Author's Accepted Manuscript

Comparison of the nature of interactions oftwo sialic acid specific lectins Saraca indicaand Sambucus nigra with N-acetylneuraminicacid by spectroscopic techniques

Shuvendu Singha, Partha P. Bose, Tapan Gang-uly, Patricia T. Campana, Rina Ghosh, Bishnu P.Chatterjee

PII: S0022-2313(14)00691-7DOI: http://dx.doi.org/10.1016/j.jlumin.2014.11.041Reference: LUMIN13041

To appear in: Journal of Luminescence

Received date: 18 July 2014Revised date: 30 October 2014Accepted date: 24 November 2014

Cite this article as: Shuvendu Singha, Partha P. Bose, Tapan Ganguly, Patricia T.Campana, Rina Ghosh, Bishnu P. Chatterjee, Comparison of the nature ofinteractions of two sialic acid specific lectins Saraca indica and Sambucus nigrawith N-acetylneuraminic acid by spectroscopic techniques, Journal ofLuminescence, http://dx.doi.org/10.1016/j.jlumin.2014.11.041

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1

Comparison of the nature of interactions of two sialic acid specific

lectins Saraca indica and Sambucus nigra with N-

acetylneuraminic acid by spectroscopic techniques

Shuvendu Singhaa,b

, Partha P. Bosec, Tapan Ganguly

d, Patricia T. Campana

e, Rina

Ghoshb and Bishnu P. Chatterjee

a,*

aDepartment of Natural Science, West Bengal University of Technology, Kolkata 700064,

India bDepartment of Chemistry, Jadavpur University, Jadavpur, Kolkata 700032 cDepartment of Biotechnology, National Institute of Pharmaceutical Education and Research

(NIPER), Hajipur, 844101, India dSchool of Laser Science and Engineering, Jadavpur University, Jadavpur, Kolkata 700032,

India eEscola de Artes, Ciências e Humanidades, Universidade de São Paulo, 03828-000, São

Paulo, Brazil *Corresponding author. Department of Natural Science, West Bengal University of

Technology, Kolkata 700064, India, e-mail: cbishnup@gmail.com , Phone: +91-33-2321-

0731

2

Abstract

The present paper deals with the isolation and purification of a new sialic acid binding

lectin from the seed integument of Saraca indica (Ashok) and the purified lectin was

designated Saracin II. Comparative studies on the interactions of saracin II and another sialic

acid specific lectin Sambucus nigra agglutinin (SNA) with N-acetylneuraminic acid (NANA)

were made using UV-vis absorption, steady state and time resolved fluorescence along with

circular dichroism (CD) spectroscopy to reveal the nature and mechanisms of binding of

these two lectins with NANA. The experimental observations obtained from UV-vis, steady

state and time resolved fluorescence measurements demonstrated that SNA-NANA system

formed relatively stronger ground state complex than saracin II-NANA pair. CD

measurements further substantiated the propositions made from steady state and time

resolved spectroscopic investigations. It was inferred that during interaction of SNA with

NANA, the lectin adopted a relatively looser conformation with the extended polypeptide

structures leading to the exposure of the hydrophobic cavities which favoured stronger

binding with NANA.

Key words:

Saracin II, time resolved spectroscopy, circular dichroism, tryptophan, NANA

Abbreviations:

Saracin II, the second lectin from Saraca indica; SNA, Sambucus nigra agglutinin;

NANA, N-acetylneuraminic acid; DEAE, diethylaminoethyl; BSA, bovine serum albumin;

PTG, porcine thyroglobulin; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel

electrophoresis; TEMED, N,N,N´,N´-tetramethylethylenediamine; TBS, Tris-buffered saline;

TCSPC, time-correlated single-photon counting

3

1. Introduction

Lectins are proteins or glycoproteins omnipresent in nature which bind specifically

and reversibly to carbohydrate moieties of complex glycoconjugates on cell surface resulting

in their agglutination [1]. They are found to be present in viruses, bacteria, fungi, plants, and

animals [2-4]. Owing to their carbohydrate binding properties they participate in a variety of

cellular processes e.g. cell-cell recognition [5], cell adhesion [6], cell signaling [7,8],

fertilization [9], opsonisation [10], mitogenesis [11-13] and apoptosis [14,15]. Participation

of lectins in biological processes also includes host pathogen interaction, serum glycoprotein

turn over and innate immune response [1]. Most of the lectins bind to mono- and

oligosaccharides as observed by their hemagglutination-inhibition; but some of them are

specific to complex saccharides and their activity are inhibited by glycoproteins as found in

Scilla campanulata bulb lectin [16], Acacia constricta seed lectin [17], and Arisaema flavum

tuber lectin [18]. Lectins find enormous application in analytical chemistry as well as

preparative biochemistry, for example, purification and characterization of glycoconjugates

as well as in biomedical fields such as fractionation of cells and their use in bone marrow

transplantation [19]. Over the last few decades, lectins have created interest to a diverge

spectrum of researchers owing to their potential biological properties including antitumor

[20,21], immunomodulatory and anti-insect [22], antifungal [23], antibacterial [24], anti-HIV

[23,25], and mitogenic [26] activities.

Sialic acids are often found as the end-capping groups in the terminals of cell surface

glycans of vertebrates and are very important sugar molecules in life processes since they

play significant regulatory and protective roles in cell biology [27-29]. There are a large

number of sialic acid-binding lectins that have been isolated from microorganisms, plants,

and animals [28]. With growing evidence of the crucial lectin-sugar interactions in many

important life processes the study of the binding events of lectins with sugars have received

impetus in recent years [30-32].

Previously we isolated a monomeric lectin, saracin (molecular mass ~12 kDa) from

Saraca indica (Ashok) seed integument extract by tandem purification steps of PTG-

sepharose 4B, gel filtration chromatography on Sephadex G-50 column and PAK 300 SW

column in HPLC [33-35]. Importantly, saracin was found to be a weak mitogen for normal T

cells and can induce apoptosis on the activated T cells in vitro [34]. A second new lectin

saracin II has recently been isolated from seed integument of Ashok tree (Saraca indica) by

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the combination of DEAE-ion exchange chromatography, gel filtration chromatography on

Superose 6 and PTG-sepharose affinity chromatography. Like saracin, saracin II has no

specificity for human blood group erythrocytes and to be constituted with a single

polypeptide chain of apparent molecular mass of 75 kDa by SDS-PAGE.

The present paper describes the purification and physico-chemical characterization of

a new lectin, saracin II from S. Indica seed integument. Transitions in the tryptophan

microenvironment and global structural changes in terms of secondary structural elements

upon binding of an inhibitor NANA with saracin II have been described by steady state and

time resolved fluorescence and circular dichroism study. A comparative study of another

sialic acid specific plant lectin, Sambucus nigra agglutinin (SNA1) with saracin II has also

been made under the treatment of same inhibitor, NANA.

2. Materials and Methods

2.1. Materials

Acrylamide, N,N´-methylenebisacrylamide, ammonium persulfate, TEMED, sodium

dodecyl sulfate, BSA, Tris, CNBr (cyanogen bromide) activated-Sepharose 4B, N-

acetylneuramic acid, porcine thyroglobulin (PTG), DEAE-cellulose, transferrin,

ceruloplasmin, fetuin, γ-globulin, haptoglobin and Sambucus nigra agglutinin (SNA) were

purchased from Sigma, USA. Precision Plus Protein Standards (Protein markers) was

procured from BioRad, USA. Superose 6 10/300 GL gel filtration column was purchased

from Pharmacia Biotech, Uppsala, Sweden. Gel filtration markers for protein molecular

weights (Carbonic anhydrase from bovine erythrocytes, ovalbumin from chicken, BSA,

alcohol dehydrogenase from yeast and β-amylase) were purchased from Sigma. All other

reagents were obtained from commercial sources and were of highest purity grade.

2.2. Purification of lectin

Saraca indica seed integument extract (20% w/v) was prepared by soaking in Tris-

buffered saline (10 mM Tris, 150 mM NaCl, pH 8) for 5 hrs at 4˚C. The extract after

filtration through Whatman filter paper No. 1 was dialysed, concentrated by Amicon Ultra

Centrifugal filters, and stored in 10 ml aliquots in –20˚C for further use. Lectin from the

extract (0.8 mg/ml, 2 ml) was purified subsequently by anion-exchange chromatography on

5

DEAE-cellulose column (1 x 20 cm) attached to FPLC system (Akta Prime Purifier, GE

Healthcare). The column washed with 10 mM Tris-HCl, pH 8, and equilibrated with the same

buffer. The bound protein was obtained using gradient elution with 1 M NaCl. The agglutinin

from DEAE-cellulose column was subjected to gel filtration chromatography on a Superose 6

10/300 GL column (1 x 30 cm). The column was equilibrated with TBS (10 mM Tris, 150

mM NaCl, pH 8). The added sample was washed with the same buffer. Further purification

was achieved by affinity chromatography using PTG-sepharose 4B matrix (1 x 2 cm). The

concentrated active fraction from gel filtration column was loaded on to affinity column,

percolated well and kept overnight at 4 ˚C. The unbound fractions were eluted with TBS and

the bound fractions were eluted with glycine-HCl buffer (0.1 M, pH 3.5) and immediately

neutralized with saturated sodium bicarbonate solution. After each purification step the

activity was checked by hemagglutination assay with 2% pronase-treated human

erythrocytes.

2.3. Protein estimation

Protein content of all the extract and stepwise purified lectin fractions were

determined by Bradford method using BSA as the standard [36].

2.4. Hemagglutination and hemagglutination-inhibition assay

The hemagglutinating activity of the extract and the stepwise purified lectin fractions

was determined by incubating a 2-fold serially diluted sample (25 µl) in TBS with an equal

volume of 2% (v/v) pronase-treated human B erythrocytes suspension in saline for 1 h at 25

ºC in a 96-well polystyrene plate. Hemagglutination titer is defined as the reciprocal of the

highest dilution showing visible hemagglutination after 1 h [37].

The hemagglutination-inhibition assays were performed by preincubating 25 µl of

two hemagglutination doses of saracin II with known concentration of serially diluted

saccharides and glycoproteins (25 µl) in 96-well polystyrene U-bottomed microtitre plate for

2 h at 25 ºC. Pronase-treated human B erythrocytes suspension (25 µl, 2% w/v) was added to

each well and the results were recorded after 1 h. Controls contained 25 µl TBS instead of the

inhibitor solution. The inhibitory activity of saccharide or glycoprotein is defined as the

minimum concentration (mg/ml) required for complete inhibition of two hemagglutination

doses of the lectin.

6

2.5. Gel electrophoresis

The homogeneity and molecular weight of saracin II was determined by 10% SDS-

PAGE with and without 2-mercaptoethanol according to Laemmli using precision plus

protein standards (BioRad) [38]. The gel was stained with Coomassie Brilliant Blue G-250.

2.6. Physico-chemical property

Aliquots of saracin II (0.5 ml) in TBS was incubated at different temperatures (30-

80ºC) for 10 min and cooled in ice. The activity of the aliquots was tested with pronase-

treated human B erythrocytes.

The effect of pH on hemagglutinating activity of saracin II was studied in the pH

range of 3.5 to 10 using different buffers such as glycine-HCl (100 mM, pH 3.5), sodium

acetate (100 mM, pH 5), citrate phosphate (150 mM, pH 6), sodium phosphate (100 mM, pH

7), Tris-buffered saline (20 mM, pH 7.5), Tris-HCl (150 mM, pH 8) and glycine-NaOH (100

mM, pH 10). The lectin was dialyzed against desired buffer at 4ºC for 6 h and then

hemagglutination assay was performed.

2.7. UV-vis, steady state and time resolved fluorescence spectroscopy

Steady-state UV–vis absorption and fluorescence emission spectra of dilute solutions

(10-4 - 10-6 mol dm-3) of the lectin samples were recorded at 25 ºC using 1 cm path length

rectangular quartz cells by means of an absorption spectrophotometer (Shimadzu UV-1800)

and Perkin Elmer LS 55 fluorescence spectrophotometer (Hitachi) respectively.

Fluorescence lifetime measurements were carried out by the time-correlated single-

photon counting (TCSPC) method using a Horiba Jobin Yvon Fluorocube. For fluorescence

lifetime measurements the samples were excited at 375 nm using a picosecond diode (IBH

Nanoled-07). The emission was collected at a magic-angle polarization using a Hamamatsu

microchannel plate photomultiplier (2809U). The TCSPC setup consists of an Ortec 9327

CFD (constant fraction discriminator) and a Tennelec TC 863 TAC (Time to amplitude

converter). The data is collected with a PCA3 card (Oxford) as a multichannel analyzer. The

typical full width at half-maximum (fwhm) of the system response is about 80 ps. The

7

channel width is 12 ps /channel. The fluorescence decays were deconvoluted using IBH

DAS6 software. The goodness of fit has been assessed over the full decay including the rising

edge with the help of statistical parameters χ2 and Durbin Watson (DW) parameters. All the

solutions for room temperature measurements were deoxygenated by purging with argon gas

stream for about 30 mins.

2.8. Circular dichroism (CD) spectroscopy

CD spectra were recorded in a Jasco J-600 spectropolarimeter (Jasco Inc., Japan) in

the far UV region (250-190 nm) at 25 ºC. Saracin II (3.8 x 10-6 M) and SNA (4.3 x 10-6 M) in

a 1-mm rectangular quartz cell were used in the experiment. The above experiment was

conducted for both the lectins in the presence of different concentrations of NANA. All

spectra were recorded after accumulation of three runs and smoothened using a fast Fourier

transform filter to minimize background effects. The data were expressed in terms of molar

ellipticities (θ) in deg.cm2.dmol-1. Quantitative prediction of the secondary structure was

performed by deconvolution of the CD spectra using CONTINLL, CDSSTR, and SelCon

programs [39]. The better results were achieved from CONTINLL program, with a root mean

square difference between the experimental and calculated curves (RMSDExp-Calc) lower

than 5 % for all deconvolutions.

3. Results

3.1. Purification and characterisation of saracin II

Purification of S.indica seed integument lectin involved three-steps: ion-exchange

chromatography on DEAE-cellulose, gel filtration chromatography on Superose 6 10/300 GL

and affinity chromatography on PTG-sepharose 4B column. The seed integument extract by

ion-exchange chromatography separated into four fractions (Supplementary Fig. S1). The

fourth fraction (peak IV) having hemagglutination activity (sp. activity 246) resolved into

two fractions when passed through gel filtration column (Supplementary Fig. S2). Only

fraction I (peak I) showed hemagglutination (sp. activity 941), which was further purified by

affinity column (Fig. 1A) and the purification achieved was 145 fold. The purified lectin was

designated saracin II and its homogeneity was tested by 10 % SDS-PAGE under both

reducing and non-reducing conditions. It was found to be homogenous with an apparent

molecular mass of 75 kDa (Fig. 1B). The native molecular mass of saracin II was determined

8

by gel filtration chromatography on calibrated Superose 6 10/300 GL column was 74.13 kDa

(Fig. 1C) and hence it is monomer. The purification profile is shown in Table 1. The

hemagglutination titre was found to be increased with pronase-treated erythrocytes and in the

presence of Ca2+; the property being common to lectins as a whole. Saracin II exhibited

maximum activity from 30 to 40 °C; its activity gradually decreased with rise in temperature

and persisted up to 80 oC (titer 2). It showed activity at pH between 3.5 and 10 being

maximum between pH 7.5 and 8. The lectin saracin from the seed integument of S. indica

earlier isolated by consecutive chromatography on PTG-sepharose, gel filtration on Sephadex

G-50, and Protein PAC 300 SW column had molecular mass of 12 kDa and was a monomer.

However, the purification fold of saracin achieved was 182 [33]. The effect of temperature

and pH on saracin characterized earlier was identical to that of saracin II [33]. Like saracin no

mono- and oligosaccharides including N-acetylneuraminic acid (NANA), neuraminyl lactose

[NeuAc-α-(2→6)/(2→3)-D-Gal-β-(1→4)-Glc], lactosylamine [D-Gal-β-(1→4)- GlcNAc]

(50 mM each) and [D-Gal-β-(1→3)-GalNAc] (100 mM) inhibited saracin-II induced

hemagglutination. However certain sialo glycoproteins containing NeuAc-α-(2→6)/(2→3)-

Gal unit were inhibitory as shown in Table 2. Among them porcine thyroglobulin (PTG) was

found to be the most potent (0.078 mg/ml) inhibitor whereas its asialo counterpart did not

inhibit at all. Fetuin, ceruloplasmin, transferrin, haptoglobin inhibited the hemagglutination

fairly well; α1-acid glycoprotein showed least inhibitory effect.

3.2. UV-vis absorption and steady state fluorescence emission spectra of SNA-NANA and

saracin II-NANA systems

Steady-state UV-vis absorption spectra of SNA in 150 mM NaCl at pH 7.5, at 25 ºC

was measured and the effect of increasing concentration of NANA on SNA was examined.

With gradual addition of NANA, the entire absorption spectrum underwent a hypochromic

effect without any noticeable spectral shift (Fig. 2A). It is to be mentioned that in the

absorption spectral region of SNA, the addition of NANA with varying concentrations used

in absorption measurements did not show any significant absorbance. It is apparent that the

observed hypochromic effect in UV-vis spectra could be due to ground state complex

formation between SNA and NANA. Further, Benesi–Hildebrand (BH) plot (inset in Fig.

2A) constructed by using Equation 1:

1/( ε0 – εc) = 1/( ε0 – εb) + 1/( ε0 – εb)(1/K)(1/C). (1) [40]

9

shows clearly the linear relation between 1/(ε0 – εc) vs 1/C, where ε0 and εc are the respective

molar extinction coefficients of SNA in the absence and presence of NANA having the

concentration C. εb denotes the molar extinction coefficient for the complex molecule. The

observed linearity corroborates the formation of a 1:1 adduct (complex) between the sugar

and the lectin. The association constant of the SNA-NANA complex was measured from the

BH plot and its value was found to be 4.4 (±0.2) ×104 M-1.

Steady state fluorescence emission spectra of SNA in 150 mM NaCl were recorded in

the presence of different concentrations of NANA (Fig. 2B) at pH 7.5 at 25 ºC using the

excitation wavelength at 280 nm. It is noteworthy that at ~ 280 nm only SNA was excited but

not NANA as the latter sample was transparent in this region even at the maximum

concentrations used in the fluorescence quenching measurements. The emission band of SNA

was found to be quenched regularly with increasing concentration of NANA. The spectra

were analyzed with the help of the Stern–Volmer (SV) relation represented by the Equation 2

(Fig. 2C) [41-44].

F0/F = 1+ KSV [Q] (2)

Where F0 and F denote the steady-state fluorescence emission intensities in the absence and

presence of the quencher, respectively, KSV is Stern Volmer constant and [Q] is the

concentration of the quencher NANA.

As SV plot, measured from steady state fluorescence emission intensities of SNA (~

5.7 × 10-7 M) in the absence and presence of NANA, shows linearity (Fig. 2C) it indicates

that the nature of the quenching either is of static type or of dynamic one [45]. As the

fluorescence lifetimes (details given in next section) of SNA remained unchanged i.e., did not

quench in presence of NANA, the quenching mode should be, in high probability, static in

nature. Thus, the ground state complex formation between SNA and NANA appears to be

primarily responsible for the observed quenching phenomena. The value of KSV was

computed from the slope of the straight line and found to be 1.55 (± 0.2) x 106 M-1.

Steady state UV-vis absorption spectra of the other lectin saracin II in 10 mM TBS,

pH 8, at 25 ºC was recorded and the effect of increasing the NANA concentration on the

spectra was examined (Fig. 3A). With the addition of NANA, the absorption spectrum of

saracin II like SNA underwent similar hypochromic effect but without any noticeable spectral

shift (Fig. 3A) throughout the entire spectral envelop. The linear BH plot (inset in Fig. 3A)

10

suggested the formation of 1:1 complex between saracin II and NANA in the ground state,

similar to the situation observed in SNA-NANA system.

Steady-state fluorescence emission spectra produced by the excitation of wavelength

λex ~ 280 nm of saracin II (~ 5.6 × 10–6 M) in 10 mM TBS, pH 8 were measured in the

presence of varying concentrations of NANA at 25 ºC (Fig. 3B). It was observed that at ~ 280

nm only saracin II was excited but not NANA as the latter sample was found to be

transparent in this region even at its maximum concentrations used in the fluorescence

quenching measurements. The fluorescence of saracin II was found to be quenched regularly

without any spectral shift with the addition of the quencher NANA. The fluorescence data

were analyzed by the well-known SV relation (shown in Equation 2).

The linear SV plot obtained by using the Equation 2 is reproduced in the inset of Fig.

3B. From the time resolved spectroscopic measurements it was apparent, similar to the

situation observed for the other lectin SNA and NANA system, that the fluorescence lifetimes

of saracin II was not affected much with addition of NANA. Thus, the quenching observed

here along with the linearity in SV plot should be primarily due to the presence of static

quenching mode. KSV value was determined from the plot which was found to be (5.14

(±0.2) x 104 M-1) nearly two order of magnitude less than the corresponding value observed

for SNA-NANA system. This indicated weaker ground state complex formation in the case of

the saracin II-NANA pair. To substantiate the possibility of the stronger bonding in SNA-

NANA system, the binding sites and binding constants were determined for the two lectins

bound with NANA, which has been discussed below.

3.3. Number of binding sites and binding constant for SNA-NANA and saracin II-NANA

systems

When small molecules bind independently to a set of equivalent sites of a

macromolecule, the equilibrium between the free and bound molecule is given by the

following generalized Equation 3,

Log [(F0–F)/F] = n log Kb - n log [1/{[Q] - (F0-F)[Pt]/F0}] (3)

Where “n” represents the binding site and “Kb” is the binding constant.

From the linear plot of Log [(F0–F)/F] versus log [1/{[Q] - (F0-F)[Pt]/F0}] for SNA-NANA

system (Fig. 4A), the observed values of n and Kb were found to be 1.1 and 3.31 (±0.3)

11

x 106 M-1 respectively. The Kb value was observed to be very similar to the magnitude of KSV

(~1.55 X 106 M-1) obtained for this system from linear SV plot. From the corresponding

linear plot of saracin II-NANA system (Fig. 4B), respective values of n and Kb were

estimated to be ~1.28 and 7.29 x 104 M-1. The Kb and KSV (5.14x 104 M-1) values were nearly

similar, as expected. Both the Kb and KSV values were found to be two orders of magnitude

larger in the case of SNA- NANA system, relative to the corresponding values observed for

saracin II-NANA pair. However, in both the cases the nature of the complex was found to be

1:1, as evidenced from the values of n (binding sites). The possibility of formation of 1:1

ground state complex between the present lectins and the sugar ligand (NANA) was also

inferred from the linear BH plots measured from the electronic (UV-vis) absorption spectra

(Insets of Fig. 2A and 3A).

All the above experimental observations, made from UV-vis and steady state fluorescence

measurements demonstrated that SNA-NANA system forms relatively stronger ground state

complex than saracin II-NANA pair, as evidenced from the observed larger KSV and Kb

values resulted from the former binding interactions. Apart from the steady state

observations, it is felt that time resolved measurements through fluorescence lifetime

determination may provide further useful evidences in favour of formations of ground state

binding within SNA (or saracin II) when interacts with NANA.

3.4. Fluorescence lifetime measurements

Time resolved measurements of SNA in 150 mM NaCl, pH 7.5 and saracin II in 10

mM TBS, pH 8 were carried out in the presence of different concentrations of NANA. To

substantiate the static nature of quenching, fluorescence lifetime measurements (Fig. 4C)

were carried out by using time correlated single photon counting (TCSPC) set up. From the

analysis of the fluorescence decays of both SNA and saracin II, it is apparent that the three

exponential fit was the best fit, as inferred from the observed values of χ2 and Durbin-Watson

parameters (DW). Both the lectins exhibit primarily the three lifetimes which are of the

values of 90 ps, 2 ns and 5.7 ns for SNA and 70 ps, 2 ns, 5.0 ns for saracin II. In both

lectins the three fluorescence lifetimes remained unaltered in presence of the quencher

NANA. According to our earlier observations on fluorescence lifetime values of different

proteins [46], it is apparent that the lifetime corresponding to picosecond (ps) order should

12

correspond to a tryptophan (Trp) residue (Trp14) and 5.0 ns should be for unquenched

tryptophan in the system.

The average fluorescence emission lifetime, < τ>, was computed from the expression

[46],

<τ> = ∑ ai τi2 / ∑ ai τi

where ai and τi represent the preexponential factor and the corresponding lifetime,

respectively. The average decay time obtained by using the above equation reveals that the

magnitudes of the lifetimes of both the lectins, SNA and saracin II do not change

significantly with addition of NANA. This observation further confirms that the quenching

of fluorescence of SNA or saracin II in presence of the quencher NANA is of static in nature

and the quenching is primarily due to the ground state complex formations between the

lectins and NANA. Thus it corroborates the views made from the steady state measurements

as discussed above.

3.5. Circular dichroism (CD) spectroscopy and secondary structure

Plant lectins from different families do not possess identical three dimensional

structures. Nevertheless they share some common structural features to uphold the specificity

in sugar binding. In lectin, occurrence of β-sheets is predominant, if not exclusive, in

different three dimensional organizations. These β-sheets connected by turns or loops provide

a rigid structural scaffold in lectin’s structures. Carbohydrate-binding sites are mostly

concave pockets surrounded by these β-sheet scaffolds converging together by loops or turns

[47]. This crucial quaternary arrangement subscribes the specific binding of lectins with

sugars. The binding affinity varies due to the presence of different amino acid residues in the

binding sites with different relative stereochemical arrangements [48,49]. With regard to

sugar specificity of lectins, sialic acid specific lectins have been one of the most extensively

studied family in the area of structural biology of lectins [50,51].

The circular dichroism spectra of two lectins, S. nigra and saracin II in the absence

and presence of NANA measured at 25 ºC are presented in Fig. 5A and 5B in the far UV

region at 190–250 nm providing information about the secondary structure of these proteins.

From Fig. 5A it is observed that SNA native spectrum presents a minimum negative peak at

208 nm and a positive peak around 193 nm. The spectral deconvolution of SNA gave 18% of

helix content (9% regular and 9% distorted), 55 % of beta elements (21% of regular, 17% of

13

distorted beta forms and 17% of turns) and 25% of unordered forms (Supplementary Table

T1).

According to crystallographic structure of native SNA (described as containing 10

helix and 22 strands, turns and unordered forms are not described), SNA presents beta

distorted strands forming sheets (yellow) together with some distorted helical content (pink)

(Supplementary Fig. S3). Hence, the results from both CD spectrum and the secondary

structure content calculated for native SNA are in agreement with the crystallographic

structure, in which the proportion of beta elements is around the triple of the helical elements

proportion. When NANA was added, the structure seemed to be changed. With the addition

of 4.2 µM NANA a slight shoulder at 220 nm has been observed suggesting an increase in

secondary order, as shown in Fig. 5A (red spectrum). The deconvolution of SNA with 4.2

µM NANA resulted in 18% of helix content (9% regular and 9% distorted), 60 % of beta

elements (23% of regular, 17% of distorted beta forms and 20% of turns) and 22% of

unordered forms. Indeed, the regular content increased around 3%, suggesting small changes

in the secondary overall structure as expected for the binding of small ligands

(Supplementary Table T1).

From the complex formed with the addition of 23 and 86 µM NANA respectively , it

is not clear whether additional changes occur, and the spectra shapes are similar to the first

one, except the difference in intensity, although the deconvolution of these spectra showed a

slight decrease in unordered forms and the increase in regular content in comparison to the

native SNA. This increament, mainly in beta forms with the addition of 23 µM NANA (8%

helical forms: 2% regular and 6% distorted; 69% beta forms: 29% of regular, 18% of

distorted and 22% of turns) and in helical forms with 86 µM NANA addition (20% helical

forms: 11% regular and 9% distorted; 57% beta forms: 23% of regular, 16% of distorted and

18% of turns), suggests strong binding for NANA, generating distortion effect at spectra

intensities more than modifications at secondary structure levels (Supplementary Table T1).

Saracin II, on the other hand, seems to be all beta. Its native CD spectrum presents a

negative minimum about 222 nm and a positive maximum near to 200 nm, suggesting a high

content of beta elements. Indeed, when this spectrum is deconvoluted no regular helix content

was found (only 3% of distorted helix). On the other hand, a considerable content of beta

sheet (42%) and turns (21%) were found. Concerning the unordered content, 34% was found.

Saracin II structure has found to be sliglty changed by the addition of NANA. In all cases, the

14

addition of 2.8 µM, 42 µM, and 120 µM NANA caused a positive maxima and negative

minima red shift, suggesting in a similar way to NANA addition to SNA causing distortion

effects due to strong binding. These results have been confirmed by the percentages

calculated from CD spectra deconvolution, that shows the same content as already found for

saracin without addition of NANA : 42% beta sheet, 21% turns and 34% unordered content.

Again, the differences in intensity on these spectra can be due to distortion effects at spectra

intensities, secondary structure reorganization or localized changes that can not be seen in CD

at this wavelength range (Supplementary Table T1).

Therefore, to elucidate the trend of secondary structural change, molar ellipticity

values at 217 nm within the same concentration window of added NANA in both the lectins

was plotted. In both the cases there were significant drops in the molar ellipticity values with

increasing concentration of NANA (Supplementary Fig. S4). It was observed that the two

lines depicting the molar ellipticity changes against concentration (for two lectins, SNA and

aracin-II) of added NANA were nearly parallel and the differences of molar ellipticity for two

consecutive concentrations of NANA were nearly the same. Therefore it validates the

observation from fluorescence experiments that both the lectins form ground state complex

with NANA in similar stoichiometry (at least within the current window of concentrations).

Upon addition of NANA there has been no substantial shifts in the CD profiles of any of the

lectins that corroborates retyention of their native structures as a whole. To measure the effect

of complexation on the over all compactness of secondary structural elements of the lectin

molecules before and after the hosting of NANA molecules, the molar ellipticities just

beyond (2 nm in higher energy side) the corresponding positive branch (at 192 nm for SNA

and 202 nm for saracin II). It was calculated and this has been a measure of overall

randomness imposed in the lectin structure after hosting NANA. The difference in molar

ellipticities between the two situations, lectins with higest amount of added NANA and the

lectins in native states produced moderately different values with SNA and saracin II

(Supplementary Fig. S5; ∆θC1-C4). The difference in values (4673 for SNA and 3748 for

Saracin) suggested that upon NANA binding to SNA recorded more effect on secondary

structural level and the structure became more randomized after hosting NANA than that in

saracin II. NANA binding to SNA or saracin II is presumed to be mediated by salt-bridge

formation between the NANA carboxylate ion and positively charged amino acids side chains

in lectin’s binding pockets that might be expected important for recognition. However the

15

published data on the binding of sialic acid specific lectins already pointed out that with the

exception of polyoma virus [52], the carboxylate moiety of the guest interacted with main-

chain amide groups, polar side chains (mostly serine), and ordered water molecules rather

than fully charged side chains of amino acids by hydrogen-bond interactions [53,54]. The

difference in binding constants as observed by fluorescence studies could be ascribed by the

fact that SNA-NANA complex may very well employ H-bonding interactions as evident from

similar kind of binding episodes already established [53,54] between NANA carboxylate and

backbone amide or other ionizable side chains like OH- of serine directly but in saracin II-

NANA some of this crucial salt bridge H-bondings might seek the intermediacy of bound

water structure within the pocket, so in the later case the imposed randomization was less as

observed in the CD (Supplementary Fig. S5) upon NANA binding. The effect on the peptide

back bones might also be less as they were not in direct interplay with the guest NANA (only

via ordered water structure) [55,56]. Because of these two different modes of binding in two

cases, the complex with saracin II was expected to be little labile than the former which was

evident from our fluorescence study.

4. Discussion

From the above experimental findings, both from steady state and time resolved

spectroscopy as well as CD measurements, the possible mechanisms of the interactions

between the two lectins and NANA have been revealed. The observed steady state ,time

resolved and CD spectroscopic studies demonstrate that of the two lectins studied, SNA

forms stronger binding with NANA. Steady state and Time resolved spectroscopic

(fluorescence lifetime measurements) results indicate that tryptophan (Trp) residues of SNA

plays significant role in binding. Following earlier observations on some other protein-

carbohydrate systems, it may be concluded that such binding originates from hydrogen

bonding interactions between amino acid Trp and the hydroxyl groups of the sugar. From the

experimental observations, it indicates that in SNA, the Trp14 residue, which was

predominantly buried in the hydrophobic core of the lectin, gets exposed to the aqueous

environment in the presence of NANA. It may be presumed that during interaction of SNA

lectin with NANA, the lectin adopts a labile conformation with the extended polypeptide

structures. This conformational change results in leading to the exposure of the hydrophobic

cavities. Thus a favourable condition for the binding of SNA-NANA is established. On the

other hand the lectin saracin II may not undergo favorable conformational changes for the full

16

exposure of Trp residue. It may be possible that NANA might affect only the partially

exposed Trp which results in weaker bonding, as evidenced from the observed values of KSV

and Kb.

CD measurements further substantiated the propositions made from steady state and time

resolved spectroscopic investigations. It was inferred that during interaction of SNA with

NANA, the lectin adopted a relatively looser conformation with the extended polypeptide

structures leading to the exposure of the hydrophobic cavities which favoured stronger

binding with NANA.The experimental findings from CD measurements throw light on the

lectin-carbohydrate interactions process of two sialic acid specific lectins (SNA/saracin II-

NANA). Given the ever growing interest of sialic acid in the context of various disease and

host pathogen interaction, the new lectin, saracin II and the spectroscopic studies of two

lectins as described can pave the way for the new generation of lectin based-therapeutics and

small molecular devise to specifically interfere with the various crucial lectin-sialic acids

interaction relevant to a diverse biomedical perspective [57,58].

5. Conclusions

From the steady state and time resolved spectroscopic investigations along with

CD measurements it was inferred that during interaction of SNA with NANA, the lectin

adopted a relatively looser conformation with the extended polypeptide structures leading to

the exposure of the hydrophobic cavities which favoured stronger binding with NANA. The

difference in binding constants for the lectins SNA and saracin II as observed by fluorescence

studies could be ascribed by the fact that SNA-NANA complex may very well employ H-

bonding interactions as evident from similar kind of binding episodes. The present findings

demonstrate the efficacy of biomedical and pharmacological applications of the lectins, SNA

and Saracin II, and their possible applications as pharmaceutics.

Acknowledgements

The authors are thankful to Dr. Urmimala Chatterjee and Dr. Gautam Mondal for

providing assistance in this study. This work was jointly supported by financial grants from

National Academy of Sciences (Scheme No. NAS/1075/3/9011-12), India and CSIR, New

Delhi (Scheme No. 38 (1256)/10/EMR-II) provided to B. P. Chatterjee. Shuvendu Singha

17

acknowledges Council of Scientific and Industrial Research (CSIR) for fellowship. Tapan

Ganguly thanks the Department of Science and Technology (DST), New Delhi, India, for his

project in SERC scheme (SR/S2/CMP-0045/2012) and the Nano-Mission project (no.

SR/NM/NS-51/2010) for supporting the various research works conducted in his

photophysics and photochemistry group. Tapan Ganguly gratefully acknowledges All India

Council for Technical Education (AICTE), New Delhi for awarding the Emeritus fellowship

and providing the contingency grant for research purpose.

Supplementary Information

Results of deconvolution of CD spectra, elution profiles in DEAE-cellulose anion

exchange and Superose- 6 size exclusion chromatography; X-ray crystallographic structure of

SNA (pdb file: 3CA1), various plots of molar ellipticity values with increasing concentration

of NANA with respect to two lectins.

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Table 1: Purification scheme of Saraca indica seed integument lectin (saracin II)

Lectin

fraction

Protein

(mg/ml)

Hemaggl-

tination titer*

Specific

activity†

Purification

fold

Protein

Recovery (%)

Crude seed

integument

extract

0.8 16 20 1 100

DEAE

anion exchange

chromatography

0.13 32 246 12 16.25

Size exclusion

chromatography on

Superose 6

0.034 32 941 47 4.25

PTG-sepharose 4B

affinity

chromatography

0.022 64 2909 145 2.75

23

*Hemagglutination was determined with pronase-treated human B erythrocytes. †Expressed as titer/mg-protein/ml.

Table 2. Hemagglutination-inhibition assay by carbohydrates and glycoproteins

Glycoprootein Minimum inhibitory

concentration of glycoprotein*

(mg/ml)

Transferrin 0.625

Ceruloplasmin 0.312

Fetuin 0.156

Porcine thyroglobulin 0.078

γ-globulin

Haptoglobin

α1-acid glycoprotein

1.25

12.5

2.5

*Required for complete inhibition of two haemagglutinating doses of lectin against human pronase-treated

erythrocytes. D-Gal, D-Glc, D-Man, D-GlcNAc, D-GalNAc, α-Me-D-Gal, β-Me-D-Gal, α-Me-D-Glc, α-Me-D-

Man, lactose, melibiose. N-acetylneuraminic acid were noninhibitors up to 50 mM. LacNAc, neuraminyl lactose

was noninhibitor up to 100 mM.

Table 3. Fluorescence lifetimes and associated fractional contributions (fi) of SNA (λex

~280 nm, λem ~340 nm ) and saracin (λex ~295 nm, λem ~375 nm )in the presence of

different concentrations NANA

Lectins Conc of

NANA (M)

f1 τ1 (ns) f2 τ2 (ns) f3 τ3 (ns) χ2

SNA 0 0.30 0.09 0.57 1.82 0.13 5.71 1.13

24

(conc. 5.68 × 10-7 M) †4.1 x 10-7-

2.0 x 10-6

0.10 0.06 0.75 2.10 0.15 6.02 ~1.11

Saracin II

(conc. 2.24 × 10–6 M)

0 0.23 2.01 0.37 5.02 0.40 0.065 1.12

‡ 5.0 x 10-6-

1.1x10-5

0.27 2.11 0.34 5.20 0.39 0.074 ~1.11

†Range of NANA concentrations used. As nearly similar f and similar τ values of SNA are

observed within this range of concentrations of NANA, average values are shown.

‡Range of NANA concentrations used. As nearly similar f and similar τ values of saracin are

observed within this range of concentrations of NANA, average values are shown.

Figures with legends

Figure 1 (A) Elution profile of protein fraction (I) from Superose 6 10/300 GL by affinity

chromatography on PTG-sepharose 4B affinity column. (B) SDS-PAGE of the the PTG-

sepharose purified Saraca indica seed integument with and without 2- mercaptoethanol. (C)

Standard curve representing native molecular weight of saracin II using superpose 6 10/300

GL column along with using standard markers carbonic anhydrase, ovalbumin, BSA, alcohol

dehydrogenase and β-amylase. The plot of the gel phase coefficient (Kav) vs. log (Mr) is

25

linear between 200 kD (β-amylase) to 29 kD (Carbonic anhydrase), where Kav = (Ve-Vo)/(Vt-

Vo), Ve = elution volume, Vo = column void volume, Vt = total column bed volume, Mr =

molecular weight.

Figure 2 (A) UV-vis absorption spectra of SNA (~ 1.8 × 10–6 M) in the presence of NANA at

concentration (M): (1) 0, (2) 8 × 10-6, (3) 1.2 × 10-5, (4) 1.74 × 10-5, (5) 2.0 × 10-5, (6)2.17 ×

10-5, at 25 ºC in 150 mM NaCl at pH 7.(Inset) Benesi–Hildebrand plot for SNA-NANA

complex. (B) Fluorescence emission spectra of SNA (~5.7 × 10-7 M) (λex~ 280 nm) in the

presence of different concentrations of NANA (M): (1) 0, (2) 7 × 10-8, (3) 1.2 × 10-7 , (4) 1.7

× 10-7 , (5) 2.0 × 10-7 , (6) 2.34 × 10-7 ,(7) 2.74 × 10-7, (8) 3.4 × 10-7 , (9) 4.1 × 10-7 M 25 ºC in

150 mM NaCl at pH 7.5 (C) Stern–Volmer (SV) plot from steady-state fluorescence emission

intensity measurements of SNA in the presence of NANA in 150 mM NaCl at pH 7.5 at the

25 ºC KSV = 1.55 (± 0.2) x 106 M-1

26

Figure 3 (A) UV-vis absorption spectra of saracin II (~ 2.42 × 10–6 M) in the presence of

NANA at the concentration (M): (1) 0, (2) 1.0 × 10-6, (3) 2.13 × 10-5, (4) 1.0 × 10-5, (5) 1.0 ×

10-4 at 25 ºC in 10 mM TBS, pH 8. (inset) Benesi–Hildebrand plot for saracin II-NANA

complex, K = 4.529 × 105 M-1 (B) Fluorescence emission spectra of saracin II (~ 5.6 × 10-6

M) in the presence of NANA at different concentrations (M): (1) 0, (2) 2.1 × 10-6, (3) 5.0 ×

10-6, (4) 8.0 × 10-6, (5)1.1× 10-5 M, at 25 ºC in 10 mM TBS, pH 8, (λex ~ 290 nm) (inset)

SVplot from steady-state fluorescence emission intensity measurements of saracin in the

presence of NANA, KSV = 5.14 (±0.2) x 104 M-1

27

Figure 4 (A) Plot using the general form Log [(F0–F)/F] = n log Kb - n log[1/{[Q] - (F0-

F)[Pt]/F0}] for SNA and NANA (B) Plot using the general form Log [(F0–F)/F] = n log Kb

- n log[1/{[Q] - (F0-F)[Pt]/Fo}] for saracin II and NANA for λex ~ 290 nm. (C) The

fluorescence decay profile associated with impulse response function (IRF) of SNA in 0.9 %

NaCl, pH 7.5 environment has been carried out in the presence of different concentrations of

NANA. The curves were found to overlap with one another showing the similar magnitudes

of the fluorescence life times of SNA in presence of the different concentrations of NANA

(χ2 ~ 1.15). The similar observations were made for another lectin saracin from time resolved

measurements.

Figure 5 (A) CD spectra of the SNA-NANA system at pH 7 with SNA (4.3 x 10-6 M) and

increasing of NANA concentration (M) in (1) 0, (2) 4.2 x 10-6, (3) 2.3 x 10-5 and (4) 8.6 x 10-

5 (B) CD spectra of the saracin II-NANA system at pH 8 with saracin II (3.8 x 10-6) and

increasing of NANA concentration (M) in (1) 0, (2) 2.8 x 10-6, (3) 4.2 x 10-5 and (4) 1.2 x 10-

4.

28

Highlights

� Of the two lectins, stronger binding of SNA with NANA is observed.

� Full exposure of the hydrophobic cavities of SNA favors the stronger interactions.

� Saracin II can be used for the new generation of lectin based-therapeutics.