Structural elucidation of a heteropolysaccharide from the wild mushroom Marasmiellus palmivorus and its immune-assisted anticancer activity

Accepted Manuscript Title: Structural elucidation of a heteropolysaccharide from the wild mushroom Marasmiellus palmivorus and its immune-assisted anticancer activity Authors: Hemanta Kumar Datta, Debsankar Das, Andreas Koschella, Tapoti Das, Thomas Heinze, Subrata Biswas, Sujata Chaudhuri PII: DOI: Reference:

S0144-8617(19)30146-8 https://doi.org/10.1016/j.carbpol.2019.02.011 CARP 14584

To appear in: Received date: Revised date: Accepted date:

5 September 2018 20 December 2018 2 February 2019

Please cite this article as: Kumar Datta H, Das D, Koschella A, Das T, Heinze T, Biswas S, Chaudhuri S, Structural elucidation of a heteropolysaccharide from the wild mushroom Marasmiellus palmivorus and its immune-assisted anticancer activity, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.02.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Structural elucidation of a heteropolysaccharide from the wild mushroom Marasmiellus palmivorus and its immune-assisted anticancer activity

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Hemanta Kumar Dattaa, Debsankar Dasb, Andreas Koschellac, Tapoti Dasa, Thomas Heinzec, Subrata Biswasd and Sujata Chaudhuria*

a

Mycology & Plant Pathology Laboratory, Department of Botany, University of Kalyani, Nadia,

Department of Chemistry, Prabhat Kumar College, Contai, Purba Medinipur, 721404, West

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b

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West Bengal, 741235, India.

Friedrich Schiller University of Jena, Institute for Organic Chemistry and Macromolecular

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c

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Bengal, India

Chemistry, Center of Excellence for Polysaccharide Research, Humboldtstraße 10 D-07743

a*

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Department of Chemistry, University of Kalyani, Nadia, West Bengal, 741235, India

Corresponding Author

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d

D

Jena, Germany

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[email protected], Ph. No.- +919339797481 [email protected]; [email protected]

b

[email protected]

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a

c

[email protected]; [email protected]

d

[email protected]

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Highlights A water soluble heteropolysaccharide was isolated from Marasmiellus palmivorus Its approximate molar weight was 1,45,000 g/mol

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The structure was comprehensively determined by means of state-of-the art methods It was bioactive and activated the anticancer immune surveillance system

Abstract

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The biological activity of macrofungal polysaccharides (MFPS) depends on their structural

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features and is a topic of keen interest for researchers since long time. In this communication, we

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report a water soluble macrofungal heteropolysaccharide (MFPS1) with a molar weight of

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~1,45,000 g/mol, obtained through alkali extraction, of the wild mushroom, Marasmiellus

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palmivorus, with significant immunomodulatory properties. In cancer, after the induction of

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metastasis, the anticancer immune system becomes unresponsive. By studying cytokine secretion and immune phenotyping, it was observed that MFPS1 reactivated the anticancer immune

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surveillance system. MFPS1 executed T-cell maturation and activation via M1Φ; and also

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stimulated natural killer (NK) cell and B-cell population. The entire immune activation pathway corroborates its anticancer activity. The RP-HPLC analysis of hydrolyzed MFPS1 showed

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arabinose, glucose, galactose and mannose as monosaccharide units. The proposed structure of repeating unit was established from methylation analysis, 1D- and 2D NMR study, HR-MS and MALDI-TOF MS analysis. Key

Words:

Marasmiellus

palmivorus;

Macrofungi;

Immunomodulation; Anticancer activity 2

Heteropolysaccharide;

Structure;

1. Introduction Bioactive polysaccharides from macrofungi are an area of deep interest and research since long time. These polysaccharides exhibit antitumor, immunomodulation and antioxidant activities

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(Lemieszek & Rzeski, 2012; Ooi, 2008; Sánchez, 2017). The bioactivity is dependent on the structure of the polysaccharides and the activity varies depending on the molecular weight (Mw), monosaccharide units and linkage types. Different types of polysaccharide, ranging from those with high to low molecular weights and from homopolysaccharides to heteropolysaccharides with different type of linkages are found in fungal cell (Huang & Nie, 2015; Meng, Liang & Luo,

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2016).

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In the late cancer stage, the tumor cells enter into the blood and circulate to initiate metastasis.

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Hence, this stage is very critical and it is necessary to target the spreading of tumor cells through

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blood circulation, which has access to all organs and cells of the human body. Although the blood circulation system also contains different components of the immune system, which

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initially act as an anticancer immune surveillance circuit, however, in the later stages of cancer,

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the immune system gets biased and cannot recognize cancer cells. (Grivennikov, Greten &

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Karin, 2010; Dunn, Old & Schreiber, 2004; Almand et al., 2000; Gabrilovich, 2004). To reinstall the unbiased immune system with anticancer property an external stimulation is necessary.

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Macrofungal polysaccharides have been reported to work as an external stimulator under these situations (Wasser, 2002; Schepetkin & Quinn, 2006; Moradali, Mostafabi, Ghods &

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Hedjaroude, 2007) and can reinstall the anticancer immune surveillance system and eliminate cancer cells from body. Majority of the findings pertaining to the immunomodulatory activity of the macrofungal polysaccharides (MFPS) has been focused on homopolysaccharides from edible mushrooms. In

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the present study, we focused on the structural characterization of a novel macrofungal heteropolysaccharide (MFPS1) from a wild non-edible mushroom and its immune assisted anticancer activity. In this investigation, we also tried to unravel the immune pathway(s) associated

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with the anticancer property of MFPS1. 2. Materials & Methods 2.1.

Materials and Chemicals

The DNA primers, PCR reaction mixture, Taq DNA polymerase and solvents used in this study were purchased from Merck (India). All standard monosaccharides including other standards for

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biochemical estimation and chromatography were obtained from Sigma-Aldrich Chemical Co.

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(St. Louis, MO, USA). Dialysis membrane;1-phenyl-3-methyl-5-pyrazolone (PMP); 2,5-

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dihydroxy benzoic acid (DHB) matrix; deuterium oxide (D2O); trifluoroacetic acid (TFA); N,O-

fetal

Bovine

Serum

(FBS),

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bis (trimethylsilyl) trifluoracetamide (BSTFA), Dulbecco's Modified Eagle's medium (DMEM), penicillin,

streptomycin,

histopaque

solution,

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methylthiazolyldiphenyl-tetrazolium bromide (MTT), Cisplatin and bacterial lipopolysaccharide

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(LPS) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Cell lines were

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obtained from National Centre for Cell Science (NCCS), Pune, India. ELISA kit for cytokine assay and fluorescent tagged monoclonal antibody for immunophenotyping were obtained from

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Invitrogen, Thermo Fisher Scientific (USA). 2.2.

Collection and identification of mushroom

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Carpophores of M. palmivorus, growing on forest floor Hijuli Forest, Ranaghat (Lat: 23°11'04.0"N Long: 88°34'39.3"E) in Nadia District of West Bengal, India, were collected during the monsoon.

4

Genomic DNA was isolated by cetyl trimethyl ammonium bromide (CTAB) method (Gawel & Jarret, 1991). The mushroom was identified by morphological and ribosomal DNA (rDNA) sequencing. The 28S rDNA or large subunit (LSU) [primer LROR-LR5 (Forward: 5′-

transcribed

spacer

(ITS)

region

(primer

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ACCCGCTGAACTTAAGC-3′ & Reverse: 5′-TCCTGAGGGAAACTTCG-3′)] and internal ITS5,

ITS4

Forward:

5′-

TCCTCCGCTTATTGATATGC-3′ & Reverse: 5′-GGAAGTAAAAGTCGTAACAAGG-3′) were amplified by PCR (Schoch et al., 2012). Sequencing was done by Sanger dideoxy method and the fungus was identified by NCBI nBLAST homology matching. Extraction and purification of polysaccharide

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2.3.

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The method by Mandal et al., (2010) with minor modifications, was used for polysaccharide

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extraction. Mushroom fruiting bodies were washed with sterile double distilled water, crushed

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and incubated at 90 °C for 6 h in 4 % NaOH. The extract was filtered through Whatman No.1 paper and the filtrate was mixed with 80 % ethanol (1:5 v/v) and allowed to stand at 4 °C

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overnight to precipitate the polysaccharides. The precipitated crude polysaccharides were

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collected by centrifugation at 8000 rpm for 40 min at 4 °C and washed sequentially with 80:20,

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90:10 ethanol-water and absolute ethanol followed by acetone and dissolved in sterile HPLC grade water. The pH of the collected crude polysaccharide solution was highly alkaline (pH-

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11.0). It was dialyzed with a membrane having molecular weight cut off 10,000 g/mol against double distilled water for 3 days at 4 °C to remove the low molecular weight compounds and

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excess NaOH to neutralize the pH (7.0-7.2). During dialysis, the alkali soluble part precipitated while water soluble fraction remained in solution. Centrifugation afforded the water soluble fraction (MFPS1) which was collected and lyophilized prior to further experiments. 2.4.

Analysis of the polysaccharide

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Total polysaccharide content was estimated by the phenol-sulphuric acid method (Dubois, Gilles, Hamilton, Rebers & Smith, 1956) using glucose as standard. Total phenol and protein content were also quantified with the Folin–Ciocalteau- (Kaur & Kappor, 2002) and Bradford methods,

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(Bradford, 1976) respectively. Bovine serum albumin and gallic acid were used as standard compounds in protein and phenol determination, respectively. For further confirmation, the UVVIS spectrum of MFPS1 in water was recorded in the scanning range from 190 nm to 1100 nm. Molar mass and molar mass distribution of MFPS1 were determined by advanced Polymer Chromatography (APC) (Acquity Waters, USA) equipped with ACQUITY APC AQ Column

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(waters) with dimension (i.d. x length) of 4.6 x 75 mm and the pore size is 450 Å. The column

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material was ethylene bridged hybrid (BEH) particles. T-Dextran series (Mw: 18,300; 35,600;

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10, 000,0; 23,600,0; 33,300,0 g/mol) was used for molar mass determination. Water was used as

2.5.

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solvent at 0.5 ml/min flow rate and detected by refractive index (R.I.) detector. Monosaccharide analysis of MFPS1

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The monosaccharide composition of MFPS1 was analyzed by RP-HPLC using a HITACHI

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Chromstar System (Japan) equipped with HITACHI LaChrom C18 column (250x4.6mm), UV-

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VIS detector 5420 (at 245 nm), auto sampler 5210, and pump 5110 (Honda et al, 1989). Each of the standard monosaccharides (2 mg each) and MFPS1 were hydrolyzed with 4 M TFA (1 ml) at

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120 ºC for 2h. Excess TFA was removed by co-distillation with 2-propanol. All of the standard monosaccharides including the hydrolyzed MFPS1 were derivatized by incubating at 70 ºC for

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2h with 0.5 M PMP (20µl). Each (20 µl) were injected into HPLC column in two separate runs. The solvent system consisted of 80 % 0.1 M phosphate buffer (pH 7.0) and 20 % acetonitrile, and the flow rate was 1ml/min. 2.6.

Determination of absolute configuration

6

The absolute configurations of sugar units were determined by the method of Gerwig, Kamerling & Vliegenthart (1978). MFPS1 (2.0 mg) was hydrolyzed with 4 M TFA, and the acid was evaporated. 0.625 M HCl in R-(+)-2-butanol (250µl) was added to it and heated at 80 ˚C for 16

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h. The reactants were removed by evaporation and the sample was derivatized with N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) followed by GC-MS analysis using HP-5 column (7000C Triple Quadrupole GC/MS, Agilent, USA). The oven temperature for GC was 150 °C to 210 °C with temperature ramp of 3 °C/min. The 2,3,4,6-tetra-O-trimethylsilyl- (+)-2-butyl glycosides were recognized by comparison with standard

GC-MS analysis

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2.7.

and L-enantiomers of different

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monosaccharides.

D-

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The methylation of MFPS1 was done by the method of Ciucanu & Kerek (1984). 5 mg of

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MFPS1 was methylated with methyl iodide (CH3I) and hydrolyzed by 4 M TFA. The hydrolyzed methylated monosaccharides were extracted with chloroform. The organic layer was collected

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and washed with water three times and dried. The methylated products were then formolyzed

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with formic acid and further reduced by sodium borohydride. The hydroxyl groups were

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acetylated with 1:1 acetic anhydride–pyridine. Partially methylated alditol acetates were analyzed by GC-MS (HP-5 column) (7000C Triple Quadrupole GC/MS, Agilent, USA), using

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the following temperature program- two minutes at an initial temperature of 80 °C, increased to 170 °C at 3 °C/min, then to 240 °C at 4 °C/min, and kept for 5 min at 240 °C. The mass spectra

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were recorded continuously by scanning from 70 to 300 m/z. The ionization voltage was 70 eV, scan rate 1100 atomic mass unit (amu s–1), electron multiplier energy 1600 V, and ion source temperature 200 °C. 2.8.

HR-MS of MFPS1

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The mass of the MFPS1 fragments was studied by HR-MS (XEVO G2S QTOF, Waters, USA) analysis. 2 mg of MFPS1 were dissolved in water (pH-7.2) and scanned in the range of 50-2000 m/z in positive ion mode. MALDI-TOF MS of MFPS1

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2.9.

The mass of the MFPS1 fragments was analyzed by MALDI-TOF MS. Two mg of 2,5dihydroxy benzoic acid (DHB) was dissolved in 200 µL of the matrix solvent (2:1 acetonitrile– water, v/v) and 1% sodium trifluoroacetate was added. One µl of the solution mixture containing MFPS1 was spotted for MALDI-TOF MS analysis. MALDI-TOF MS was performed on a

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Voyager-DE PRO (Applied Biosystems, USA). The instrument was calibrated with myoglobin

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prior to analysis and the spectra were recorded in positive ion mode. The scan range was 200-

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5000 m/z.

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2.10. NMR analysis

The NMR experiments were carried out in Agilent DD2-700 MHz, four channel NMR

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spectrometer at 25 °C, using D2O as solvent. Acetone was used as internal standard (δ 31.05

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ppm) for 13C NMR spectroscopy. For 1H NMR spectroscopy, relaxation delay was 1.5 sec with

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the pulse of 90 º. Mixing time was 60 ms for heteronuclear HSQC. The TOCSY experiment was recorded at 150 ms mixing time. For complete assignment, several TOCSY experiments were

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carried out with mixing times ranging from 60 to 300 ms. The delay time in the HMBC experiment was 80 ms. The mixing delay was 300 ms for NOESY and ROESY.

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2.11. Cell lines & culture

RAW 264.7 (Mice macrophage), A549 (human lung Carcinoma) and SW620 (human colorectal adenocarcinoma) were cultured in DMEM supplemented with 10% FBS, 1% penicillin and 1% streptomycin.

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Fresh blood sample was aseptically collected in EDTA coated vials from a healthy male (age- 32 years) human donor who had no history of disease or recent administration of any drug. The blood sample was collected from the volunteer, who read and singed an informed consent. The

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study was approved by the Institutional Ethics Committee; College of Medicine & JNM Hospital, Kalyani, West Bengal (REF. NO. F-24/PRCOMJNMH/IEC/18/278) as per ICMR guideline. For the avoidance of genetic dissimilarity blood was collected from same individual each time. Peripheral blood mononuclear cells (PBMC) were isolated by histopaque solution as per the manufacturer’s instructions. All cells were maintained at 37 ˚C in 5% CO2 atmosphere.

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2.12. Cytotoxicity assay

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The cytotoxicity assay of MFPS1 was done in co-culture and individual culture models on cancer

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cells and non-cancerous cell (PBMC). In co-culture model, the cancer cells (A549 & SW620)

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were cultured with PBMC in the same well and the medium, to allow the physical interaction with each other considering the cancer cells as target cell (T) and PBMC as the effector cell (E)

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(Shi et. al., 2014; Wang, Zhu, Corral, Hong & Stein, 2005). The E:T ratio was 1:5 in the co-

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culture system. For assessment of cytotoxicity, 1x106 cells (T) were seeded in each well of a 96

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well plate. Cells were incubated with different doses of MFPS1 for 24, 48 and 72 h individually. After the specified time interval 10 µl MTT (5 mg/ml) dye was added to each well and further

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incubated for 4 h at 37 °C in 5 % CO2 condition. The medium was discarded after incubation and 100 µl of DMSO added to each well. Absorbance was taken at 570 nm (ELISA Reader -BioTek

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ELx 800, USA). Cisplatin, an anticancer drug was used as positive control. Viable cell % was calculated as per formula (1). % of viable cell =

Abs sample−Abs blank Abs control−Abs blank

x 100

(1)

(Where, blank= only DMSO; control=without any treatment) 9

2.13.

Expression of cytokines

Expression of cytokines (IL-6, IL-12, TNF-α and IFN-γ) was studied in RAW 264.7 and PBMC. The cells (1x1010) were treated with MFPS1 (100 µg/ml) and LPS (10 µg/ml) individually. LPS

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was used as positive control. After 24 h of incubation, the medium was centrifuged at 2000 rpm for 10 min. The clear supernatant was used for the further experiments. Cytokines were measured in an ELISA Reader (BioTek ELx 800, USA) following the instructions of the manufacturer. 2.14.

Immunophenotyping

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Cluster of differentiation (CD) molecules expression was studied to identify cell surface

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molecules, which target immunophenotyping of cells. PBMC (2x105) was cultured in DMEM

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medium and treated with MFPS1 (100 µg/ml) and LPS (10 µg/ml) individually. LPS was used as

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positive control. A control set without any treatment was maintained. After 24 h of incubation, the cells were harvested in ice cold PBS and incubated with monoclonal antibodies, CD45-

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PerCP, CD4-FITC, CD5-PE, CD19-APC, CD14PE, CD34-FITC, CD335-APC. After 3 h of

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incubation at 4 °C, expression dependent cell population was analyzed in BD – FACS Calibur

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Flow Cytometer, USA.

2.15. Statistical analysis

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All experiments were repeated five times and the data expressed as mean ± SD of the values of the replicates. One way ANOVA analysis was performed and differences at P < 0.05 were

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considered statistically significant by Duncan multiple range tests using GraphPad PRISM® (Ver.5.03) software.

3.

Results & Discussion

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3.1.

Identification of fungi

The PCR amplified DNA sequences of 28S (LSU) and ITS regions were approximately 1000 and 700 bp long, respectively. NCBI nBLAST of the rDNA sequences (Khaund & Joshi, 2014), The

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identified the mushroom as Marasmiellus palmivorus (Supplementary Fig. 1A &1B).

GenBank accession numbers were KX290488 (LSU) and KX290489 (ITS). The mushroom fruiting body is small in size and found mostly on the tree barks and forest litter, in humid or wet conditions, and especially during the rains (Pong, Abidin, Almaliky, Kadir, & Wong, 2012). We found it growing sparsely and in small amounts.

Extraction & detection of macrofungal polysaccharide MFPS1

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3.2.

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The rigid fungal cell wall is the prime source of different type of polysaccharides. Hence,

A

treatment with hot alkali facilitated cell wall lysis, removal of the attached cell wall proteins and

M

glycoproteins from the sugar molecules and easy extraction of the polysaccharides. Further ethanol precipitation of the alkaline extraction and dialysis eliminated all molecules including

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phenolics, monosaccharides, disaccharides, etc. Since previous reports showed that

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polysaccharides with high Mw have better immunomodulatory property with antitumor activity,

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we focused only on the water soluble fraction with Mw of >10,000 g/mol, by using an appropriate dialysis membrane (Meng et al., 2016).

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The polysaccharide content of MFPS1 was estimated to be 98.36±2.36 %. No absorbance appears at 260, 280 and 360 or 270 nm in the UV-VIS spectrum (Supplementary Fig. 2) of

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MFPS1 confirms the absence of nucleic acids, proteins, and phenolics, respectively. Thus, the present finding affirms that MFPS1 contained only carbohydrates and there was no contamination with other molecules. The unimodal molecular weight distribution curve obtained

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by advance polymer chromatography proved that MFPS1 was a homogenous mixture of polysaccharide with an approximate Mw of 1,45,000 g/mol (Fig. 1A). 3.3.

Monosaccharide units

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The quantity of different monosaccharides is a critical factor in determining the repeating units of a polysaccharide. Hydrolysis by TFA breaks the glycosidic bonds of a polysaccharide and liberates the component monosaccharides. However, since the monosaccharides do not have any absorbance, PMP labeling is required to generate a strong absorbance at 245 nm which could be analyzed by the UV detector without affecting the separation of monosaccharides (Dai et al.,

A

CC

EP

TE

D

M

A

N

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2010; Honda et al., 1989). The RP-HPLC measurement (Fig. 1B) indicated that MFPS1 was a

Fig. 1. (A) APC curve of MFPS1 with T-Dextran series. The approximate molar weight of MFPS1 was 145,000 g/mol. Where * denotes solvent front. (B) HPLC chromatogram of 12

MFPS1 after hydrolysis and labeling, where * denotes solvent front, 1- Rhamnose, 2Mannose, 3- Ribose, 4- Xylose, 5- Glucuronic acid, 6- Galacturonic acid, 7- NAGA, 8Glucose, 9- Arabinose, 10- Galactose. heteropolysaccharide with arabinose, glucose, galactose and mannose as the monosaccharide

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units in the molar ratio of approximately 3:2:2:1. Arabinose was most abundant followed by glucose and galactose while mannose was present in low amounts. 3.4.

Absolute configuration & linkage analysis

It was found that glucose, galactose, and mannose residues contained arabinose contained the

L

D

configuration whereas

configuration. The GC-MS analyses of methylated MFPS1 revealed

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the presence of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-arabinitol, 1,2,4,5-tetra-O-acetyl-3,6-di-

N

O-methyl-galactitol, 1,6,5-tri-O-acetyl-2,3,4-tri-O-methyl-glucitol, 1,3,5,6-tetra-O-acetyl-2,4-di-

A

O-methyl-mannitol, 1,5-di-O-acetyl -2,3,4,6-tetra-O-methyl-glucitol, 1,3,6,5-tetra-O-acetyl-2,4-

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di-O-methyl-galactitol, and 1,3,4-tri-O-acetyl-2,5-di-O-methyl-arabinitol in a molar ratio of approximately 2:1:1:1:1:1:1. The results thus indicated that terminal arabinopyranosyl, (1→2,4)-

D

linked galactopyranosyl, (1→6)-linked glucopyranosyl, (1→3,6)-linked mannopyranosyl,

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terminal glucopyranosyl, (1→3,6)-linked galactopyranosyl and (1→3)-linked arabinofuranosyl

3.5.

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moieties were present in a molar ratio of 2:1:1:1:1:1:1. NMR & Mass analysis of MFPS1

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The proton NMR spectrum (700 MHz, Fig. 2A) recorded at 25 °C showed signals at 4.38, 4.40, 4.86, 4.96, 5.26, and 5.71 ppm in the anomeric region. The integral values of the signals at 

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4.86, 5.26, and 5.71 ppm were nearly similar, but the total integral value of the signals  4.38 and 4.40 ppm were almost three times and that of the signal at δ 4.96 ppm was twice to that of the former. This observation indicated that the signals  4.38 and 4.40 ppm collectively corresponded to three anomeric protons signal at  4.96 ppm corresponded to two anomeric

13

protons and each of the remaining signals consisted of one anomeric proton. These signals were designated as A, B ( 4.38 ppm), C ( 4.40 ppm), D ( 4.86 ppm), E, F ( 4.96 ppm), G ( 5.26 ppm) and H ( 5.71 ppm). The 13C NMR spectrum (175 MHz, Fig. 2B) recorded at 25 °C

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showed six signals in anomeric region at δ 107.3, 103.1, 102.6, 102.4, 101.4, and 97.9 ppm. The signals at 107.3, 102.4, 101.4, and 97.9 ppm corresponded to anomeric carbons of H, E, G and D sugar units respectively whereas the signal at 103.1 corresponded to anomeric carbons of both A and B. The signal at 102.6 ppm corresponded to two anomeric carbons of C and F sugar residues. The 1H and 13C signals were assigned using COSY-, TOCSY-, and HSQC experiments

A

CC

EP

TE

D

M

A

N

U

(Table 1).

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Fig. 2. (A) 1H, (B) 13C, (C) DEPT-135 NMR spectrum of MFPS1

The residues A and B were assigned as terminal α-L-arabinopyranosyl moieties. The anomeric proton ( 4.38 ppm) and carbon ( 103.1 ppm) chemical shift values indicated that A and B were -linked anomers. The carbon chemical shifts from C-1 to C-5 corresponded nearly to the

15

standard values of methyl -L-arabinopyranosyl residue indicating their presence as terminal sugar residues. Residue C was assigned as (1→2, 4) -D-Galp. The coupling constant JC-1,H-1 ( 162 Hz) value,

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the anomeric proton and carbon chemical shifts for moiety C at  4.40 and  102.6 ppm indicated that it was -anomer. The downfield shift of C-2 (78.7 ppm) and C-4 (76.1 ppm) of residue C indicated that it was (12, 4)-linked moiety.

The coupling constants JC-1,H-1 (~171 Hz), JH-1,H-2 (~3.4 Hz), the anomeric proton ( 4.86 ppm) and carbon ( 97.9 ppm) chemical shift indicated that Residue D was -anomer. The downfield

N

U

shift ( 66.5 ppm) of C-6 (downward signal in DEPT-135 spectrum) confirmed its presence as

A

(16) linked sugar unit (Fig. 2C). Large coupling constants JH-2,H-3 (9.5 Hz) and JH-3, H-4 (9

M

Hz) for residue D indicated that it was D- glucosyl moiety. Residue E was assigned as (1 3, 6)- linked--D-mannopyranose from the anomeric proton H-1

161 Hz, high coupling

D

signal at  4.96 ppm, anomeric carbon shift at  102.4 ppm, JC-1,

TE

constants JH-1, H-2 (8 Hz) and JH-2,H-3 (10 Hz). The downfield shifts of C-3 ( 79.1 ppm) and C6 ( 69.2 ppm) signals with respect to the standard values for methyl glycosides confirmed its

EP

(13, 6)-linking-moiety. The downward signal at  69.2 ppm was observed in DEPT-135

CC

spectrum (Fig. 2C).

The assignment of the residue F as terminal α-D-glucopyranosyl moiety was done by its

A

chemical shifts of anomeric proton ( 4.96 ppm), anomeric carbon ( 102.6 ppm), C-2 ( 73.1 ppm), C-3 ( 73.5 ppm), C-4 ( 69.9 ppm), C-5 ( 72.7 ppm), and C-6 ( 60.1 ppm). Residue G was assigned as (1 3, 6)-linked-α-D-galactopyranosyl moiety because of its chemical shifts of H-1 ( 5.26 ppm) and C-1 ( 101.4 ppm), downfield resonances of C-3 (

16

75.5 ppm) and C-6 ( 69.3 ppm) as well as coupling constants, JC-1,H-1 (170 Hz) and JH-3,H-4 ( 5 Hz). The linkage at C-6 of the residue E was further confirmed by the downward signal at  69.3 ppm in DEPT-135 spectrum (Fig. 2C)

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In case of residue H, the high chemical shift values of C-1 (δ 107.3 ppm), C-2 (δ 79.3 ppm), C-3 (δ 83.9 ppm), C-4 (δ 84.3 ppm) and coupling constant JC-1,H-1 (168 Hz) indicated its αarabinofuranosyl conformation. The downfield shift of C-3 ( 83.9 ppm) with respect to the

A

CC

EP

TE

D

M

A

N

U

standard value of α -L-arabinofuranosyl residue indicated that it was (1→3)-linked moiety.

17

Fig. 3. (A) NOESY and (B) HMBC spectrum of MFPS1 The sequence of sugar residues of the polysaccharide was determined from NOESY- (Fig. 3A and Supplementary Table 1) and ROESY (Figure not shown) experiments. The NOE contacts

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AH-1 to GH-6, BH-1 to CH-4, CH-1 to EH-3, DH-1 to GH-3, EH-1 to DH-6, FH-1 to CH-2, GH-1 to HH-3, and HH-1 to EH-6 established the following sequences:

A-(1→6)-G; G-(1→3)-H; H-(1→6)-E; E-(1→6)-D; D-(1→3)-G; C-(1→3)-E; B-(1→4)-C; F(1→2)-C

The cross couplings, AH-1/GC-6, BH-1/CC-4, AC-1/GH-6, CH-1/EC-3, CC-1/EH-3, DH-1/GC-

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3, EH-1/DC-6, EC-1/DH-6, FH-1/CC-2, FC-1/CH-2, GH-1/HC-3, GC-1/HH-3, HH-1/EC-6, and

N

HC-1/EH-6 in the HMBC spectrum (Fig. 3B and Table 2) confirmed the above sequences. Hence

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D

polysaccharide was established as:

A

from NOESY, ROESY and HMBC experiments, the structure of the repeating unit of the

A

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G H E D ←3)-α-D-Galp-(1→3)-α-L-Araf-(1→6)-β-D-Manp-(1→6)-α-D-Glcp-(1→ 6 3 ↑ ↑ F C 1 1 α-L-Arap α-D-Glcp-(1→2)-β-D-Galp 4 A ↑ 1 α-L-Arap B

The proposed structure (Supplementary Fig. 3) was further supported by the HR-MS (Supplementary Fig. 4) and MALDI-TOF MS fragments (Supplementary Fig. 5) analysis. 3.6.

Cytotoxicity assessment 18

The cytotoxicity assay revealed that MFPS1 did not have any direct cytotoxic effect on cancer cells since promising cell death of cancer cells was not observed in individual cultures (Supplementary Fig. 6). However, MFPS1 showed prominent cytotoxicity on cancer cells when

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co-cultured with PBMC. The cytotoxic effect was both dose- and time dependent (Fig. 4). MFPS1 also did not exhibit cytotoxicity on PBMC (non-cancerous cells) and RAW 264.7, even after 72 h of incubation (Supplementary Fig. 7). Thus it could be inferred that cytotoxicity was executed via the immune cells, since PBMC cells are the principal component of human immune system. The physical interaction of MFPS1 with PBMC was also important because it deals with

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the immune cells and activated them. The activated immune cells, further communicated with

N

the cancer cells to promote cancer cell death. This data therefore clearly indicated that the

A

cytotoxic activity of MFPS1 was cancer cell specific. Since the cytotoxic effect was observed

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only in the presence of PBMC, it can be inferred that immune cells have a significant role in

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D

cancer cell death.

19

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Fig. 4. Cytotoxicity activity of MFPS1 on (A) SW620 & (B) A549 cells in in vitro condition after different time incubation with different dose. Data was expressed as mean ±SD (n=5) *P<0.05 (ns=non-significant). 3.7.

Immunomodulatory activity of MFPS1

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It was a very crucial factor to understand the proper immune activation system behind the cancer cell death by MFPS1. To explore the immune circuit engaged in this anticancer mechanism we studied the cytokine and immune cell specific marker expression in PBMC cell. As per previous literature (Haabeth, Bogen & Corthay, 2012) Th1 (T helper cell) lymphocytes and M1

20

macrophages play a crucial role in anticancer immune surveillance and cytotoxicity. In MFPS1 treated PBMC and RAW 264.7 cells, there was an increase in the IFN-γ and IL-12 (Supplementary Fig. 8). IFN-γ is secreted from Th1 cell and maintains M1 phenotype of

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macrophage. On the other hand IL-12 is released by M1 macrophage to sustain the Th1 phenotype (Haabeth et al., 2012). However, in PBMC, IL-12 over expression was not statistically significant while in RAW 264.7 cells it was significant. The over expression of these two cytokines indicated that a pro-inflammatory antitumor immune-surveillance was initiated by

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MFPS1 through M1Φ.

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Fig. 5. Immunophenotyping of MFPS1 (100 µg/ml) treated PBMC cell after 24 h of incubation. Total leukocyte population (CD45+) increased after MFPS1 treatment. Cells were initially gated on CD45+ cells. Data was expressed as mean ±SD (n=5) *P<0.05 (ns=non-significant).

21

It was also evident that MFPS1 stimulated IL-6 and TNF-α production (Supplementary Fig. 8). It induced the mature B-cell (CD19+) cell population and decrease in the immature B-cell population (CD19+/CD34+) (Fig. 5 & 6). These findings suggested that MFPS1 stimulated B-cell

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proliferation and differentiation via IL-6 production by M1Φ (Haabeth et al., 2012). On the other hand IL-6 stimulates TNF-α production and it can recruit NK cells. In MFPS1 treated PBMC, an increase in CD335+ cells (Fig. 5) was noticed, which is cytotoxicity activating receptor expressed in activated NK cells. Thus, this data clearly suggested that over expression of TNF-α engaged the activated NK cells against tumor cells (Burkholder et al., 2014; Vivier, Ugolini, Blaise,

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Chabannon, & Brossay, 2012). IL-6 and TNF-α both execute antitumor immunomodulation via

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M1Φ and recruit CD4+ and CD8+ cells (Burkholder et al., 2014; DeNardo, Andreu & Coussens,

A

2010). In this investigation, we could establish that MFPS1 aroused the proliferation of CD4+

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indicated activation and maturation of T-cell (CD34-/CD5+) (Fig. 6). On the other hand CD14+ up regulation specified the activation of macrophages by the polysaccharide (Fig. 6)

D

(Dobrovolskaia & Vogel, 2002).

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Anticancer and immunomodulation properties of heteropolysaccharides from mushrooms and

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other sources have been reported earlier (Maity et al. 2011; Huang et al., 2012; Yin et al., 2010). A research group (Moretão, Zampronio, Gorin, Lacomini & Oliveira, 2004) documented the

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activation of macrophage by an acidic heteropolysaccharide. Based on our work, we propose a

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possible immune assisted anticancer mechanism of the heteropolysaccharide, MFPS1 in Fig. 7.

22

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Fig. 6. Immunophenotyping of MFPS1 (100 µg/ml) treated PBMC cell after 24 h of incubation. MFPS1 can stimulate B cell (CD34-/CD19+) and T cell (CD34-/CD5+) maturation. MFPS1 activated monocytes (CD14+). Cells were initially gated on CD45+ cells. Data was expressed as mean ±SD (n=5) *P<0.05

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As per our hypothesis, MFPS1 may contribute to in the anticancer activity via immunomodulation. Since cytokine secretion is stimulated in the same manner as both the MFPS1-treated PMBC, where different types of mononuclear cell are present and murine macrophage, we assume that MFPS1 interacts with the macrophages (RAW264.7) and polarizes them.

23

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Fig. 7. Proposed immune-assisted anticancer mechanism of MFPS1.

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MFPS1 may bind to the receptor on the macrophage and is phagocytized by it. The

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internalization of MFPS1 within the cell polarizes the macrophage and stimulates it to secrete IL-

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12. IL-12 converts the naïve T cells to Th1 and Th1 cells and further secretes IFN-γ. The IFN-γ is maintained by the M1 phase of the macrophage. The M1Φ expresses IL-6. IL-6 promotes T-

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cell maturation and stimulation. A different type of polysaccharide recognizing receptor in macrophage was previously reported by other researchers (Schepetkin & Quinn, 2006). MFPS1

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promoted activation and maturation of T and B-cells via M1Φ. MFPS1 also triggered NK cells expressing natural cytotoxicity marker. Thus MFPS1 stimulated the immune system which was directed towards apoptosis of cancer cells. 4.

Conclusion

24

MFPS1, a bioactive heteropolysaccharide with a molar weight of ~1,45,000 g/mol, was isolated from the wild non-edible mushroom, Marasmiellus palmivorus. Arabinose, the dominating monosaccharide unit of MFPS1, was present in both pyranose and furanose forms. The repeating

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units of MFPS1 contained eight monosaccharides including three arabinose, two glucose, two galactose and one mannose. It was repeated almost 114 times. MFPS1 triggered both innate and adaptive immune system, induced pro-inflammatory response and activated anticancer immune surveillance system. This study indicated that MFPS1 possesses promising immune-modulatory activity and the immunomodulation leads to anticancer activity.

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The mode of cancer cell death i.e., apoptosis or necrosis and the toxicity related aspects,

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specially the allergic response of MFPS1 in in vivo system are now under further investigations.

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The synergistic effect of MFPS1 with conventional chemotherapeutic agents is also an unknown

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area and needs further research. Medicinal trial of this polysaccharide needs large quantities of it. This requires in situ synthesis of the MFPS1. However, the present study points to the fact that

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Conflict of Interest

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MFPS1 may be used as a good therapeutic agent for the treatment of late stage cancer in future.

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There is no conflict of interest.

Acknowledgement

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The first author is grateful to West Bengal Higher Education Department, for providing the fellowship. The authors are thankful to CSIR-Central Drug Research Institute (CSIR-CDRI), Indian Institute of Chemical Biology (IICB), Kolkata, and Dr. S. Ghosh, Polymer Science Unit, Indian Association for the Cultivation of Science, Kolkata, for instrumentation facility. The

25

authors acknowledge the Department of Molecular Biology & Biotechnology, Kalyani University for providing animal cell culture facilities. No agency funded this project.

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C-1/H-1

C-2/H-2

C-3/H-3

C-4/H-4

C-5/H-5

α-L-Arap-(1→ A, B

103.1/ 4.38

73.1/ 3.18

73.5/ 3.27

69.6/ 3.82

→2,4)-β-D-Galp-(1→ C

102.6/ 4.40

78.7 3.37

73.4/ 3.40

76.1/ 3.70

68.5/ 3.87, 3.55, 3.77, 3.49 76.0/ 3.59

→6)-α-D-Glcp-(1→ D

97.9/ 4.86

71.6/ 3.35

3.66

70.4 -

70.1/ 3.85

→3,6)-β-D-Manp-(1→ E

102.4/ 4.96

70.4/ 3.93

79.1/ 3.73

68.3/ 3.57

77.4/ 3.38

α-D-Glcp-(1→ F

102.6/ 4.96

73.1/ 3.77

73.5/ 3.88

69.9/ 3.29

72.7 -

→3,6)-α-D-Galp-(1→ G

101.4/ 5.26

69.6/ 3.75

75.5/ 3.78

69.6/ 3.82

68.3/ 4.09

→3)-α-L-Araf-(1→ H

107.3/ 5.71

79.3/ 4.12

83.9/ 3.68

84.3/ 3.63

61.3/ 3.62, 3.75

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N

M

D

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60.8/ 3.82, 3.91 66.5/ 3.61, 3.72 69.2/ 3.96, 4.06 60.1/ 3.67, 3.91 69.3/ 3.69, 3.72

of the 1H NMR chemical shifts were recorded with respect to the HOD signal fixed at δ 4.68 ppm at 25 °C. bValues of the 13C NMR chemical shifts were recorded with reference to the acetone as internal standard and fixed at δ 31.05 ppm at 25 °C.

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aValues

C-6/H-6

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Glycosyl residue

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Table 1 1H NMRa and 13C NMRb chemical shifts (in ppm) for MFPS1 recorded in D2O at 25 °C

31

Table 2 The connectivities observed in HMBC spectrum for the anomeric protons/carbons of the sugar residues Observed connectives Inter δ Intra δ AH-1/GC-6 69.3 AH-1/AC-2 73.1 α-L-Arap-(1→ BH-1/CC-4 76.1 A, B AC-1/GH-6 3.69 79.1 CC-1/CH-6 3.82 →2,4)-β-D-Galp-(1→ CH-1/EC-3 CC-1/EH-3 3.73 C 75.5 DH-1/DC-6 66.5 →6)-α-D-Glcp-(1→ DH-1/GC-3 D DH-1/DC-5 70.1 DC-1/DH-2 3.55 66.5 EH-1/EC-2 70.4 →3,6)-β-D-Manp(1→ EH-1/DC-6 EC-1/DH-6 3.61 EC-1/EH-2 3.93 E FH-1/CC-2 78.7 FH-1/FC-3 73.5 α-D-Glcp-(1→ FC-1/CH-2 3.37 F 83.9 GH-1/GC-5 68.3 →3,6)-α-D-Galp-(1→ GH-1/HC-3 GC-1/HH-3 3.68 GH-1/GC-2 69.6 G GC-1/GH-5 4.09 69.2 HC-1/HH-5 3.75 →3)-α-L-Araf-(1→ HH-1/EC-6 HC-1/EH-6 4.06 H

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Sugar residues

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