Đề tài Study on macrophage activation and structural characteristics of purified polysaccharide from the liquid culture broth of Cordyceps militaris

Carbohydrate Polymers 82 (2010) 982–988 Contents lists available at ScienceDirect Carbohydrate Polymers journa l homepage: www.e lsev ier .com Study on macrophage activation and structural c polysac f C Jong Seo ee, Department of public a r t i c l Article history: Received 8 Jan Received in re Accepted 15 Ju Available onlin Keywords: Cordyceps mili Immunostimu Macrophage a Random coil c des o d byD three SN Fr uction were ctive s spe ad am Fr II © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Most, if active poly cultured m known toha ing chemica no toxic sid ceps militar Ascomycete ities such as hypolipidem 2005), antit metastatic et al., 2009 2009, 2007 ily by activ such as com ∗ Correspon and Technolog Gangwon-do 2 fax: +82 33 24 E-mail add macrophage-dependent immune system responses (Lee, Cho, & 0144-8617/$ – doi:10.1016/j.not all, basidiomycetes mushrooms have biologically saccharides in the fruiting body, culture broth, and ycelia. Polysaccharides derived from mushrooms are vepotent immunomodulatingproperties. Unlike exist- l anticancer agents, polysaccharides are known to have e effects (Novak & Vetvicka, 2008). Among them, Cordy- is, an entomophathogenic fungus belonging to the class s, has been reported to have beneficial biological activ- hypoglycemic (Kiho, Yamane, Hui, Usui, & Ukai, 1996), ic (Yang et al., 2000), anti-inflammatory (Won & Park, umor (Lin & Chiang, 2008; Park et al., 2009, 2005), anti- (Nakamura et al., 1999), immunomodulatory (Cheung ; Kim et al., 2008), and antioxidant effect (Yu et al., ). Polysaccharides exert their antitumor effects primar- ating various immune system responses in the host, plement system activation (Dennert & Tucker, 1973), ding author at: College of Engineering, Department of Bioengineering y, Kangwon National University, 192-1, Hyoja-2-dong, Chuncheon, 00-701, Republic of Korea. Tel.: +82 33 250 6275; 3 6350. ress: ekhong@kangwon.ac.kr (E.K. Hong). Hong, 2009; Lee, Min, Cho, & Hong, 2009), and upregulation of interferon expression (Hamuro & Chihara, 1985). Various stud- ies have been conducted to determine the mechanism by which macrophages kill tumor cells. Activated macrophages recognize and kill tumor cells in a direct manner. However, they also play an indirect role in antitumor activity by secreting secondary com- pounds, such as tumor necrosis factor (TNF) and nitric oxide (NO), which are harmful to cancer cells, and by regulating the processing and presentation of antigens by the immune system (Medzhitov & Janeway, 2000). It has been extensively shown that the immunomodulating actions of polysaccharides are dependent on their chemical composition, molecular weight, conformation, glycosidic linkage, degree of branching, etc. (Methacanon, Madla, Kirtikara, & Prasitsil, 2005; Yadomae & Ohno, 1996). Biologically active polysaccharides are widespread among mushrooms, and most have unique structures in different species. As a result of this phenomenon, several studies have been conducted to determine accurately the structures of these different polysaccharides. The aim of this study was to better understand and character- ize the structural characteristics of the polysaccharide, CPSN Fr II, which was isolated and purified from the liquid culture broth of C. militaris by gel filtration and ion exchange chromatography. To this end, we investigated the release of NO and the produc- tion of cytokines by macrophages that were activated by this see front matter © 2010 Elsevier Ltd. All rights reserved. carbpol.2010.06.025charide from the liquid culture broth o k Lee, Jeong Seok Kwon, Dong Pil Won, Keun Eok L Bioengineering and Technology, Kangwon National University, Chuncheon 200-701, Re e i n f o uary 2010 vised form 24 May 2010 ne 2010 e 19 June 2010 taris lating polysaccharide ctivation onformation a b s t r a c t The water-soluble crude polysacchari ethanol precipitationwere fractionate This fractionation process resulted in II, and CPSN Fr III. Of the fractions, CP activated macrophages, such as prod TNF-). Its structural characteristics analyses, includingmethylation, redu (FT-IR), and gas chromatography–mas branched-glucogalactomannan that h and randomcoil conformation of CPSN respectively./ locate /carbpol haracteristics of purified ordyceps militaris Won Cheol Shin, Eock Kee Hong ∗ of Korea btained from the liquid culture broth of Cordyceps militaris by EAE cellulose and Sepharose CL-6B column chromatography. polysaccharide fractions that were termed CPSN Fr I, CPSN Fr II was able to upregulate the functional events mediated by of nitric oxide (NO) and expression of cytokines (IL-1 and investigated by a combination of chemical and instrumental cleavage, acetylation, Fourier transform infrared spectroscopy ctrometry (GC–MS). Results indicate that CPSN Fr II was a 1,6- olecularweight of 36kDa. The configuration of the-linkage were confirmedusing a Fungi-Fluor kit and congo red reagent, J.S. Lee et al. / Carbohydrate Polymers 82 (2010) 982–988 983 polysaccharide as part of the innate immune response. In addition, its chemical composition, molecular weight, conformation, degree of branching, and glycosidic linkage were determined. 2. Materials and methods 2.1. Materi The stra which was pH, and a ing 80g/l g 0.5 g/l KH2 was centrif precipitated collected b resuspende remove low (Kwon, Lee membranes trans, lipop curdlan, an cal Co. (St. were obtai macrophag Collection ( grade. 2.2. Extract polysacchar The crud tilled water a DEAE cel tral and acid onto a Seph 0.5N NaCl, charides ba derived fro endotoxin l as assessed 2.3. Cell cul RAW264 plemented 10% fetal bo 5% CO2 incu 2.4. Cell via The effe cells was de diphenyltet the reducti nase in viab cells/ml) fo was added Fifty micro added to ea incubation and the sup well were d erated was on a scanni Table 1 Primer sequences of genes investigated by RT-PCR analysis. Gene Primer sequences IL-1 a ′ ′ ard. rse. eterm r pr ach and in c ess r nedia s. T aNO -PCR valu otal as p the at − iptio MuL with rand as f he ad ermi plet ubat s [Bi mM U of lowi ing s and imer iters visu F-˛ abi 64.7 cultu tion o tional, Camarillo, CA, USA), according to the manufacturer’s tions. alysis of chemical properties total sugar content of each polysaccharide was determined the phenol–sulfuric acid method (Chaplin & Kennedy, the total protein concentration was determined using the rd method (Bradford, 1976), the hexosamine content wasals in used in this study was C. militaris KCTC 6064, cultivated for 11 days at 24 ◦C, 200 rpm, uncontrolled 2% (v/v) inoculum size in modified medium contain- lucose, 10 g/l yeast extract, 0.5 g/l MgSO4·7H2O, and PO4. After 11 days of cultivation, the culture broth uged at 5000 rpm for 20min. Polysaccharides were from the liquid culture broth using 95% ethanol, y filtration through 0.45m Whatman filter paper, d and dialyzed against distilled water for 5 days to -molecular-weight compounds, and then freeze-dried , Shin, Lee, & Hong, 2009). Dialysis tubing cellulose , DEAE cellulose, Sepharose CL-6B, standard dex- olysaccharide (LPS, Escherichia coli 0111:B4), laminarin, d congo red were purchased from Sigma Chemi- Louis, MO, USA). Fetal bovine serum and RPMI1640 ned from GIBCO (Grand Island, NY, USA). RAW264.7 es were purchased from the American Type Culture Manassa, VA, USA). All other chemicals were of Sigma ion, fractionation and purification of water-soluble ides e polysaccharides, termed CPS, was dissolved in dis- , centrifuged at 5000× g for 20min, and loaded onto lulose (Cl−) column (2.5 cm×50 cm) to separate neu- ic polysaccharides. The resulting fractions were loaded arose CL-6B column (2.3 cm×80 cm) equilibrated with then eluted with the same solution to separate polysac- sed on molecular weight. Each polysaccharide fraction, m the liquid culture broth of C. militaris, contained an evel that was below the detection limit (0.0015EU/ml) by an E-TOXATE kit (Sigma, St. Louis, MO, USA). ture .7 cells were maintained in RPMI1640 that was sup- with 100U/ml penicillin, 100g/ml streptomycin, and vine serum. Cells were grown at 37 ◦C in a humidified bator. bility ct of polysaccharides on the viability of RAW264.7 termined using the [3-(4,5-dimethylthiazol-2-yl)-2,5- razolium] bromide (MTT) assay, which is based on on of a tetrazolium salt by mitochondrial dehydroge- le cells. After pre-incubating RAW264.7 cells (1×106 r 18h, polysaccharides (1000g/ml) or LPS (2.5g/ml) and the mixture was incubated for an additional 24h. liters of the MTT stock solution (2mg/ml) was then ch well to attain a total reaction volume of 200l. After for 2h, the plate was centrifuged at 800× g for 5min ernatantswere aspirated. The formazan crystals in each issolved in 150l dimethylsulfoxide and the color gen- determined by measuring the optical density at 540nm ng multiwell spectrophotometer. TNF- GAPDH a Forw b Reve 2.5. D Afte 18h, e added Nitrite of Gri ethyle sample using N 2.6. RT To e sion, t cells w ing to stored transcr using bated first-st ture w after t were t was de the inc primer 8.3, 50 and 1 the fol anneal 72 ◦C, The pr microl gel and light. 2.7. TN The RAW2 in the centra Interna instruc 2.8. An The using 1986), BradfoF 5 -CAGATGAGGACATGAGCACC-3 Rb 5′-CACCTCAAACTCAGACGTCTC-3′ Fa 5′-TTGACCTCAGCGCTGAGTTG-3′ Rb 5′-CCTGTAGCCCACGTCGTAGC-3′ Fa 5′-CACTCACGGCAAATTCAACGGCAC-3′ Rb 5′-GACTCCACGACATACTCAGCAC-3′ ination of NO production e-incubation RAW264.7 cells (1×106 cells/ml) for polysaccharide (1000g/ml) or LPS (2.5g/ml) was the mixture was incubated for an additional 24h. ulture supernatants was measured by adding 100l eagent (1% sulfanilamide and 0.1% N-[1-naphthyl]- mine dihydrochloride in 5% phosphoric acid) to 100l he nitrite concentration was determined at 540nm 2 as a standard. ate levels of LPS or CPSN Fr II-inducible mRNA expres- RNA from CPSN Fr II-treated or untreated RAW264.7 repared by adding TRIzol reagent (Gibco-BRL) accord- manufacturer’s protocol. The total RNA solution was 70 ◦C prior to subsequent use. Semiquantitative reverse n-polymerase chain reaction (RT-PCR) was performed V reverse transcriptase. Total RNA (1g) was incu- oligo-dT15 for 5min at 70 ◦C, then mixed with a 5× buffer, 10mM dNTPs, and 0.1M DTT. The reaction mix- urther incubated for 5min at 37 ◦C, then for 60min dition of 2U of MuLV reverse transcriptase. Reactions nated by heating for 10min at 70 ◦C, and total RNA ed by addition of RNase H. PCR was performed with ion mixture (2l of cDNA, 4M forward and reverse oneer, Seoul, Korea], a 10× buffer [10mM Tris–HCl, pH KCl, 0.1% Triton X-100], 250M dNTPs, 25mM MgCl2, Taq polymerase [Promega, Madison, WI, USA]) under ng conditions: a 45 s denaturation step at 94 ◦C, a 45 s tep between 55 ◦C and 60 ◦C, a 60 s extension step at a 7min final extension step at 72 ◦C after 30 cycles. s used in this experiment are indicated in Table 1. Ten of PCRproductswere electrophoresed on a 1.2% agarose alized by ethidium bromide staining under ultraviolet production lity of CPSN Fr II to induce production of TNF- in cells was determined by dissolving the polysaccharide re medium. Supernatants were harvested and the con- f TNF- was determined using an ELISA kit (Biosource 984 J.S. Lee et al. / Carbohydrate Polymers 82 (2010) 982–988 evaluated using the Elson–Morgan method (Dische, 1962), and the uronic acid content was assessed using the Blumenkrantz method (Blumenkrantz & Asboe-Hansen, 1973). 2.9. Analysis of monosaccharide composition Monosac first hydrol (TFA) in a by repeate The hydrol and filtered hydrolysate electrochem following c (4mm×24 program: 0 B); flow ra galactose, m standards. 2.10. Deter Themol mined by g A standard the molecu 150kDa, an 2.11. Analy The con tion was de complexes. the single-s max of con tions rangin (1mg/ml) c with differe tra were re Rochester, N 1978; Ogaw 2.12. Identi To ascer uration in detected us USA). Each tion was p the additio 20min. Fun hydroxide) sample and ing with di using a UV France). 2.13. Methy CPSN Fr oped by Ciu (Ciucanu & the FT-IR sp 2.14. Determination of glycosidic linkage Permethylated CPSN Fr II was extracted in dichloromethane and reductive cleavage was performed using a combination of trimethylsi alyst as pr lowe ed b ted w lyco tus ( lum 0–25 oniza ty of tatis uden tistic ined xpre ken icate gnifi ults rifica he fi exch sep f the ed fr 34g as i arose ame .066 acrop exam bro ion o ted w n wa untr large ed p A). T ses i mine ed e sho inflam and emic tota uent ) (Tab onic tivelycharide composition and ratios were determined by yzing the polysaccharide with 2M trifluoroacetic acid sealed tube at 100 ◦C for 4h. Acid was removed d evaporation using a vacuum distillation device. ysate was then dissolved in 1.0ml of distilled water through a 0.2m PTFE membrane. The aqueous was analyzed by reverse-phase HPLC using an ED50 ical detector (Dionex, Sunnyvale, CA, USA) under the onditions—column: CarboPac PA10 Analytical Column 0mm); solvent: A, deionized water, B, 200mM NaOH; –20min (8% B), 20–40min (25% B), 40–70min (8% te: 0.9ml/min; column oven temp.: 30 ◦C. Glucose, annose, and fucose were used as monosaccharide mination of molecular weight ecularweight of the polysaccharide fractionswas deter- el filtration using a Sepharose CL-6B packed column. curve was prepared based on the elution volume and lar weight. Standard dextrans (MW: 670kDa, 410kDa, d 25kDa) were used for calibration. sis of helix–coil transition formational structure of the polysaccharides in solu- termined by characterizing congo red-polysaccharide The transition from a triple-helical arrangement to tranded conformation was examined by measuring the go red-polysaccharide solutions at NaOH concentra- g from0.01N to 0.5N. Polysaccharide aqueous solutions ontaining 100l of 0.5mg/ml congo red were treated nt concentrations of NaOH. Visible absorption spec- corded with a UV/vis spectrophotometer (Milton Roy, Y, USA) at each alkali concentration (Ogawa & Hatano, a, Tsurugi, & Watanabe, 1973). fication of anomeric configuration tain the presence or absence of the  or  config- each polysaccharide, -linked polysaccharides were ing a Fungi-Fluor Kit (Polysciences, Warrington, PA, sample was dissolved in distilled water and the solu- laced on a slide and dried in an oven. Following n of methanol, each sample dried for an additional gi-Fluor Solution A (cellufluor, water, and potassium was used as a dye. A few drops were added to each the mixtures were incubated for 3min. After wash- stilled water, the fluorescence level was determined Illuminator (Vilber Lourmat, Marne La Vallee Cedex 1, lation of CPSN Fr II II was methylated according to the method devel- canu and Kerek, using powdered NaOH in Me2SO–MeI Kerek, 1984). Methylation was confirmed by measuring ectrum. was al quench separa lated. G appara 5MS co and 18 lows: i intensi 2.15. S ASt the sta determ were e were ta in tripl cally si 3. Res 3.1. Pu In t of ion used to yield o obtain and 0.0 CPSN w a Seph tions, n Fr III (0 3.2. M To culture activat incuba ductio by the duced trigger (Fig. 2 respon to exa increas iments of pro (Fig. 2B 3.3. Ch The constit (6.12% and ur respeclylmethanesulfonate and trifluoride etherate as the cat- eviously described (Rolf & Gray, 1982). The reaction d to proceed for 8–12h at room temperature, then was y addition of sodiumbicarbonate. The organic layerwas ith a syringe and products were isolated and acety- sidic linkage was analyzed by GC–MS on a Micromass Waters Corp., Milford, MA, USA) equipped with an HP- n and a temperature program of 120–180 ◦C at 5 ◦C/min 0 ◦C at 2 ◦C/min). The mass conditions were set as fol- tion mode with EI, ionization energy of 70eV, a current 500A, and ion source temperature at 250 ◦C. tical analysis t’s t-test andaone-wayANOVAwereused todetermine al significance of the differences between the values for the various experimental and control groups. Data ssed as means± standard errors (SEM) and the results fromat least three independentexperimentsperformed . P-values of 0.05 or less were considered to be statisti- cant. tion and fractionation rst stage of purification and fractionation, the method ange chromatography on DEAE cellulose column was arate neutral polysaccharides from acidic fractions. The neutral fraction (CPSN) and the acidic fraction (CPSA) om the crude polysaccharide extract CPS was 0.328g/g /g, respectively (Fig. 1A). The molecular distribution of nvestigated using gel filtration chromatography with CL-6B column, resulting in three polysaccharide frac- ly CPSN Fr I (0.077g/g), CPSN Fr II (0.153g/g), and CPSN g/g) (Fig. 1B). hage activation by polysaccharides ine whether polysaccharides purified from the liquid th of C. militaris were able to stimulate the functional f macrophages, macrophage-like RAW264.7 cells were ith 1000g/ml of each polysaccharide and NO pro- s measured and compared to the amount produced eated control group. Polysaccharide-treated cells pro- r amounts of NO than untreated cells, and CPSN Fr II roduction of the most NO among the polysaccharides o address whether CPSN Fr II elicits innate immune n macrophages, RT-PCR and ELISA assays were used induction of transcriptional gene upregulation and xpression of proinflammatory cytokines. These exper- wed that CPSN Fr II strongly triggers the expression matory cytokines TNF- and interleukin-1 (IL-1) C). al properties and monosaccharide composition l sugar content of CPSN Fr II was 92.45%. Its major sugar s are mannose (65.12%), galactose (28.72%) and glucose le 2 and Fig. S1). The contents of proteins, hexosamine acid of this polysaccharide are 0.20%, 0.06% and 0.29%, (Table 2). J.S. Lee et al. / Carbohydrate Polymers 82 (2010) 982–988 985 Fig. 1. Isolationandpurificationofpolysaccharides extracted fromthe liquid culture broth of C. militaris. (A) Ion exchange chromatogram of the crude polysaccharides, CPS, on a DEAE cellulose column. (B) Gel filtration chromatogram of the neutral polysaccharide fraction, CPSN, on a Sepharose CL-6B column (fraction number of ion exchange chromatography: 14–28). 3.4. Homogeneity and molecular weight The homogeneity of CPSN Fr II was confirmed by refractiona- tion throughgel filtration chromatographyusing a SepharoseCL-6B packed column (Fig. 3A). The molecular weight of this fraction was then determined by gel filtration chromatography to be 36kDa using dextrans as standards (Fig. 3B). 3.5. Identification of helix–coil transition A shift in the visible absorption maximum of congo red is induced by the presence of polysaccharides and can thus be used to provide conformational information. The absorption maximum of dextran, which has a random coil conformation, was around 450nm (Fig. 4). Curdlan exhibits a triple-helical conformation, which was demonstrated by the shift in the absorption maximum at 0.24M NaOH. However, the absorption maximum of laminarin, which has a different triple-helical conformation, was around 560nm. Based on this analysis, CPSN Fr II was found to exhibit a random coil conformation similar to that of dextran. 3.6. Identification of anomeric configuration To ascertain the presence or absence of the  or  configuration in CPSN Fr II, the Fungi-Fluor Kit was used. The Fungi-Fluor staining solution, cellufluor, binds nonspecifically to -linked polysaccha- rides, thus enabling their rapid detection. While dextra