Characterization of Immunomodulatory Activities of Honey

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Characterization of Immuno-modulatory Activities of Honey Glycoproteins and Glycopeptides M. Ahmed Mesaik, Nida Dastagir, Nazim Uddin, Khalid Rehman, and M. Kamran Azim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505131p • Publication Date (Web): 12 Dec 2014 Downloaded from http://pubs.acs.org on December 17, 2014

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Journal of Agricultural and Food Chemistry

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Characterization

of

Immuno-modulatory

Activities

of

Honey

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Glycoproteins and Glycopeptides

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M. Ahmed Mesaik@1,2, Nida Dastagir@1, Nazim Uddin1, Khalid Rehman1 and M. Kamran Azim1*

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1

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Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. 2Faculty of

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Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala

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Lumpur, Malaysia.

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*

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Address for correspondence: Dr. M. Kamran Azim, International Center for Chemical and

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for

Corresponding author; @These authors contributed equally.

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Biological Sciences, University of Karachi, Karachi-75270, Pakistan.

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Tel. +9221-111222292 ext. 236

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Fax +9221-3498018

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Email: [email protected]; [email protected]

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1 ACS Paragon Plus Environment


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ABSTRACT

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Recent evidences suggest an important role for natural honey in modulating immune response.

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To identify active components responsible, we investigated the immuno-modulatory properties

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of glycoproteins and glycopeptides fractionated from Ziziphus honey. Honey proteins/peptides

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were fractionated by size exclusion chromatography into 5 peaks with molecular masses in the

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range of 2-450 kDa. The fractionated proteins exhibited potent, concentration-dependent

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inhibition of reactive oxygen species production in zymosan-activated human neutrophils (IC50

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6-14 ng/mL) and murine macrophages (IC50 2-9 ng/mL). Honey proteins significantly suppressed

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the nitric oxide production by LPS-activated murine macrophages (IC50 96-450 ng/mL).

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Moreover, honey proteins inhibited the phagocytosis latex beads macrophages. The production

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of proinflammatory cytokines IL-1β and TNF-α by human monocytic cell line in presence of

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honey proteins was analyzed. Honey proteins did not affect the production of IL-1β, however

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TNF-α production was significantly suppressed. These findings indicated that honey

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glycoproteins and glycopeptides significantly interfere with molecules of the innate immune

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system.

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Keywords: Honey, glycoprotiens, immunomodulatory, phagocytosis, oxidative burst

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INTRODUCTION

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Natural honey is a well-known neutraceutical which is mainly composed of fructose, glucose,

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sucrose and maltose. Small amount of oligosaccharides, proteins, amino acids, minerals, trace

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elements, vitamins, aroma compounds and polyphenols are also presentreviewed in 1. Honey exerts a

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number of pharmacological effects including antimicrobial, immunomodulatory, prebiotic,

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antinematodal, anti-inflammatory and anti-nociceptive activities1-9. It showed both bacteriostatic

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and bactericidal potentials against pathogenic bacteria including drug-resistant strains5. Honey

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was found to exert anti-inflammatory effects via inhibiting (a) thrombin induced reactive oxygen

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species (ROS) production6 and (b) myeloperoxidase dependent oxidative burst by professional

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phagocytes7. It inhibited the nitric oxide production in carrageenen induced inflammatory rat

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modal10. In addition, Manuka, Pasture and Jelly bush honeys were found to reduce reactive

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oxygen species (ROS) production11 and stimulate monocytes (MonoMac-6 cell line) to produce

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tumor necrosis factor–α (TNF-α), Interleukin (IL)–1β and IL-6, which are known to play a vital

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role in tissue repair12. The effect of honey on cytokine production was found to be associated

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with a 5.8 kDa component isolated from manuka honey which interacted with TLR-413. Natural

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honey exhibited anti-tumor activity by inhibiting the growth of Lewis Lung Carcinoma/2 cells14.

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Immuno-stimulatory effect of honey on antibody production against thymus-dependent (sheep

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red blood cells) and thymus-independent (E. coli) antigens in mice has also been observed15.

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Likewise, honey also inhibited mitogen activated T cells proliferation in vitro16. Moreover,

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honey regulates the homeostasis by inhibiting the human platelet aggregation and blood

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coagulation proteins17.

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Natural honeys have low protein content which varies from type to type. Characterization of

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honey proteins from various sources was found to be valuable in determining the floral and 3 ACS Paragon Plus Environment


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geographical origin18. A number of reports pointed out immuno-modulatory potential of honey

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proteins. Four immunologically characterized IgE-binding glycoproteins (belongs to major royal

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jelly protein-1 (MRJP-1) family) were identified in honey and N-linked glycosylation in these

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proteins played an important role in binding of IgE19,21. Ultracentrifugation of royal jelly (RJ)

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revealed that the MRJP1, the most abundant protein of RJ present in monomeric (55 kDa) as

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well as oligomeric (ca 450 kDa) forms20. Honey contains MRJP1 protein as well which exerts its

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immuno-stimulatory effect by stimulating TNF-α production in murine peritoneal macrophages

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and the protein moiety of this glycoprotein was found be responsible for this property22.

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Recently, highly glycosylated Arabinogalactan proteins (AGPs) with molecular mass of ~110

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kDa have been characterized from New Zealand Kanuka honey23. AGPs are proteoglycans which

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contain type II arabinogalatans and 2-10% covalently attached protein that is typically rich in

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hydroxyproline and serine. AGPs are compact molecules consisting of a protein core to which

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highly branched polysaccharide chains are attached24. AGPs are found in the female sexual

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tissues of flowers, and thus could be a potential source of AGPs in honey. AGPs and type II

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arabinogalactans from different sources have been shown to exert immuno-modulatory

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properties25. As a continuation of our research, we have elaborated the role(s) of honey

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glycoproteins and glycopeptides in modulation of innate immune system.

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MATERIALS AND METHODS

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FRACTIONATION AND CHARACTERIZATION OF HONEY PROTEINS

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Honey samples: Monofloral Ziziphus species honey samples produced by Apis mellifera in bee

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farms located in Himalayan and Pothohar plateau regions in northern Pakistan were used during

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this study.


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Ammonium Sulphate Precipitation: Honey samples were diluted in buffer A (20 mM Tris-

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HCl, 150 mM NaCl, and 0.05% sodium azide, pH 7.4) and centrifuged at 2500 xg for 20 min at 4

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o

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processed for ammonium sulphate precipitation at 4 oC with 80% saturation. The solution was

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kept on stirrer for 1-2 hours followed by centrifugation at 8300 xg at 4 oC. The pellets were

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dissolved in buffer and stored at -20 oC till use.

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Gel Filtration Chromatography (GFC): Gel filtration chromatography crude honey proteins

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was carried out by HiloadTM 16/60 SuperdexTM 200 Prep Grade column (GE Healthcare,

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Uppsala, Sweden) with buffer A as mobile phase. Chromatography was done using AKTA

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FPLC system (GE Healthcare, Uppsala, Sweden) at the flow rate of 1 mL/min flow rate and

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fractions of 2 mL were collected. The resultant five chromatographic peaks were termed as GFC-

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P1 to GFC-P5. Fractions containing high molecular mass peak, GFC-P1 were pooled and treated

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with 8M urea (Sigma-Aldrich, Steinheim, Germany) followed by re-chromatography in the same

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conditions. The peaks GFC-P1 to GFC-P3 were concentrated by ultrafiltration (MWCO 10 kDa,

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Millipore, Darmstadt, Germany). Whereas peaks GFC-P4 and GFC-P5 was freeze dried

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(ALPHA 1-2/LD plus, Suarlee, Belgium) and resuspended in buffer A. Protein estimation was

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carried out by modified Lowry’s protein assay kit (Thermo Scientific, Rockford, USA)26. The

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molecular masses of proteins were estimated by Gel Filtration Calibration Kit HMW (GE

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Healthcare BioSciences AB, Sweden) which contains five proteins and Blue dextran 2000

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(Ovalbumin Mr 43,000, Conalbumin Mr 75,000, Aldolase Mr 158,000, Ferritin Mr

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440,000,Thyroglobulin Mr 669,000 and Blue dextran 2000 Mr 2,000,000).

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Reverse Phase HPLC of GFC Peaks: The gel chromatography peaks GFC-P1 and GFC-P5

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were subjected to reverse phase HPLC using C-18 column (Purospher STAR, RP-18 endcapped

C in order to remove particulates, particularly pollens. The supernatants were collected and



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(5 µm, 150-4.6, MERCK, Darmstadt, Germany). Column was equilibrated with 0.1% TFA and

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100 µL of filtered protein samples were applied with flow rate of 1.0 mL/min. Gradient elutions

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were made with acetonitrile as 5min/0%, 40min/70% and 45min/0%. Peaks were collected,

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concentrated and stored at -20oC till next use.

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Mass Spectrometry: MALDI-TOF analysis of peaks obtained by RPLC was carried out as

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follows. 0.5 µL of peptide suspension was mixed with 0.5 µL of matrix solution (Sinapinic acid

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in 1:1 0.1% TFA/Acetonitrile). The matrix/analyte solution was applied to sample plate. Mass

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spectra were recorded on a Bruker Ultraflex III TOF/TOF spectrometer. Two hundered shots of

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light from smart beam laser were irradiated to record mass spectra. Bioinformatics analysis of

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mass spectrometric data was done by using Glycosuite Database and GlycoMod tool of ExPASy

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server (www.expasy.org).

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IMMUNOMODULATORY ACTIVITY ASSAYS

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Isolation of Phagocytic Cells: Polymorph nuclear leukocytes (PMNLs) were collected from

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human blood by using ficoll paque gradient centrifugation. For this purpose, blood was drawn

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from healthy human volunteer (36-45 years old who did not receive any medication for one

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week). Blood was mixed with equal volume of lymphocyte separation medium (MP Biomedical,

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Inc, Kaysersberg, France) and hank’s balanced salt solution without calcium and magnesium

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(Sigma-Aldrich, Steinheim, Germany). The diluted blood was kept undisturbed at room

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temperature for 30-45 min. When red blood cells settled down, supernatant was layered onto 5

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mL of LSM in separate centrifuge tube and centrifuged at 400 g for 20 min at 4oC. Supernatant

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was discarded and pellet was resuspended in HBSS free from calcium and magnesium and

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washed at 300 g for 15 min at 4oC. In presence of RBCs contamination, RBCs were lysed with


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sterile distilled water; finally pellet was resuspended in HBSS containing calcium and

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magnesium. Cells viability was determined using trypan blue dye and cell count was adjusted to

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one million cells per mL.

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In addition, murine peritoneal macrophages were collected after injecting 10 mL sterile

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phosphate buffer saline (PBS) into mouse peritoneum (immunized with 1 mL sterile fetal bovine

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serum 72 hrs earlier). Cells were spun at 300 g for 15 min at 4oC. Pellet was resuspended in 1

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mL sterile PBS, viability was checked with trypan blue exclusion method (cells were >95%

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viable) and count was adjusted as one million cells per mL.

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Chemiluminescence Assay for Oxidative Burst: The effect of honey protein fractions on

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reactive oxygen species (ROS) production was measured on polymorph nuclear leucocytes

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(PMNLs) and mouse peritoneal macrophages. In brief, cells were plated in flat bottom 96-well

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plates and activated by serum opsonized zymosan-A (Sigma-Aldrich, Steinheim, Germany) at

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250 µg/mL final concentration. Proteins/peptides/glypeptides samples added at various

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concentrations (0.125-2.0 µg/mL) and plates were incubated for 20 min at room temperature.

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Luminol (3-Aminophthalhydrazide) (Alfa Aesar, Karlsruhe, Germany) was added at 1.5 µg/mL

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final concentration. Plates were immediately kept in the thermostated chamber of Luminoskan

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and chemluminescence kinetics of ROS production was monitored as relative light units [RLUs]

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for 50 min in Luminoskan (Labsystem, Helsinki, Finland) with repeated scan27.

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Nitric Oxide Assay: The murine macrophage cells (J774.2 cell line) were purchased from

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European Collection of Cell Cultures (Salisbury, UK). Cells were cultured in complete DMEM

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(Sigma-Aldrich, Steinheim, Germany) supplemented with 10% heat inactivated fetal bovine

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serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. The effect of honey proteins on



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nitric oxide production was monitored according to Inoue et al.28. Briefly cells were seeded in a

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96-well plate as 1.8x105 cells/well and activated by 30 µg/mL of LPS (E. coli serotype 0111:B4,

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DIFCO Laboratories, Michigan, USA) for the production of nitric oxide. Protein samples were

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added at various concentrations and plate was incubated at 37 oC for 48 hours. Supernatants (50

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µL) from each well were mixed with an equal amount of griess reagent (0.1% naphthyl ethylene

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diamine dihydrochloride in distilled water and 1% sulfanilic acid in 5% H3PO4 in 1:1 ratio)

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(Fisher Scientific, Leicestershire, UK) and absorbance was recorded at 550 nm using Spectra

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Max plus 340 (Molecular Devices, CA, USA).

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Phagocytosis Assay: Phagocytosis assay was performed according to Foukas et al. with some

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modifications29. The murine macrophage cells (J774.2 cell line) were cultured in complete

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DMEM and seeded into 96-well tissue culture treated plate with 5x104 cells/well. Culture

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reaction plate was incubated in CO2 environment at 37 oC with 5% CO2 for 24 hour. Next day,

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latex beads (fluorescent coated carboxylated modified polystyrene beads, 1 µm, Sigma-Aldrich,

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Steinheim, Germany) were added as 100 beads/cell. Protein samples were added as 800 ng/mL

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final concentration and plate was further incubated for 24 hours at 37 oC in CO2 environment.

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Afterward, supernatant was removed and wells were washed twice by gently inverting the plate

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with 1X PBS. 1X trypan blue (50 µL) was added to each well and plate was kept at room

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temperature for 1 minute. The trypan blue was removed and fluorescence was recorded at 575-

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610 nm using fluorescence reader (Molecular Devices, CA, USA).

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Cytokine (TNF-α and IL-1β) Production Assay: THP-1 cell line (human monocytic leukemia

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cells) was purchased from European Collection of Cell Cultures (Salisbury, UK). Cells were

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cultured in 75 mm2 tissue culture flask with complete RPMI-1640 medium (MP Biomedical,

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Ohio, USA) supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L8 ACS Paragon Plus Environment

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glutamin, 5.5mM D-glucose, 50 µM β-mercaptoethanol, and 1% penicillin & streptomycin. Cells

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were seeded into a 24-well polystyrene sterile culture plate with 20 ng/mL PMA (SERVA,

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Heidelberg, Germany), which cause differentiation of monocytes to macrophages. The culture

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plate was incubated at 37 oC for 24 hours. Next day, cells were activated with 50 ng/mL LPS,

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protein samples were added in various concentrations and plate was incubated at 37 oC with 5%

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CO2. Supernatants were collected after 6 hours and level of TNF-α and IL-1β were estimated

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using ELISA kit (R & D System, Minneapolis, USA). Absorbance was recorded at 450 nm using

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microplate reader, (DIA Reader, Gmblt, Wr. Neudorf, Austria)30.

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Statistical Analysis: All experiments were run in triplicates and data were presented as

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statistical mean ± standard deviation (STD). A one-way Analysis of Variance (ANOVA) was

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applied to measure the significant difference between the results of test proteins and controls.

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Significance level was set as P

Characterization of Immunomodulatory Activities of Honey

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