by the increase in NaCl concentration from 0.0 to 0.5 M in 10 mM Tris/HCl buffer, pH 8.2. The eluted fraction was dialysed against distilled water and was mixed with an equal volume of 20 mM Tris/HCl buffer, pH 8.2 containing 1.0 M (NH4)2SO4 and subjected to hydrophobic chromatography on a HiTrap phenyl HP column (5 ml), which was previously equilibrated with the same buffer. The elution was performed by a decrease in ammonium sulfate concentration in the same buffer from 0.5 to 0.0 M. The eluted fraction was dialysed against distilled water and 10 mM Tris/HCl buffer (pH 8.2) and subjected to anion-exchange chromatography on a HiTrap Q FF column (5 ml) that was previously equilibrated with the same buffer. The elution was performed by an increase in salt concentration from 0 to 0.5 M NaCl in 10 mM Tris/HCl buffer (pH 8.2). The purity was checked by using SDS/PAGE in 15% (w/v) polyacrylamide gel as described by Laemmli [18]. The subunit content was checked on the same gel in the presence and absence of 2-mercaptoethanol. The glycoprotein was detected by PAS (periodate–Schiff) staining of the SDS/PAGE gel [13]. The protein elution profiles were monitored at 280 nm. The purified lectin was designated as NNTL (N. nouchali tuber lectin).

Molecular mass determination

The molecular mass of the purified protein was determined by SDS/PAGE using 15% (w/v) polyacrylamide gel. BSA (67 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa) and lysozyme (14.6 kDa) were used as marker proteins.

Erythrocytes

Blood group specificity was tested using different types of human blood groups collected from six donors and blood of chicken was collected from a slaughter house. All the blood samples were collected in saline and centrifuged at 1500 g/min for 10 min. The erythrocyte pellet was washed thrice and resuspended in the same saline to make a 2% erythrocyte suspension.

Haemagglutination assay

The haemagglutination assay was performed in a 96-well microtitre U-bottomed plates in a final volume of 100 μl containing 50 μl of protein solution serially diluted with an equal amount of haemagglutination buffer (20 mM Tris/HCl buffer, pH 7.8 containing 0.9% NaCl and 10 mM CaCl2) and 50 μl of 2% suspension of albino rat erythrocytes previously washed with 0.15 M NaCl. After gently shaking, the plate was kept at room temperature (20°C) for 30 min. The visual agglutination titre of the maximum dilution giving positive agglutination was recorded. Inhibition of haemagglutinating activity was examined by adding a serial dilution of the following sugars (D-glucose, D-galactose, D-raffinose, L-rhamnose, D-melibiose, maltose, lactose, L-fucose, inositol (meso) inactive, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-xylose, o-nitrophenyl β-D-glucopyranoside, o-nitrophenyl β-D-galactopyranoside, methyl α-D-glucopyranoside, methyl α-D-mannopyranoside, methyl α-D-galactopyranoside, methyl β-D-galactopyranoside, 4-nitrophenyl α-D-galactopyranoside, L-arabinose, D-mannose, 4-nitrophenyl α-D-mannopyranoside, 4-nitrophenyl α-D-glucopyranoside, 4-nitrophenyl β-D-mannopyranoside, 4-nitrophenyl β-D-glucopyranoside and methyl β-D-glucopyranoside) and 4 mg/ml of glycoprotein (fetuin) to the incubation mixture.

Effect of temperature and pH on haemagglutination activity

To examine the thermostability, NNTL [0.5 mg/ml in TBS (Tris/HCl-buffered saline), pH 7.8] was heated in a water bath for 30 min at different temperatures in the range of 30–90°C and cooled to room temperature. The thermal inactivation was done using serially diluted 50 μl of NNTL with an equal amount of TBS, pH 7.8, and the haemagglutination titre was assayed as explained above. A non-heated lectin sample was used as control, which was taken as 100% activity. pH stability was determined by incubating NNTL solutions (0.25 mg/ml) against different buffers (pH value ranging from 3 to 11) containing 0.15 M NaCl for 8 h at room temperature. After 8 h, the lectin solutions were dialysed against 20 mM Tris/HCl buffer, pH 7.8, containing 0.15 M NaCl for 12 h. The following buffers were used for pH stability assay: 0.1 M sodium acetate (pH 3–6), 0.1 M phosphate (pH 7.0), 0.1 M Tris/HCl (pH 8.0), and 0.1 M glycine/NaOH (pH 9–11).

Estimation of protein and sugar content

Protein was estimated by the Lowry method [19] using lipid-free BSA as the standard. The sugar content of NNTL was determined according to the phenol–sulfuric acid method [20] using D-glucose as standard.

N-terminal sequence determination

The purified lectin was first subjected to SDS/PAGE and then the lectin was immobilized on a PVDF membrane by electroblotting. After that the protein band was excised from the membrane. Finally, the N-terminal sequence was determined by Edman degradation using an amino acid analyser and the N-terminal sequence homology was analysed using the BLAST database search.

Amino acid analysis

The amino acid analysis was carried out in acidic condition with a Biochrom 20 Plus Amino Acid Analyzer after sample hydrolysis in a sealed, evacuated ampoule at 110°C with 6 M HCl for 24 h.

Bacterial agglutinating activity

The bacterial agglutinating activity was performed by using Bacillus cereus, Staphylococcus aureus, B. subtilis, B. megaterium, Sarcina lutea, Escherichia coli, Shigella shiga, Shigella dysenteriae, Shigella sonnei, Salmonella typhi, and Klebsiella sp. Bacteria were grown at 37°C overnight in nutrient broths (liquid nutrient medium), then the bacteria were collected by centrifugation at 4000 rev./min for 3 min, washed with 10 mM Tris/HCl buffer saline, pH 7.8, and re-suspended in the same buffer with an attenuance of 2.0 (640 nm). Then 50 μl of each bacterial suspension was mixed with serial dilution of NNTL to a final volume of 100 μl in 96-well microtitre plates. The plates were agitated for 2 min and the mixture was kept at room temperature for 60 min. Finally, bacterial agglutinating activity was monitored using a light microscope.

Brine shrimp nauplii lethality assay

Lethality assay was studied by using brine shrimp (Artemia salina L) nauplii. 25, 50, 100 and 200 μl of NNTL (4 mg/ml) were taken in different vials and ten brine shrimp nauplii in artificial sea water was added to each vial. Artificial sea water was prepared by dissolving 38 g of NaCl in 1 litre of distilled water and sodium tetraborate was added to adjust the pH to 7.0. Finally, the volume of each vial was adjusted to 4 ml by adding artificial seawater. All tests were performed at room temperature (~30°C), under a continuous light. Three replicates were used for each treatment and control. From these data, the percentage of mortality of the nauplii was calculated for each concentration and the LC50 values were determined using Probit analysis as described by Finney [21].

Determination of EAC (Ehrlich ascites carcinoma) cell growth inhibition

The EAC cells were propagated intraperitoneally in our Departmental Research Laboratory biweekly and the cells were collected from a donor Swiss albino mouse bearing 6–7-day-old ascites tumour. The collected cells were diluted with normal saline and the cell number adjusted to 3 × 106 cells/ml by counting with the help of a haemocytometer. Tumour cells that showed 90% viability were injected (0.1 ml) intraperitoneally to each Swiss albino mouse. After 24 h, the mice were randomly distributed into three groups with at least five mice per group. Two groups of mice treated for 5 days with NNTL at a concentration of 3 and 1.5 mg·kg−1 of body weight·day−1 and the remaining group was used as control. Mice in each group were killed on the sixth day and the total intraperitoneal tumour cells were harvested into normal saline and counted with a haemocytometer. The total number of viable cells in every mouse of the treated groups was compared with those of the control (EAC treated only). The percentage of inhibition was calculated by the following equation:

 
formula

Animal experiments were approved by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) for Experimentations on Animal, Human, Microbes and Living Natural Sources (286/320-IAMEBBC/IBSc), Institute of Biological Sciences, University of Rajshahi, Rajshahi, Bangladesh.

Effect of detergents and metal ions on lectin-induced haemagglutination activity

For detecting the effect of denaturants, NNTL in 0.1 M TBS was incubated at room temperature with 50 mM DTT (dithiothreitol) and 4 M urea for 12 h. NNTL in the same buffer without denaturants was used as control and its activity was considered as 100%. To determine the dependency of haemagglutination activity on divalent cations, NNTL was incubated with 0.1 M EDTA for 2 h at room temperature. Then NNTL solution was dialysed against 20 mM TBS buffer, pH 7.8 for 12 h at 4°C and then subjected to the haemagglutination assay in the presence or absence of each Ba2+, Ca2+ and Mg2+ in the haemagglutination buffer.

Fluorescence spectroscopy

Fluorescence measurements of NNTL were performed at a protein concentration of 50 and 40 μg/ml on a Shimadzu Spectrofluorometer RF-5301 PC at room temperature. The native and treated samples (with Ca2+, o-nitrophenyl β-D-galactopyranoside and urea) were placed in a 1 cm × 1 cm × 4.5 cm quartz cuvette for measurement. Samples were λex = 280 nm and the λem was recorded in the range of 300–400 nm and widths for the excitation and emission monochromators were maintained at 5 nm.

RESULTS

Purification and molecular mass determination

A lectin was purified from White water lily tuber crude extract followed by anion-exchange chromatography on DEAE-cellulose column, hydrophobic chromatography on a HiTrap Phenyl HP column and then reapplying to anion-exchange chromatography on a HiTrap Q FF column. The DEAE-cellulose column (1.5 cm × 12 cm) gave two major peaks as shown in Figure 1(A). Unbound fraction (results not shown) as well as bound fraction, F-2 did not give any haemagglutination activity. Only the bound fraction, F-1, showed haemagglutination activity and it was subjected to hydrophobic chromatography on a HiTrap Phenyl HP column (5 ml) previously equilibrated with 10 mM Tris/HCl buffer, pH 8.2 containing 0.5 M (NH4)2SO4. Elution was performed with the same buffer without (NH4)2SO4. Two fractions, bound (B) and unbound (UB), were collected as shown in Figure 1(B). The unbound fraction that showed agglutination activity was subjected to an anion-exchange chromatography on a HiTrap Q FF column (5 ml) for further purification. Two major peaks were obtained as shown in Figure 1(C). Only fraction QB showed haemagglutinating activity and migrated on SDS/PAGE as a single band with an apparent molecular mass of 27.0 ± 1.0 kDa in the presence and absence of 2-mercaptoethanol (Figure 1D). Approx. 30 mg of NNTL was obtained from 13.7 g of crude protein that was extracted from 1 kg of N. nouchali tubers and the purification procedure of this lectin is summarized in Table 1.

Elution profile of NNTL

Figure 1
Elution profile of NNTL

(A) Ion-exchange chromatography of NNTL. Crude protein was applied to a DEAE-cellulose column (2.5 cm × 12 cm) previously equilibrated with 10 mM Tris/HCl buffer (pH 8.2). Proteins were eluted with the same buffer with the gradually increase of NaCl gradient from 0.0 to 0.4 M. The elution profiles were monitored at 280 nm. Fractions (2.5 ml/tube) were collected at a 1 ml/min flow rate. (B) Fraction 1 was applied to a HiTrap Phenyl HP column (5 ml) previously equilibrated with the 10 mM Tris/HCl buffer containing 0.5 M (NH4)2SO4. The bound fraction was eluted by the same buffer without (NH4)2SO4 at a 1 ml/min flow rate. (C) The unbound fraction was dialysed against 10 mM Tris/HCl (pH 8.2) and then applied to a HiTrap Q FF column (5 ml) previously equilibrated with the same buffer. Proteins were eluted with 10 mM Tris/HCl (pH 8.2) with a gradual increase in NaCl gradient from 0.0 to 0.5 M. Fractions were collected at a 1 ml/min flow rate. (D) SDS/PAGE of NNTL on 15% polyacrylamide gel. Lane 1: NNTL in the presence of 2-mercaptoethanol; lane 2: NNTL in the absence of 2-mercaptoethanol; lane 3: marker proteins; lane P: PAS staining of the purified lectin.

Figure 1
Elution profile of NNTL

(A) Ion-exchange chromatography of NNTL. Crude protein was applied to a DEAE-cellulose column (2.5 cm × 12 cm) previously equilibrated with 10 mM Tris/HCl buffer (pH 8.2). Proteins were eluted with the same buffer with the gradually increase of NaCl gradient from 0.0 to 0.4 M. The elution profiles were monitored at 280 nm. Fractions (2.5 ml/tube) were collected at a 1 ml/min flow rate. (B) Fraction 1 was applied to a HiTrap Phenyl HP column (5 ml) previously equilibrated with the 10 mM Tris/HCl buffer containing 0.5 M (NH4)2SO4. The bound fraction was eluted by the same buffer without (NH4)2SO4 at a 1 ml/min flow rate. (C) The unbound fraction was dialysed against 10 mM Tris/HCl (pH 8.2) and then applied to a HiTrap Q FF column (5 ml) previously equilibrated with the same buffer. Proteins were eluted with 10 mM Tris/HCl (pH 8.2) with a gradual increase in NaCl gradient from 0.0 to 0.5 M. Fractions were collected at a 1 ml/min flow rate. (D) SDS/PAGE of NNTL on 15% polyacrylamide gel. Lane 1: NNTL in the presence of 2-mercaptoethanol; lane 2: NNTL in the absence of 2-mercaptoethanol; lane 3: marker proteins; lane P: PAS staining of the purified lectin.

Table 1
Purification scheme of NNTL from N. nouchali tubers (1 kg)
Purification stepFractionsTotal protein (mg)103 × Total activity (titre/mg)Recovery of activity (%)
Crude  13700 1080 100 
Anion-exchange chromatography on DEAE-cellulose F-1 210 537 49.7 
Hydrophobic chromatography on HiTrap Phenyl HP Unbound fraction (NNTL) 105 269 23.7 
Anion-exchange chromatography on HiTrap Q FF QA fraction (NNTL) 30 153.6 14.2 
Purification stepFractionsTotal protein (mg)103 × Total activity (titre/mg)Recovery of activity (%)
Crude  13700 1080 100 
Anion-exchange chromatography on DEAE-cellulose F-1 210 537 49.7 
Hydrophobic chromatography on HiTrap Phenyl HP Unbound fraction (NNTL) 105 269 23.7 
Anion-exchange chromatography on HiTrap Q FF QA fraction (NNTL) 30 153.6 14.2 

Haemagglutination assay and carbohydrate specificity of the purified protein

The minimum agglutinating activity of the NNTL was found to be 8 μg/ml for rat erythrocytes, 16 μg/ml for different groups (A+, B+, O+, AB+, AB and O) of human and chicken blood as shown in Table 2. The carbohydrate-binding specificity was evaluated by inhibiting the agglutination of rat erythrocytes using different sugars. The best inhibitor for NNTL was found to be o-nitrophenyl-β-D-galactopyranoside followed by o-nitrophenyl-β-D-glucopyranoside and N-acetyl-D-galactosamine as presented in Table 3.

Table 2
Agglutination of different blood types by NNTL
GroupNNTL (μg/ml)*
Human blood A+ 16 
 B+ 16 
 O+ 16 
 O 16 
 AB 16 
 AB+ 16 
Chicken blood − 16 
Albino rat blood − 
GroupNNTL (μg/ml)*
Human blood A+ 16 
 B+ 16 
 O+ 16 
 O 16 
 AB 16 
 AB+ 16 
Chicken blood − 16 
Albino rat blood − 
*

Minimum NNTL concentration required for a visible agglutination.

Table 3
Inhibition of haemagglutination activity of NNTL by mono- and oligo-saccharides

+, Haemagglutination activity; −, no haemagglutination activity; o, was not checked at that concentration.

Haemagglutination
SugarConcentration (mM)…200100502512.5
o-Nitrophenyl β-D-galactopyranoside  − − − − 
o-Nitrophenyl β-D-glucopyranoside  − 
N-Acetyl-D-galactosamine  − 
N-Acetyl-D-glucosamine  
D-Xylose  
L-Fucose  
D-Mannose  
L-Arabinose  
D-Raffinose  
L-Rhamnose  
D-Melibiose  
Maltose  
D-Glucose  
Lactose  
D-Galactose  
Inositol (meso)inactive  
Methyl α-D-glucopyranoside  
Methyl α-D-mannopyranoside  
Methyl α-D-galactopyranoside  
Methyl β-D-galactopyranoside  
4-Nitrophenyl α-D-galactopyranoside  
4-Nitrophenyl α-D-mannopyranoside  
4-Nitrophenyl α-D-glucopyranoside  
4-Nitrophenyl β-D-mannopyranoside  
4-Nitrophenyl β-D-glucopyranoside  
Methyl β-D-glucopyranoside  
Fetuin (glycoprotein) (4 mg/ml)      
Haemagglutination
SugarConcentration (mM)…200100502512.5
o-Nitrophenyl β-D-galactopyranoside  − − − − 
o-Nitrophenyl β-D-glucopyranoside  − 
N-Acetyl-D-galactosamine  − 
N-Acetyl-D-glucosamine  
D-Xylose  
L-Fucose  
D-Mannose  
L-Arabinose  
D-Raffinose  
L-Rhamnose  
D-Melibiose  
Maltose  
D-Glucose  
Lactose  
D-Galactose  
Inositol (meso)inactive  
Methyl α-D-glucopyranoside  
Methyl α-D-mannopyranoside  
Methyl α-D-galactopyranoside  
Methyl β-D-galactopyranoside  
4-Nitrophenyl α-D-galactopyranoside  
4-Nitrophenyl α-D-mannopyranoside  
4-Nitrophenyl α-D-glucopyranoside  
4-Nitrophenyl β-D-mannopyranoside  
4-Nitrophenyl β-D-glucopyranoside  
Methyl β-D-glucopyranoside  
Fetuin (glycoprotein) (4 mg/ml)      

Effect of temperature and pH on haemagglutination activity

Thermal inactivation of NNTL was investigated by incubating the lectin at different temperatures for 30 min and assaying the haemagglutination activity. Higher lectin activity was observed between 40 and 60°C (Figure 2A), while activity decreased to almost 50% at the temperature range between 70 and 90°C. NNTL was observed to be stable between pH 3 and pH 11 and showed the maximum activity between pH 5 and pH 9 (Figure 2B).

Effects of temperature and pH on haemagglutination activity of NNTL

Figure 2
Effects of temperature and pH on haemagglutination activity of NNTL

(A) Effect of heat on NNTL activity. Equal amounts of sample were heated from 40 to 90°C for 30 min. The highest agglutination activity observed at room temperature was taken as 100% activity. (B) pH stability of NNTL. Equal amounts of sample were incubated with buffers of pH 3–11. After adjusting the pH to 7.8 haemagglutination activity was measured. Percentage activity was calculated assuming the maximum agglutination activity to be 100%.

Figure 2
Effects of temperature and pH on haemagglutination activity of NNTL

(A) Effect of heat on NNTL activity. Equal amounts of sample were heated from 40 to 90°C for 30 min. The highest agglutination activity observed at room temperature was taken as 100% activity. (B) pH stability of NNTL. Equal amounts of sample were incubated with buffers of pH 3–11. After adjusting the pH to 7.8 haemagglutination activity was measured. Percentage activity was calculated assuming the maximum agglutination activity to be 100%.

N-terminal sequence determination

Except for the first residue, the N-terminal sequence of NNTL was determined to be -PEEADYLTE. The N-terminal sequence homology of NNTL with other proteins is given in Table 4. The result of the amino acid analysis of NNTL is shown in Table 5.

Table 4
Sequence homology of NNTL with other proteins
ProteinsSequenceNCBI accession number
NNTL 10  
Putative dehydrogenase 182 P E E A D Y L T 189 ZP_01898845.1 
Clostripain 213 P E A D Y L T E 221 YP_504219.1 
Protein rocB 11 P E A Y L T E 19 ZP_03228294.1 
NAD+-dependent epimerase/dehydratase 319 E E A D Y L T 326 YP_003798514.1 
Reticulocalbin 2 253 P E E D Y T E 261 EDL95577.1 
Taipoxin-associated calcium-binding protein 49 184 P E E D Y T E 192 AAC05132.1 
TonB family protein 65 P E E A D Y L 73 YP_001002515.1 
Permease 200 P E E A D Y T E 208 YP_003406033.1 
Zinc-binding dehydrogenase family oxidoreductase 182 P E E A D Y L 188 YP_259548.1 
3-Deoxy-D-manno-octulosonate 8-phosphate phosphatase, YrbI family 143 E E A D Y T E 150 YP_003828733.1 
Thioredoxin reductase 160 E E A D Y L T 166 CBL04572.1 
Putative ATP-binding protein 870 P E A D Y L E 878 ZP_06198416.1 
Prolyl aminopeptidase 110 P E E A D Y L T 118 ZP_04700458.1 
ProteinsSequenceNCBI accession number
NNTL 10  
Putative dehydrogenase 182 P E E A D Y L T 189 ZP_01898845.1 
Clostripain 213 P E A D Y L T E 221 YP_504219.1 
Protein rocB 11 P E A Y L T E 19 ZP_03228294.1 
NAD+-dependent epimerase/dehydratase 319 E E A D Y L T 326 YP_003798514.1 
Reticulocalbin 2 253 P E E D Y T E 261 EDL95577.1 
Taipoxin-associated calcium-binding protein 49 184 P E E D Y T E 192 AAC05132.1 
TonB family protein 65 P E E A D Y L 73 YP_001002515.1 
Permease 200 P E E A D Y T E 208 YP_003406033.1 
Zinc-binding dehydrogenase family oxidoreductase 182 P E E A D Y L 188 YP_259548.1 
3-Deoxy-D-manno-octulosonate 8-phosphate phosphatase, YrbI family 143 E E A D Y T E 150 YP_003828733.1 
Thioredoxin reductase 160 E E A D Y L T 166 CBL04572.1 
Putative ATP-binding protein 870 P E A D Y L E 878 ZP_06198416.1 
Prolyl aminopeptidase 110 P E E A D Y L T 118 ZP_04700458.1 
Table 5
Amino acid composition of NNTL
Amino acidComposition (mol%)
Asp 6.4 
Thr 5.1 
Ser 6.1 
Glu 7.5 
Pro 0.3 
Gly 10.9 
Ala 5.5 
Cys 0.4 
Val 4.1 
Met 11.1 
Ile 7.0 
Leu 15.2 
Tyr 7.5 
Phe 7.5 
His 1.9 
Lys 2.8 
Arg 0.7 
 100.0 
Amino acidComposition (mol%)
Asp 6.4 
Thr 5.1 
Ser 6.1 
Glu 7.5 
Pro 0.3 
Gly 10.9 
Ala 5.5 
Cys 0.4 
Val 4.1 
Met 11.1 
Ile 7.0 
Leu 15.2 
Tyr 7.5 
Phe 7.5 
His 1.9 
Lys 2.8 
Arg 0.7 
 100.0 

Protein and sugar analysis

Staining with PAS revealed that the lectin was a glycoprotein (Figure 1D). Moreover NNTL gave an orange–yellow colour when it was subjected to the phenol–sulfuric acid method and the neutral sugar content of NNTL was estimated to be 8%. The absorbance (A) of 1.0 at 280 nm for NNTL corresponded to 0.25 mg of protein as determined by the Lowry method.

Bacterial agglutinating assay

NNTL agglutinated B. cereus, Staphylococcus aureus, B. subtilis, B. megaterium, Sarcina lutea, Klebsiella sp., E. coli, Shigella shiga, Shigella dysenteriae, Shigella sonnei and Salmonella typhi. The minimum concentration of NNTL solution needed for the agglutination of each bacterium is summarized in Table 6.

Table 6
Minimum concentrations of NNTL needed for agglutination of some pathogenic bacteria
BacteriumNNTL (μg/ml)
Bacillus cereus 9.7 ± 2.5 
Staphylococcus aureus 3.7 ± 1.5 
Bacillus subtilis 0.42 ± 0.17 
Bacillus megaterium − 
Sarcina lutea 1.0 ± 0.39 
Klebsiella sp.  40 ± 10.3 
Escherichia coli 39.2 ± 11.5 
Shigella shiga 1.1 ± 0.43 
Shigella dysenteriae − 
Shigella sonnei 2.2 ± 0.86 
Salmonella typhi  40 ± 10.3 
BacteriumNNTL (μg/ml)
Bacillus cereus 9.7 ± 2.5 
Staphylococcus aureus 3.7 ± 1.5 
Bacillus subtilis 0.42 ± 0.17 
Bacillus megaterium − 
Sarcina lutea 1.0 ± 0.39 
Klebsiella sp.  40 ± 10.3 
Escherichia coli 39.2 ± 11.5 
Shigella shiga 1.1 ± 0.43 
Shigella dysenteriae − 
Shigella sonnei 2.2 ± 0.86 
Salmonella typhi  40 ± 10.3 

Brine shrimp nauplii lethality assay

The toxic effect of NNTL against brine shrimp nauplii was determined. The mortality rate of brine shrimp nauplii was found to be increased with the increase of concentration of the sample as shown in Figure 3 and the LC50 value for NNTL was calculated as 120 ± 29 μg/ml.

Percentage mortality of brine shrimp nauplii treated with NNTL solution after exposure for 24 h

Figure 3
Percentage mortality of brine shrimp nauplii treated with NNTL solution after exposure for 24 h
Figure 3
Percentage mortality of brine shrimp nauplii treated with NNTL solution after exposure for 24 h

EAC cell growth inhibition

Proliferation of EAC cells was effectively inhibited by NNTL. At the dose of 1.5 mg·kg−1·day−1, the EAC cell growth inhibition was 56% but when the concentration increased to 3 mg·kg−1·day−1 the cell growth inhibition was increased to 76% as shown in Figure 4.

Number of EAC cells counted by light microscope in the presence and absence of NNTL in mouse

Figure 4
Number of EAC cells counted by light microscope in the presence and absence of NNTL in mouse
Figure 4
Number of EAC cells counted by light microscope in the presence and absence of NNTL in mouse

Effect of denaturants and divalent ions on NNTL activity

In the presence of 50 mM DTT and 4 M urea the lectin lost its activity by 50% and 77.5% respectively compared with the control. Furthermore, EDTA-treated NNTL did not show any agglutination activity when divalent ions were absent in the haemagglutination buffer. But after addition of 10 mM of Ba2+, Ca2+ or Mg2+ to the haemagglutination buffer NNTL showed strong agglutination activity.

Fluorescence measurement of NNTL in the presence and absence of Ca2+

Structural changes of the NNTL were measured on its binding to Ca2+ and o-nitrophenyl β-D-galactopyranoside by measuring fluorescence emission spectra with λex = 280 nm as shown in Figure 5(A). The fluorescence spectrum of NNTL had a peak at 330 nm, mainly from tryptophan residues in the protein molecule. In the presence of 0.5 and 1 mM CaCl2, the fluorescence intensity increased significantly upon binding with Ca2+ and after raising the CaCl2 concentration more than 1 mM, it did not affect the intensity. It was found that the nature of changes in the spectra was different when NNTL was complexed with o-nitrophenyl β-D-galactopyranoside. In this case, a slight decrease in intensity was observed in the presence of 15 μg/ml o-nitrophenyl β-D-galactopyranoside. But a very large fall in the fluorescence intensity was observed when the concentration of the sugar was raised to twice the present concentration. Further augmentation in sugar concentration did not affect the fluorescence intensity.

Fluorescence spectra of NNTL under different conditions

Figure 5
Fluorescence spectra of NNTL under different conditions

(A) Fluorescence emission spectra of 50 μg/ml NNTL. NNTL in TBS (-●-), TBS containing 0.5 mM CaCl2 (−), TBS containing 1.0 mM CaCl2 (-○-), TBS containing 15 μg/ml o-nitrophenyl β-Dgalactopyranoside and 1.0 mM CaCl2 (−−−), TBS containing 30 μg/ml O-nitrophenyl-β-D-galactopyranoside and 0.1 mM CaCl2 (-Δ-). (B) Fluorescence emission spectra of 40 μg/ml NNTL. TBS containing 1 mM CaCl2 and 4 M urea incubated for 90 min (−−−), TBS containing 1 mM CaCl2 and 4 M urea incubated for 8 h (−), TBS containing 4 M urea incubated for 90 min (ooo), TBS containing 4 M urea incubated for 8 h (+++).

Figure 5
Fluorescence spectra of NNTL under different conditions

(A) Fluorescence emission spectra of 50 μg/ml NNTL. NNTL in TBS (-●-), TBS containing 0.5 mM CaCl2 (−), TBS containing 1.0 mM CaCl2 (-○-), TBS containing 15 μg/ml o-nitrophenyl β-Dgalactopyranoside and 1.0 mM CaCl2 (−−−), TBS containing 30 μg/ml O-nitrophenyl-β-D-galactopyranoside and 0.1 mM CaCl2 (-Δ-). (B) Fluorescence emission spectra of 40 μg/ml NNTL. TBS containing 1 mM CaCl2 and 4 M urea incubated for 90 min (−−−), TBS containing 1 mM CaCl2 and 4 M urea incubated for 8 h (−), TBS containing 4 M urea incubated for 90 min (ooo), TBS containing 4 M urea incubated for 8 h (+++).

Ca2+ stabilization of NNTL was further examined by incubating the lectin solution with 4 M urea in the presence or absence of 1 mM CaCl2 during a shorter time period (90 min) and a longer time period (8 h) at room temperature as shown in Figure 5(B). When NNTL was incubated with 4 M urea in the presence of CaCl2 for 90 min and 8 h, no significant change in intensity was found. On the other hand, when NNTL was incubated with 4 M urea in the absence of CaCl2, a noticeable change occurred after 90 min and the difference was very significant after 8 h.

DISCUSSION

A lectin (NTTL) was purified from N. nouchali tuber and was a potent agglutinin for rat erythrocytes. The minimum concentration of NNTL for haemagglutination activity of rat erythrocytes was 8 μg/ml, i.e. within the range of other tuber lectins. DB1 (Dioscorea batatas protein 1) and DB2 (Dioscorea batatas protein 2) from D. batatas tuber [10] agglutinate rabbit erythrocytes at 2.7 and 3.9 μg/ml respectively; whereas AJL (Arisaema jacquemontii lectin) from A. jacquemontii tuber [5] required 11.5 μg/ml. NNTL showed no human blood group specificity as it agglutinated A+, B+, O+, AB+, AB and O, all the blood groups tested. This behaviour was observed in other lectins also, for example, EspecL (Erythrina speciosa lectin) and BBL (Belamyia bengalensis lectin) can agglutinate all human blood groups [22,23]. A different result was also observed for HTTL (Helianthus tuberosus lectin), which could not agglutinate any human blood group [11]. Haemagglutination inhibition studies revealed that NNTL was an o-nitrophenyl β-D-galactopyranoside sugar-specific lectin and the minimum inhibitory concentration was 25 mM. o-Nitrophenyl β-D-galactopyranoside-specific lectins were also purified from edible mushroom Pleurotus citrinopileatus and samta tomato with an apparent molecular mass of 32.4 and 79 kDa [24,25] respectively. The molecular mass of NNTL, however, was 27.0 ± 1.0 kDa, which is also different from that of other lectins purified from A. jacquemontii [5], A. helleborifolium [6] A. maculatum [7], D. japonica [8,9], H. tuberosus L. [11] and S. tuberosum L. tubers [1214].

The N-terminal sequence of NNTL, -PEEADYLTE was determined and the homology sequence was searched by using BLAST. The sequence did not match with any other lectin; the only similarity was found with the regions of some proteins other than N-terminal sequence as shown in Table 4. This result suggested that NNTL is a novel lectin. The amino acid composition analysis showed that NNTL was rich in leucine, methionine and glycine residues.

In the present study, NNTL was a glycoprotein in nature and the sugar content was 8%, whereas potato lectin contains 50% sugar on a weight basis [13], but AHL (Arisaema helleborifolium lectin) [6], HTTL [11] and AJL [5] contained only 3.4, 5.3 and 3.4% sugars respectively. The heat stability of NNTL was found to be similar to or higher than that of some other tuber lectins from different sources. The activity of NNTL was stable at 40–60°C but the activity decreased to approx. 50% when incubated at 70–90°C. This result showed good agreement with that of AJL [5], AHL [6] and HTTL [11], which are stable up to 60°C but higher than that of DB3 (Dioscorea batatas protein 3) [10] which is stable up to 50°C. Above and below the optimum pH values (pH 5–9), the presently purified lectin lost its activity significantly, indicating the changes in structure of the lectin-binding sites or ionization of the group(s) associated with the sugar binding. Again the haemagglutinating activity of NNTL was observed to be higher at pH 11 than at pH 3, indicating that the lectin may be more active at basic pH than at acidic pH. The pH stability of NNTL is found to be very close to that of AJL [5] and HTTL [11] but lower as compared with that of DB3 [10] and AHL [6], and higher than that of DB1 [10].

Some lectins are toxic and show toxicity against brine shrimp nauplii. Our present data showed the mortality rate of brine shrimp nauplii was increased with the increase of concentration of the lectin. In the present study, 50% mortality of nauplii occurs (LC50) at 120 ± 29 μg/ml concentration of NNTL, whereas LC50 of MSL (mulberry seed lectin) is 21.87 μg/ml [26]. This result revealed that NNTL is less toxic than that of MSL. Agglutination of several bacterial strains confirmed the interaction between the lectins and the strains. From the present result it has been proved that B. subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei were more sensitive to NNTL and the minimum agglutination concentrations were 0.42 ± 0.17, 1.0 ± 0.39, 1.1 ± 0.43 and 2.2 ± 0.86 μg/ml respectively. But Shigella dysenteriae and B. megaterium did not agglutinate in the presence of NNTL. It was reported that potato lectin interacts with some phytobacteria [27]. Strong agglutination of Helicobacter pylori coccoids was observed with mannose-specific concanavalin A (Con A), fucose-specific Tetragonolobus purpureas (lotus A) and N-acetyl-D-glucosamine-specific Triticum vulgaris [WGA (wheatgerm agglutinin)] lectins [28]. Concanavalin A also aggregates a variety of Gram-negative bacteria specifically Salmonella typhimurium [29,30]. It was also reported that EuniS lectin agglutinate Staphylococcus aureus, Streptococcus sp., Klebsiella sp. and Pseudomonas aeruginosa [31]. The bacterial agglutination might be due to the presence of cognate glycan antigen on the cell surface of induced bacteria. An inhibition study suggested that bacteria agglutination occurs by the interaction of lectin with bacterial surface carbohydrates [27].

Several kinds of plant lectins have been reported to stop multiplication of cancer cells [16,17] and due to the differences in their sugar specificity each lectin exhibits a difference in their antiproliferative effect against tumour cell lines [15]. NNTL showed 56% and 76% EAC cells growth inhibition in vivo in mice at 1.5 and 3 mg·kg−1·day−1 dose respectively. EAC cell growth was also studied in vivo in mice by using jackfruit lectin that inhibited 21.8, 40.2, 57.5 and 83% of growth when administrated as 50, 100, 150 and 200 μg/day respectively [32]. A lectin from Curcuma amarissima rhizomes also showed in vitro antiproliferative activity against a breast cancer cell line (BT 474) and showed the high antiproliferative activity with an IC50 of approx. 21.1 μg [16]. Recent studies on laboratory mice have shown that plant lectins might be employed in the formulation of novel cancer diagnostic and therapeutic approaches in pre-clinical stages [33]. Several typical lectins such as mistletoe lectin, ricin and Wheat germ agglutinin have been reported to possess remarkable antitumour activities by inducing apoptosis in cancer cells [17]. Animals with cancer showed an almost complete inhibition of tumour growth when mistletoe lectins were applied to the right side [34].

The denaturation of NNTL by urea and DTT indicates the globular nature of lectin that was stabilized mainly by hydrogen bonding and hydrophobic interactions [35]. Demetallization and remetallization of NNTL suggest that the lectin activity was dependent on the presence of metal ions. The agglutination activity of NNTL recovered 100% by the addition of 5 mM Ca2+, Ba2+ or Mg2+. A similar result was obtained when DB3 [10] treated with the chelating agent EDTA. On the other hand, EDTA treatment did not affect the agglutination activity of DB1 [10], AJL [5] and HTTL [11]. Furthermore, to check the Ca2+-induced structural stability of NNTL, measurement of fluorescence spectra were done at different states as shown in Figure 5. The fluorescence intensity of NNTL at 330 nm was increased remarkably in the presence of Ca2+ salt. Furthermore, on addition of o-nitrophenyl β-D-galactopyranoside to the lectin solution, the fluorescence intensity decreased remarkably as compared with that in the presence of Ca2+ salt. From this finding it may be suggested that the changes in the fluorescence spectra induced by Ca2+ and o-nitrophenyl β-D-galactopyranoside might be attributed to the change in the environment of the tryptophan residue(s) at or near the carbohydrate-binding site of NNTL [36]. No remarkable change was found when NNTL in TBS was incubated with 4 M urea and 1 mM CaCl2 for 90 min and 8 h. But fluorescence intensity decreased remarkably when NNTL in TBS was incubated with 4 M urea in the absence of CaCl2 for 90 min and the decrease in intensity was very large when the incubation period was increased to 8 h compared with that of NNTL-containing urea and CaCl2. This result indicates that Ca2+ induces a conformational change of NNTL molecule that stabilized the lectin structure.

Abbreviations

     
  • AHL

    Arisaema helleborifolium lectin

  •  
  • AJL

    Arisaema jacquemontii lectin

  •  
  • BBL

    Belamyia bengalensis lectin

  •  
  • DB1

    Dioscorea batatas protein 1

  •  
  • DB3

    Dioscorea batatas protein 3

  •  
  • DTT

    dithiothreitol

  •  
  • EAC

    Ehrlich ascites carcinoma

  •  
  • HTTL

    Helianthus tuberosus tuber lectin

  •  
  • MSL

    Mulberry seed lectin

  •  
  • NNTL

    Nymphaea nouchali tuber lectin

  •  
  • PAS

    periodate–Schiff

  •  
  • TBS

    Tris/HCl-buffered saline

  •  
  • WGA

    wheatgerm agglutinin

AUTHOR CONTRIBUTION

Syed Rashel Kabir is the first author who contributed most to this research and wrote the manuscript; Md. Abu Zubair, Md. Nurujjaman, Md. Azizul Haque, Imtiaj Hasan, Md. Tanvir Hossain and Md. Farhadul Islam were involved in the specific works and helped the first author. Md. Anowar Hossain and Yoshinobu Kimura determined the N-terminal sequence of the lectin; Md. Abdur Rakib performed the amino acid analysis; Md. Taufiq Alam, Ranajit Kumar Shaha, Md. Tofazzal Hossain and Nurul Absar provided technical support.

We thank Dr Parvez Hassan, Institute of Biological Sciences, Rajshahi University, for his kind suggestions and help.

FUNDING

This work was partially supported by the Faculty of Science, Rajshahi University.

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