Accepted Manuscript

Title: Purification, characterization and biological significance

of mannose binding lectin from Dioscorea bulbifera bulbils

Author: Mamta Sharma H. Vishwanath Reddy B.R. Sindhura

A.S. Kamalanathan Bale M. Swamy Shashikala R. Inamdar

PII: S0141-8130(17)30798-5
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.04.085
Reference: BIOMAC 7449

To appear in: International Journal of Biological Macromolecules

Received date: 3-3-2017

Revised date: 21-4-2017
Accepted date: 24-4-2017

Please cite this article as: M. Sharma, H.V. Reddy, B.R. Sindhura, A.S. Kamalanathan,
B.M. Swamy, S.R. Inamdar, Purification, characterization and biological significance
of mannose binding lectin from Dioscorea bulbifera bulbils, International Journal of
Biological Macromolecules (2017), http://dx.doi.org/10.1016/j.ijbiomac.2017.04.085

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Purification, characterization and biological significance of mannose binding lectin

from Dioscorea bulbifera bulbils

Mamta Sharma1, Vishwanath Reddy H, Sindhura BR, Kamalanathan A. S2, Bale M Swamy,

Shashikala R Inamdar*
1
Department of Studies in Biochemistry, Karnatak University, Dharwad-580003, India

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2
Centre for Bioseparation Technology, VIT University, Vellore 632014, India

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*Corresponding author. Tel.: +91 836 2215243; Fax +91 836 2747884

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E-mail address: srinamdar2009@gmail.com

ABSTRACT
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Dioscorea bulbifera or air potato has been used as a folk remedy to treat cancer. A mannose
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binding lectin from bulbils of Dioscorea bulbifera was purified in a single step by affinity

chromatography on mucin coupled Sepharose 4B column, determined by its fine sugar

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specificity by glycan array analysis and studied for its clinical potential in cancer and HIV

research.
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SDS-PAGE showed that lectin is a monomer of Mr 24 kDa. DBL agglutinated only rabbit
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erythrocytes and was inhibited by mucin, asialomucin, fetuin, asialofetuin and transferrin but

not by any monosaccharides. Glycan array analysis of DBL revealed its affinity towards high
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mannose N-linked glycans with enhanced affinity for terminal mannose including N-linked

glycans of HIV envelope glycoprotein gp120 and has strong anti-reverse transcriptase

activity. DBL showed strong binding to non metastatic human colon epithelial cancer HT 29,

metastatic SW 620 and hepatocellular HepG2 cell lines. DBL showed dose and time

dependent growth inhibitory effects on all the three cell lines HT 29, SW 620 and HepG2

with IC50 of 110g, 9.8g, 40g respectively at 72h. Inhibitory effect of DBL was effectively

Page 1 of 35
blocked in presence of competing glycans like mucin. DBL has promising clinical potential

both in cancer and HIV research.

Key words: - Dioscorea bulbifera lectin, monocot mannose binding lectin, glycan microarray,

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antiproliferative effect, anti-reverse transcriptase activity.

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1. Introduction

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Alternative medicines are gaining popularity in recent years to treat any disease

including cancer because of their lesser side effects. In this context research in identifying the
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bioactive molecules and their integration with conventional medicine is encouraging [1].

Dioscorea bulbifera commonly called as air potato, a species of yam family- Dioscoreaceae
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is native to Africa and Southern Asia. D. bulbifera has two types of storage organs namely

bulbils in the leaf axils of the stem and tubers [2]. Bulbils have been used as a folk remedy to
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treat conjunctivitis, diarrhea and the extracts of the plant is known to exhibit anti-tumor
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activity making it worth to explore it for detecting the presence of lectin with possible clinical

applications [2, 3].

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Lectins comprise a group of proteins with at least one non-catalytic domain and the

ability to bind reversibly to simple or complex carbohydrates through noncovalent

interactions [4]. Lectins are chief bioactive molecules which could initiate biological signals

by their interaction with glycans expressed on many receptors [5]. Plant lectins constitute

major part of tuber storage proteins, exhibit many biological activities which could contribute

to the resistance to pests, pathogens or abiotic stress [6]. Amongst plant lectins, in recent

Page 2 of 35
years, monocot mannose-binding lectins are gaining much interest because of their exclusive

specificity towards high mannose N-glycans of clinical importance [7]. Due to their exclusive

carbohydrate specificity towards mannose, they are referred to as monocot mannose-binding

lectins (MMBLs) and recently as GNA related lectins [8]. Many of them exhibited weak

affinity towards mannose, but strong affinity towards oligomannosides and high mannose

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containing N-glycans [9]. MMBLs have been purified, characterized, reported from

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Amaryllidaceae [7, 10] Alliaceae [11], Araceae [12], Orchidaceae [13], Liliaceae [14] and

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their sugar binding specificities are being exploited for insecticidal applications [15]. Here we

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report the purification, physicochemical characterization, fine sugar specificity of a novel

MMBL from bulbils of Dioscorea bulbifera (DBL) and its possible clinical applications.
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DBL is purified in single step by affinity chromatography, it is a monomer of Mr 24 kDa, has

strong affinity towards high mannose N-linked glycans with enhanced affinity for terminal
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mannose. DBL shows dose and time dependent growth inhibitory effects on human colon

epithelial cancer and hepatocellular cell lines. DBL has promising clinical potential both in
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cancer and HIV research.

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2. Materials and methods

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Sepharose 4B was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden.

Cyanogen bromide, calcein AM, mucin (porcine stomach, type III), fetuin (fetal calf serum),
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N-hydroxysuccinimido biotin and fluorescein isothiocyanate (FITC), fine sugars and MS

grade formic acid and acetonitrile were procured from Sigma Chemical Co., St. Louis, USA.

Standard protein molecular weight marker was procured from Merck, India. A Milli-Q

system (Millipore, India) was used to prepare deionized water for liquid chromatography

mobile phases.

Page 3 of 35
DMEM, trypsin and EnzCHEK reverse transcriptase assay kit were purchased from

Invitrogen, USA. Cell culture flasks and 96 well plates were procured from NUNC

(Denmark). Rabbit blood was collected from local animal house. Asialofetuin, asialomucin

were prepared from fetuin and mucin as described by Spiro and Bhoyroo [16]. Mucin-

Sepharose 4B affinity matrix was prepared by coupling mucin to cyanogen bromide activated

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Sepharose 4B according to the method of March et al [17]. All other chemicals used were of

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analytical grade.

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2.1 Cell culture

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The human colon cancer HT 29 cells, were procured from the European Cell Culture

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Collection via the Public Health Laboratory Service (Porton Down, Wiltshire, UK). Human

colon cancer SW 620 and Hepatocellular carcinoma HepG2 cells were a kind gift
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respectively from Dr. D. Karunagaran from IIT Madras and Dr. Milind Vaidya from

ACTREC Navi Mumbai. SW 620, HT 29 and HepG2 cells were cultured in DMEM,
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supplemented with 10% fetal bovine serum (FBS), 100g/ml streptomycin and 100 units/ml

penicillin. Cells were incubated at 37 C in 5% CO2 and 95% humidified air.

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2.2 Dioscorea bulbifera bulbils

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Dioscorea bulbifera bulbils were collected from Western Ghat region of Southern
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India, Uttara Kannada, Karnataka state during June to August and stored at 20C for further

use in airtight containers.

2.3 Isolation and purification of Dioscorea bulbifera bulbils lectin (DBL)

Dioscorea bulbifera bulbils (10g) were homogenized in 100 ml of extraction buffer

TBS (25mM Tris buffer saline pH 7.5) with 1% w/v insoluble PVP at 4C overnight. The

extract was filtered through muslin cloth and centrifuged at 10,000 rpm for 20 min at 4C.

Page 4 of 35
Supernatant was filtered through glass wool to remove insoluble PVP. Crude extract was

treated with ammonium sulphate to 70% saturation. Precipitate was collected by

centrifugation (10,000 rpm for 20 min at 4C), dissolved in 4 ml of TBS and dialyzed

extensively against same buffer. The dialysate was loaded to mucin-sepharose 4B affinity

column which was pre-equilibrated with TBS. Unbound proteins were washed off with

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washing buffer (TBS) till absorbance of eluting fractions read zero at 280 nm. Affinity bound

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protein was eluted with 100mM Glycine-HCl buffer pH 2.0, containing 500mM NaCl.

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Fractions with high lectin activity were pooled and dialyzed extensively against TBS, pH 7.5.

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Purified DBL stored at -20 C for further use.

2.4 Preparation of biotinylated and FITC conjugated DBL

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Fluorescein isothiocyanate-conjugated DBL (FITC-DBL) required for binding studies
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and biotinylated DBL required for glycan array analysis was prepared as described by

Goldman [18] and Duke et al respectively [19].

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2.5 SDS PAGE

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To check the homogeneity, the purified DBL was subjected to SDS PAGE by using

12% (w/v) acrylamide gel both in reducing and non reducing condition as described by
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Laemmli [20]. The gel was stained with Coomassie brilliant blue and the molecular mass of
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the lectin was estimated from the standard calibration curve using standard protein molecular

weight markers.

2.6 ESI-QToF

Due to its high resolution efficiency and mass accuracy, Top-down proteomics using ESI-Q-

ToF instrument was employed to determine the intact mass of the DBL. ESI-Q-ToF

instrument was employed and Liquid Chromatography- Mass Spectrometry (LC-MS) was

Page 5 of 35
carried out using 1290 infinity LC system comprising of a binary pump, a temperature

controlled autosampler, a column thermostat, a diode array detector connected to 6540 UHD

accurate mass Q-ToF MS with Agilent Jet Stream ESI source (Agilent Technologies, Santa

Clara, CA, USA).

Purified DBL(1 g) was injected onto (2.1 x 100 mm, 1.8 particle size) C18 column

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(Zorbex Eclipse Plus, Agilent Technologies, USA). The mobile phase solvents, A: water with

0.1% formic acid and B: acetonitrile (ACN) with 0.1% formic acid, respectively, were used at

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a flow rate of 0.2 mL/min and at a column temperature of 40C. The elution was carried out

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by following gradient : 0 min 2% B, 0-3min 20% B, 3-10 min 60% B, 10 -15 min 80% B and

15-18 min 100% B.

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The ESI-Q-ToF was operated in positive ion polarity and in the MS acquisition mode
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with a scan rate of 1 spectra/s and mass range between 200-3000m/z. The source parameters

were set as follows - gas temperature 200C, gas flow 6L/min, nebulizer 40 psi, sheath gas
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temperature 350C, capillary voltage 3500 V, nozzle voltage 500V and fragmentor 200 V.

Data acquisition was done with inbuilt Agilent MassHunter workstation B.04.00 and Agilent
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MassHunter Qualitative analysis B.07.00 Bioconfirm software was used for data analysis and
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deconvolution.
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2.7 Hemagglutination and Hapten inhibition assay

Hemagglutination assay was performed in 96 well U bottom microtitre assay plates to

monitor lectin activity during all the purification steps by using serial two fold dilution

technique [21]. The highest dilution of the lectin sample causing visible agglutination was

Page 6 of 35
regarded as the titre value and the minimum concentration of the protein required for

agglutination (MCA) as one hemagglutination unit. The specific hemagglutinating activity is

expressed as activity unit per mg of protein.

Carbohydrate binding specificity of the purified lectin was determined by hapten

inhibition assay. Lectin (25 l) with hemagglutination activity of 8 units was mixed with an

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equal volume of serially diluted sugar (200 mM) or glycoprotein (1 mg/ml). The plate was

incubated at 37 C for 30 min, prior to the addition of trypsinised erythrocytes (50 l) to each

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well and hemagglutination was observed. The lowest concentration of the sugar/

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glycoprotein, which inhibited the hemagglutination, was considered as the inhibitory titre of

the hapten.
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Protein concentration was estimated by the method of Lowry [22] using bovine serum
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albumin as standard. Total sugar content in proteins was estimated by phenol-sulphuric acid

method [23] using glucose as a standard.

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2.78 Effect of temperature and pH

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In order to determine the thermostability, DBL (200 g/ml) in TBS pH- 7.5 was

incubated at different temperatures (20C to 90C) for 20 min in temperature controlled

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The stability of the DBL at various pH was determined by testing the hemagglutination
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activity of the lectin samples incubated in buffers of different pH (2 to 12). DBL (200 g/ml)

was incubated with an equal volume of 100 mM buffers of pH 2.0 (Glycine-HCl), pH 4.3

(Acetate buffer), pH 7.0-8.0 (Tris-HCl buffer) and pH 9.0-12.0 (Carbonate buffer) for 24h at

4 C. After incubation samples were neutralized to pH 7.0 by adding 0.1N NaOH or 0.1N HCl

before testing hemagglutination activity.

2.9 Glycan array analysis

Page 7 of 35
 The carbohydrate-binding specificity of DBL was determined by glycan microarray

analysis on printed glycan array slides at the Consortium for Functional Glycomics, USA

(www.functionalglycomics, CFG#2921, v 5.2). Microarray slides (printed array version 5.2)

containing 609 different naturally occurring and synthetic glycans which are covalently

attached on NHS activated slides were used for the binding analysis. The analysis involved

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replicates of six printed slides as described by Blixt et al. [24]. In the binding assay,

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preprinted microarray slides were incubated with biotinylated DBL of three concentrations

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(2, 20, 200 g/ml), for 1 h at room temperature in a humidified chamber away from light. The

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slide was washed first with the binding buffer without Tween-20 and then with distilled

water. After washing, the slide was incubated with 100 l of streptavidin-FITC conjugate (10
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g/ml) in binding buffer at room temperature for 30 min. After incubation, slide was washed

thrice with wash buffer and finally dried under a stream of nitrogen. An image of the bound
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fluorescence was obtained using a confocal microarray scanner (Perkin Elmer Microscan XL

4000) and the integrated spot intensities were determined using IMAGENE image analysis
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software (Bio Discovery, EI Segundo CA, USA). The data acquired was stored and relative

fluorescence units (RFU) for DBL binding to each of the glycans were plotted using
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Microsoft EXCEL software.

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2.10 Binding of DBL to HT 29, SW 620 and HepG2 cells by flowcytometry

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The surface binding of DBL to human colon cancer epithelial HT 29, SW 620 and

hepatocellular carcinoma HepG2 cells in presence or absence of competent glycoconjugates

were analyzed by flow cytometry. Cells (0.2 x 106) were incubated with 3% BSA for 1 h to

block nonspecific sites and then stained with FITC conjugated DBL (5g/ml) for 1h at 4C.

Carbohydrate-mediated binding was analysed by pre-incubating the FITC-DBL with 100

g/ml of competing glycoconjugates, mucin, asialomucin, fetuin and asialofetuin for 1 h at

37 C before cell staining. Data were acquired for 10000 events using FC500 flow cytometer

Page 8 of 35
(Beckman Coulter, USA) or BD Accuri and were analysed by CXP analysis or

BD_AccuriTM_C6 softwares respectively. Unstained cells processed similarly were used as

negative controls.

2.11 Effect of DBL on human colon cancer HT 29 cell growth by Calcein AM method

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Effect of DBL on HT 29 cell proliferation was studied by Calcein AM method.

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Briefly, subconfluent HT 29 cells were seeded at 5 104 cells/ml in 96-well plates in DMEM

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complete medium for 48h. The medium was replaced with serum-free DMEM containing

0.5% BSA (w/v) and the cells were incubated with various concentrations of lectin (10 to 80

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g/ml) for 48 and 72 h in presence or absence of competing glycoproteins for different time

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intervals at 37C. After the specified time point the cells were labeled with 10 M Calcein

AM at 37C for 30 min in a CO2 incubator, lysed with100l lysis buffer containing 50 mM
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TrisHCl, pH 6.8 containing 5% SDS and 2% mercaptoethanol. The fluorescent intensity was

read with Tecan i-200 Pro microplate reader with an excitation wavelength of 485 nm and
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emission wavelength of 535 nm. The percentage viability of cells treated with lectin was

compared with untreated control cells.

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2.12 Effect of DBL on colon cancer SW 620 and hepatocellular carcinoma HepG2 cell
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growth by MTT Assay

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Subconfluent SW 620 and HepG2 cells were seeded at 5 104 cells/ml in a 96 well

plate and grown for 48 h. Cells were then treated with different concentrations (5 to 80g/ml)

of DBL for 48 and 72 h. After each time point 50 l of MTT (5 mg/ml) in DMEM was added

to each well and incubated for 2h at 37C and formazan crystals formed were dissolved in

100l of DMSO. Absorbance was measured at 595 nm using Tecan i- 200Pro microplate

reader. The percentage viability of cells treated with lectin was compared with untreated

control cells.

Page 9 of 35
2.13 HIV-1 reverse transcriptase activity

Reverse transcriptase assay was performed in order to test the ability of DBL to

inhibit HIV- 1 reverse transcriptase activity by using EnzChek kit. A fixed amount (46ng)

of recombinant HIV-1 reverse transcriptase, which generates long RNA-DNA heteroduplexes

from a mixture of a long poly (A) template, an oligo-dT primer and dTTP was used for the

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assay. The RNA-DNA heteroduplexes formed were detected by the PicoGreen reagent. The

assay was performed in presence of different concentration of DBL or its absence and the

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inhibitory activity of the lectin were expressed as percent inhibition as compared to control.

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Statistical analysis

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Each experiment was performed at least two times, each time in triplicate. Data

represent Mean SD of triplicate determinations from two different assessments by one

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way ANOVA and data considered significant with students t-test when *p < 0.05; **p <

0.01;***P < 0.001.

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3. Results
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3.1 Isolation and purification of Dioscorea bulbifera lectin

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Dioscorea bulbifera lectin was purified by 21.0 fold using ammonium sulphate

precipitation followed by affinity column chromatography on mucin coupled Sepharose 4B

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(Fig.1). The fold purification and the total % recovery of the lectin purified from 10 g bulbils

are summarized in Table 1.The minimum concentration of the protein required for

agglutination (MCA) was found to be 0.195g for the crude extract and 0.00898 g for the

purified lectin. Homogeneity of purified lectin was analysed by SDS-PAGE in 12% gel

(Fig.1Inset).The lectin content in Dioscorea bulbifera bulbils constituted approximately

Page 10 of 35
0.778 % of the total protein. DBL contains 52 % of carbohydrate as determined by phenol

sulfuric acid method.

3.2a Molecular Mass Estimation by SDS

The eluted lectin was found to be homogenous as revealed by single band of subunit

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molecular mass of 24 kDa on SDS-PAGE under both reduced and non reduced conditions

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(Figure 1, Inset) revealing that DBL is a monomer of single poly peptide chain with Mr of 24

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kDa.

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3.2b Molecular Mass Estimation by Liquid Chromatography- Mass Spectrometry

Mass spectrometry plays an important role for the fine characterization of proteins
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and peptides and also for identification of post-translational modifications. The Base Peak

Chromatogram (BPC) revealed two peaks at 1.5 and 6.5 min RT. These peaks were
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deconvoluted using the Agilent Bioconfirm software using a maximum entropy algorithm

and with a mass step of 1Da. Interestingly, deconvolution results of the second peak
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represented a monoisotopic mass of 24.49 KDa (Fig 2). This result concords the molecular

weight observed on the SDS-PAGE and suggested that the recovered protein to be
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homogenous.
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3.3 Hemagglutination and Hapten inhibition assay

DBL showed hemagglutination activity with trypsinized rabbit erythrocytes, however

no activity was observed with either trypsinized or untrypsinized human A, B and O

erythrocytes. Hapten inhibition studies showed that the hemagglutinating activity of DBL

was not inhibited by any of the simple sugars or oligosaccharides including mannose

indicating that it has complex sugar specificity. Activity of DBL was inhibited by

Page 11 of 35
asialofetuin, fetuin, mucin, asialomucin and transferrin (Table 2). Transferrin exhibited a

strong inhibitory effect (MIC-1.56g/50 l) compared to fetuin or asialofetuin (MIC-

3.125g/50 l), and none of the simple sugars tested inhibited hemagglutination of DBL.

DBL does not recognize mannose but strongly bind to asialofetuin and transferrin which are

known for containing high mannose N-glycans. DBL was thermostable in neutral pH up to

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80C and also the lectin showed tolerance under wide ranges of pH 2.0 to 9.3. Observed

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stability of Dioscorea bulbiferia lectin to heat and wide ranges of pH is greater than the

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lectins reported in the yam family Dioscoriaceae [25, 26].

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3.4 Fine sugar specificity of Dioscorea bulbifera lectin by Glycan array analysis:

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As the hapten inhibition assay showed complex specificity for DBL, the glycan array

analysis was performed using a panel of 609 different naturally occurring and synthetic
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glycans to identify glycans that selectively and specifically bind to DBL (Fig.4). DBL

recognizes a tri-mannosyl pentasaccharide core (G#51), with highest mean RFU of 79 %.

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DBL binding is greatly enhanced by trimannosyl pentasaccharide core in the presence of

terminal mannose moiety at non-reducing end (G# 50, 51, 216,210, 215) compared with the
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glycans in which mannose is masked by GlcNAc/Gal/GalNAc etc. (G #363&353) (Table 3).

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Interestingly, DBL also strongly binds to core fucosylated structure (G#483 with RFU of

65%) with high affinity but presence of fucose is not mandatory for binding (G# 51).
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Extensive fucosylation at either of the arms of trimannosyl pentasaccharide core reduces the

DBL binding drastically as seen in glycan (G# 444 and G# 445). DBL binds to tri-mannose

(G#213) with greater affinity having a mean RFU of 24% compared with disaccharide with a

mean RFU of 3.5%) (G#310), and these glycans together forms trimannosyl pentasaccharide

core, essential for DBL binding with highest RFU (G# 51). The difference in SP (spacer arm)

in (G# 51) and (G# 50) does not make much difference in the DBL binding however the

presence of GlcNAc on trimannosyl pentasaccharide core of G# 50 and G# 51 drastically

Page 12 of 35
reduces the binding of DBL( results in G# 52 and G# 53). DBL doesnt bind to any simple

sugars such as Mannose, Glucose, Galactose etc. or O-linked glycans. These results observed

for DBL are in agreement with those reported for the majority of monocot-mannose binding

lectins which strongly interact with both high-mannose and complex N-glycans [12, 27]

3.5 Cell-surface binding of DBL to HT 29, SW 620 cells and HepG2

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DBL binding to HT 29, SW 620 and HepG2cells was determined by staining cells

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with FITC-DBL, followed by flowcytometric analysis. A total of 99.5% (HT 29), 33.7% (SW

620) and 94.62% (HepG2) cells, were positive for DBL binding with MFI of 25.3, 47.5 and

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23.2 respectively compared to unstained cells that had MFI of 0.3, 0.2 and 0.4 respectively.

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There was a significant reduction in binding of FITC- DBL to HT 29, SW 620 and HepG2

cells in presence of competing glycoconjugates. MFI decreased much effectively for HT 29

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cells in presence of mucin (98.3%), asialomucin (98.11%), fetuin (98.31%) and asialofetuin

(98.39 % ) respectively (Fig. 5A2) as compared to SW 620 cells where the haptens results in
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the inhibition by 66%, 60%, 73%,65% and for HepG2 cells by 47%, 44%, 37%, 66%

respectively (Fig. 5B2 and C2). These results indicate the carbohydrate mediated binding of
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DBL to all the three cells (Fig 5 A, B and C).

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3.6 Effect of DBL on human colon cancer HT 29, SW 620 and hepatocellular carcinoma
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HepG2 cell growth:

DBL induced growth inhibitory effects on HT 29, SW 620 and HepG2 cells in both

dose and time dependent manner. DBL inhibits the growth of HT 29, SW 620 and HepG2

cells with IC50 of 110 g, 9.8 g, 40 g respectively after 72 h. At 48 h, a growth inhibitory

effect of DBL on SW 620 cells was effectively blocked (by 60 %) in presence of competing

Page 13 of 35
glycoprotein mucin.. These results show that DBL induces cell death in these cancer cells by

interaction with cell-surface glycoproteins. (Fig. 6 A, B and C)

3.7 HIV-1 Reverse Transcriptase inhibitory activity

In order to test the inhibitory effect of DBL to HIV- 1 reverse transcriptase, assay

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was performed which shows that DBL inhibits the HIV-1 reverse transcriptase activity

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effectively in a dose dependent manner with IC50 of 1.3 g (Fig. 7).

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4. Discussion

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A mannose binding lectin from bulbils of Dioscorea bulbifera, that are used as a folk

remedy to treat cancer, was purified in a single step by affinity chromatography,

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characterized, its fine sugar specificity determined by glycan array analysis. DBL is studied
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for exploring its clinical potential in cancer, using human colon cancer HT 29, SW 620 and

hepatocellular HepG2 cells and HIV research testing its anti-reverse transcriptase activity.
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Lectins have been purified and studied from various Dioscorea species, including D.

japonica [28], D. cayenesis [29], D. batatas [25], D. alata [30], D. polygonoides [31], D.
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rotundata [32] and D. opposita [26]. Most of these lectins reported from this family are of
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tuber origin, where as DBL, is purified from the bulbils rather than tubers. The occurrence of

multiple lectins is common feature observed in most of the members of Dioscorea or yam
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family, but unlike this D. bulbifera bulbil lectin is a single lectin and a monomer of Mr 24

kDa suggesting the presence of single lectin in D. bulbifera bulbils. Top-down proteomics

using ESI-MS/MS revealed intact mass of the DBL as 24.49 kDa. Though, the BPC revealed

two peaks, the intensity of the second peak was high and based on the hydrophobicity, in

general, the glycan or glycoform proteins are well known to be retained on a reverse phase

(C18) column. Hence the second peak, was largely focused and subjected to deconvolution,

Page 14 of 35
which revealed an accurate mass of the isolated lectin, DBL as 24.49 KDa. Nevertheless, the

analysis of the first peak provided an worthwhile information of the protein. The

deconvulation of the I peak depicted a monoisotopic mass of 23.30 kDa protein (Fig 3[A1]).

We anticipate this to be deglycosylated protein form of DBL. This view is also

semiquantiatively supported, by both the retention time and the mass difference of ~1189.49

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Da between the two peaks, which shall correspond to the glycan moiety. In addition, the

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elobrate studies about glycoproteins and glycopeptides support our view [33,34, 35]. Thus,

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MS analysis of DBL confirms that the isolated protein to be a lectin with Mr of 24.49 KDa.

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Recently, Nagaimo lectin from Dioscorea opposita tubers has been reported as a

single protein and is a dimer with a Mr of 70-kDa consisting of two subunits each of 35-kDa
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[26]. In contrast, DBL is a monomer of single poly peptide chain with Mr 24 kDa.
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Similar to other monocot mannose binding lectins, DBL agglutinates only rabbit

erythrocytes but not human A, B and O red blood cells. DBL exhibit higher thermostability
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and pH stability than any other lectins reported from Dioscoreaece family. DBL is stable

upto 80oC in neutral pH and is active over a wide range of pH at room temperature. Although
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the Amaryllidaceae, Alliaceae, Araceae, Liliaceae, Orchidaceae and Dioscoriaceae families

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belong to same group of monocots, the lectins purified from Amaryllidaceae, Alliaceae,

Araceae, Liliaceae, Orchidaceae have fine sugar specificity directed only towards complex
Ac

high mannose N-glycans where as the lectins reported from Dioscoriaceae shows specificity

towards high mannose, N-glycans and also for maltose and galactose. For example, lectins

from D. batatas are mannose- and maltose-specific [25], while Nagaimo lectin from

Dioscorea opposita is specific to galactosides/galactose [26].Unlike this DBL shows affinity

towards high mannose N-linked glycans with enhanced affinity for terminal mannose and

does not recognize any of the simple monosaccharides. Interestingly, some of the glyans

recognized by DBL are naturally occurring and are of biological significance. For example

Page 15 of 35
DBL strongly binds to trimannosyl pentasaccharide (G # 51), normally found in insect pests

[36, 37], viral pathogens [38] and also in cancer associated antigens [39, 40]. G#215

recognized by DBL is a part of well known ovarian epithelial cancer marker CA-125 [41,

42]. G#216 and G# 210 are found in envelope glycoproteins of pathogenic viruses like HIV,

Influenza which are known to be essential for initiating viral infection [43]. G# 483 and 216

t
are known to be present on gut epithelial cells of insect pests [37].

ip
The exquisite sugar binding specificity of the Dioscoreaece family lectins reported

cr
are being exploited for their applications. For instance D. batatas lectin was studied for its

us
insecticidal activity against Helicoverpa armigera [44]. Nagaimo lectin from D. opposita

exhibit anti-proliferative activity on several types of cancer cells tested, like breast cancer
an
MCF7 cells, hepatoma HepG2 cells and nasopharyngeal carcinoma CNE2 cells [26].
M
Glycan array analysis of DBL showed its affinity for N linked glycans, expressed on

cancer cell surface which was demonstrated by binding of DBL to hepatoma HepG2 cells and
ed

colon HT 29, SW 620 cells, which are known to express altered N-glycans [45,46]. DBL

binds to both hepatoma HepG2 and colon HT 29 cells and induce growth inhibitory effect.
pt

Many lectins are known to induce apoptosis in tumor cells due to their carbohydrate binding
ce

capability [5]. In this regard it is similar to Nagaimo lectin from D. opposita which has anti-

proliferative activity on several cancer cells [26]. Similar to other MMBLs, DBL has anti
Ac

antireverse transcriptase activity against HIV.

These results reveal the potential of this novel lectin DBL from medicinal plant both

in cancer and AIDS research and are worth to be explored further.

Acknowledgement

Page 16 of 35
SRI would like to thank Core H facility, Consortium for functional glycomics, USA for the

glycan array analysis of DBL. The work was supported by the funding from UGC under UPE

(F.NO.14-3/2012 (NS/PE) and CPEPA (F.3-2/CPEPA/KU/UGC-SWRO/ 2015).

t
Author Contributions: Conceived and designed the experiments: SRI and BMS, Performed

ip
the experiments: MS and VRH contributed equally; and

cr
SBR ,Analyzed the data: SRI, Contributed

us
reagents/materials/analysis tools: SRI, KAS and BMS. Wrote the paper: SRI and MS.

Conflicts of interest
an
Authors dont have any conflict of interest to declare concerning to the present work.
M
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Page 22 of 35
Figure legends

Fig.1- Purification of Dioscorea bulbifera lectin (DBL): A. Purification of Dioscorea

bulbifera lectin (DBL) by affinity chromatography using mucin Sepharose-4B column

(1.510 cm) equilibrated in TBS and the bound lectin was eluted with 100 mM Glycine-HCl

t
ip
buffer, pH 2.0 containing 500 mM NaCl. Fractions of 3.0 ml were collected at flow rate of 15

ml /h. Inset-SDS- PAGE of DBL (affinity purified) in 12% gel. Lane1- standard protein

cr
molecular weight markers, lane 2- DBL (20 g) in reducing and lane3 DBL (20 g) in non

us
reducing conditions. The gel was stained with Coomassie brilliant blue.

an
B. Calibration curve for the estimation of molecular weight of DBL by SDS PAGE: X-axis

represents the log molecular weight and Y-axis representsRelative mobility of , marker
M
proteins; phosphorylase b (97.4 kDa), BSA (66 kDa), ovalbumin (43.0 kDa), carbonic

anhydrase (29 kDa), soyabean trypsin inhibitor (20.1 kDa), lysozyme (14.3 kDa)
ed

Fig.2- Deconvulated monoisotopic mass of the elution peak at RT6.5min. The peak
pt

corresponding to the 24492.88 Da is the mass of the DBL

Fig.3- Deconvulated monoisotopic mass of the elution peak at RT1.5min. The peak
ce

corresponding to the 23303.39 Da is the mass of the deglycosylated DBL

Ac

Fig.4- Glycan array analysis of DBL: A total of 609 glycans were screened for binding of

DBL (20g/ml) and Relative fluorescence units (RFU) for 609 glycans were plotted. X-axis

represents glycan numbers and Y-axis represents RFU. Inset highest binding glycans.

Page 23 of 35
Error bars represents the mean SD of replicates from a single experiment.

Fig 5- Binding of DBL to HT 29, SW 620 and HepG2 cells and inhibition of binding with

competing glycoconjugates by flowcytometry. DBL shows strong binding to HT 29 (A1),

SW 620 (B1) and HepG2 cells (C1): Cells were treated with FITC-labeled DBL and

subjected to flow cytometric analysis. The overlays are representative data with X-axis and

t
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Y-axis representing fluorescent intensity and cell count, respectively. A2, B2 and C2

represent inhibition of DBL binding (% MFI) in presence and absence of competing

cr
glycoconjugates.

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Fig.6- Effect of DBL on of HT 29 cell growth. A. The cells incubated with or without

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different concentrations of DBL (1080 g/ml) for 48 and 72 h before the cell viability was

measured by Calcein-AM assay. Band C: Effect of DBL on SW 620 and HepG2 cell growth.
M
SW620 (B1) and HepG2(C) cells were incubated with or without different concentrations of

DBL (580 g/ml) in presence or absence of mucin (B2) for 48h and 72 h before the cell
ed

viability was measured by MTT assay. Data represent Mean SD of triplicate determinations

from two different assessments. *P < 0.05, **P < 0.01, ***P < 0.0001 when compared to
pt

control.
ce

Fig. 7- HIV-1 Reverse transcriptase inhibitory activity of DBL

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HIV1 Reverse transcriptase assay was performed using EnzChek kit,in presence of

different concentrations of DBL and the inhibitory activity of DBL is expressed as percent

inhibition compared to control.

Page 24 of 35
 Vol a Specificb Total
Protien MCA Fold %
Sample activity Activityc
(ml) (mg) ( g) (units) (units)
purification Recovery

Crude extract 90 90 0.195 5.128103 46.10104 -- 100

t
Ammonium

ip
sulphate
4 26.07 0.128 7.8 103 20.5 104 1.53 44
precipitation
(70%)

cr
Affinity
15 0.7 0.00898 11.13104 7.79104 21.0 16.8
Chromatography

us
an
M
Table 1- Purification of lectin from Dioscorea bulbifera.
ed

a - Minimum concentration of protein required to agglutinate rabbit erythrocytes.

b - Specific activity: hemagglutinating activity/ mg protein.
pt

c -Total activity: hemagglutinating activity of lectin in total protein.

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Page 25 of 35
 t
ip
Table 2- Hapten inhibition studies of DBL

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Minimum concentration required for
Haptens tested for
inhibition

us
inhibition
in g (MIC)

Simple sugars NIL

1. D-Glucose

2. D-Galactose
an
NIL

NIL
M
3. D-Mannose NIL

4. L-Fucose NIL
ed

5. D-Lactose NIL

6. D-Maltose NIL
pt

Sugar derivatives tested

Glucosamine NIL
ce

N-Acetyl D-galactosamine NIL

N-Acetyl D-mannosamine NIL

Ac

Methyl -D-Manno NIL

Pyranoside
Methyl mucin pyranoside NIL

Oligaosaccharide tested
High mannan 1.5625
Glycoproteins tested
Transferrin 1.5625
Fetuin 3.125

Page 26 of 35
 Asialofetuin 3.125
Mucin 12.5
Asialomucin 12.5

The carbohydrate binding specificity of Dioscorea bulbifera lectin (DBL) was determined by
hemagglutination assay using simple sugars, sugar derivatives (200 mM) and glycoproteins
(1mg/ml).

t
ip
Table 3- Relative binding specificity of DBL to high mannose N glycans with mean RFUs

cr
Glycan Glycan structure 2g 20g 200g Mean
number Rank

us
G#51 Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-Sp13 94% 90% 53% 79%

Mana1-6(Mana1-3)Mana1-6(Mana1-3)Manb1-4GlcNAcb1-
G#216
4GlcNAcb-Sp12
an 56% 88% 69% 71%

G#483 Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp19 45% 100% 52% 65%

M
G#50 Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 9% 97% 57% 54%

G#210 Mana1-6(Mana1-2Mana1-3)Mana1-6(Mana1-2Mana1-3)Manb1- 46% 74% 41% 53%

ed

4GlcNAcb1-4GlcNAcb-Sp12

G#215 Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1-3)Manb1-4GlcNAcb1- 100% 33% 0.18% 44%

4GlcNAcb-Sp12
pt

G#364 Fuca1-4(Galb1-3)GlcNAcb1-2Mana1-6(Fuca1-4(Galb1-3)GlcNAcb1- 62% 32% 26% 40%

2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp22
ce

G#571 Galb1-3GlcNAcb1-3Galb1-4GlcNAcb1-6(Galb1-3GlcNAcb1-3Galb1- 54% 6.4% 18% 39%

4 GlcNAb1-2)Mana1-6(Galb1-3GlcNAcb1-3Galb1-4GlcN Acb1-
2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp24
Ac

G#356 KDNa2-6Galb1-4GlcNAc-Sp0 0.8% 0.03% 100% 33%

G#213 Mana1-6(Mana1-3)Mana-Sp9 12% 60% 0.16% 24%

G#354 (6S)GlcNAcb1-3Galb1-4GlcNAcb-Sp0 1.9% 7.9% 60% 23%

G#352 Galb1-4GlcNAcb1-2Mana1-6(Galb1-4GlcNAcb1-2 Mana1-3) Manb1- 1.39% 0.4% 67% 22.9%

4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp22

Page 27 of 35
G#570 Galb1-3GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-6 38% 17% 12% 22.5%
(Galb1-3GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAb1-2)
Mana1-6(Galb1-3GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4
GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp24

G#315 Mana1-2Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1-2Mana1- 5.4% 31% 26% 20.8%

3)Mana-Sp9

G#211 Mana1-2Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1-2Mana1- 6.7% 46% 1.0% 17.9%

3)Manb1-4GlcNAcb1-4GlcNAcb-Sp12

t
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cr
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