ZENG Yangyang, HAN Zhangrun, YU Guangli, HAO Jiejie, and ZHANG Lijuan



Polysaccharides Purified from Wild Cordyceps Activate FGF2/FGFR1c Signaling

ZENG Yangyang, HAN Zhangrun, YU Guangli, HAO Jiejie*, and ZHANG Lijuan*

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Land animals as well as all organisms in ocean synthesize sulfated polysaccharides. Fungi split from animals about 1.5 billion years ago. As fungi make the evolutionary journey from ocean to land, the biggest changes in their living environment may be a sharp decrease in salt concentration. It is established that sulfated polysaccharides interact with hundreds of signaling molecules and facilitate many signaling transduction pathways, including fibroblast growth factor (FGF) and FGF receptor signaling pathway. The disappearance of sulfated polysaccharides in fungi and plants on land might indicate that polysaccharides without sulfation might be sufficient in facilitating protein ligand/receptor interactions in low salinity land. Recently, it was reported that plants on land start to synthesize sulfated polysaccharides in high salt environment, suggesting that fungi might be able to do the same when exposed in such environment. Interestingly, Cordyceps, a fungus habituating inside caterpillar body, is the most valued traditional Chinese Medicine. One of the important pharmaceutical active ingredients in Cordyceps is polysaccharides. Therefore, we hypothesize that the salty environment inside caterpillar body might allow the fungi to synthesize sulfated polysaccharides. To test the hypothesis, we isolated polysaccharides from both lava and sporophore of wild Cordyceps and also fromcultured without or with added salts. We then measured the polysaccharide activity using a FGF2/FGFR1c signaling-dependent BaF3 cell proliferation assay and found that polysaccharides isolated from wild Cordyceps activated FGF2/FGFR signaling, indicating that the polysaccharides synthesized by wild Cordyceps are indeed different from those by the cultured mycelium.

Polysaccharide; Cordyceps;; FGF; BaF3

1 Introduction

Cordyceps, a fungus growing out of the head of a mummified caterpillar, has been extensively used as the most valued traditional Chinese medicine and nutraceuticals not only in China but also in many countries of the world. The polysaccharides of Cordycepsare considered to be one of the major pharmaceutical active components with anti-inflammatory (Won, 2005), anti-oxidative (Wang, 2012), anti-viral (Ohta, 2007), immunomodulatory (Song, 1998), hypoglycemic (Zhang, 2006), antitumor (Lin, 2008) and anti-angiogenic (Yoo, 2004) activities.Increased demand has made Cordyceps an endangered species (Cleaver, 2004; Hsu, 2002; Winkler, 2010). Many companies useortocultivate Cordyceps to meet the increased demand. However, unlike the molecular structure-defined pharmaceutical active ingredients in Cordyceps, such as cordycepin, mannitol, and ergosterols, the polysaccharide structures and biological functions from cultured mycelium may not be the same as that in wild Cordyceps since polysaccharides biosynthesis is very sensitive to environmental conditions, especially to high salt environment (Aquino, 2011).

Land animals as well as all living organisms in ocean synthesize sulfated polysaccharides. Fungi split from animals about 1.5 billion years ago (James, 2006). As early fungi (Zheng, 2011) make the evolutionary journey from ocean to land and branch off from animals, the biggest changes in their living environment may be a sharp decrease in salt concentration. It has been established that sulfated polysaccharides interact with hundreds of signaling molecules (Zhang, 2010), such as growth factors, chemokines, and cytokines, and facilitate many important signaling transduction pathways, including fibroblast growth factor (FGF) and FGF receptor (FGFR) pathway (Itoh, 2011) . The disappearance of sulfated polysaccharides in fungi and plants in land might indicate that polysaccharides without sulfation might be sufficient in facilitating protein ligand/receptor interactions in low salinity land. The presence of sulfated polysaccharides in plants in an adaptation to high salt environments was recently reported(Aquino, 2010). Therefore, we hypothesize that the high salt environment inside caterpillar bodies might allow fungi to synthesize sulfated polysaccharides and by adding salt toculturemight make the culturedsynthesize sulfated polysaccharides as well.

To test the hypothesis, we first isolated 10 polysaccharides from both lava and sporophore of wild Cordyceps. We then isolated 26 different kinds of polysaccharides fromcultured without or with different amount of added salts.An established FGF/FGFR signaling-dependent BaF3 cell proliferation assay was then employed to detect the biological activity of the isolated polysaccharides (Pan, 2010).

FGF/FGFR signaling plays a crucial role in animal development and homeostasis including embryonic development (Slack, 1987), angiogenesis (Cross, 2001), tissue regeneration (Kawai, 2000), bone regeneration (Canalis, 1988), development and maintenance of the nervous system (Timmer, 2007), stem cell self-renewal (Levenstein, 2006), and wound healing (Barrientos, 2008). More importantly, sulfated polysaccharides are required to facilitate FGF/ FGFR signaling at both cell (Yayon, 1991; Rapraeger, 1991) and tissue levels (Ornitz, 2000). To take advantage of such a property,Ornitz. transfected a lymphocyte cell line, BaF3, with FGFR1c cDNA to make a stable cell line (Ornitz, 1992). The BaF3 cells make very little sulfated polysaccharides (Zhang, 2006)and normally depend on interleukin 3 (IL-3) for growth. FGFR1c expressing BaF3 cells only proliferate when both FGF and polysaccharide are added to the growth media in the absence of IL-3 (Fig.1). By using this assay, we discovered that polysaccharides isolated from sporophore of wild Cordyceps activates FGF/FGFR signaling, indicating that the polysaccharides synthesized by wild Cordyceps are different from that of the cultured mycelium.

Fig.1 Principle of FGF and polysaccharide-dependent BaF3 FR1c cell proliferation assay.

2 Materials and Methods

2.1 Materials

, strain 11Y-6, was a generous gift from fungal specialist, Prof. X. L. Jiang (Ocean University of China, Qingdao, China). The wild Cordyceps were purchased from a reliable dealer in Qingdao, China. 1640 medium was obtained from Sigma-Aldrich (USA). Fetal bovine serum was purchased from GIBICO (USA). Fibroblast growth factor 2 (FGF2), IL-3 and G418 were purchased from Goldbio (USA). Heparin, resazurin and β-mercaptoethanol were purchased from Sigma (USA). Hyaluronic acid (HA) was purchased from Fluka (USA).

2.2 Liquid Culture of.

.was maintained on potato dextrose agar (PDA) slants and inoculated at the center of a Petri dish. The mycelium was fermentated in 200mL medium containing 6gL−1potato dextrose (PD) and 20gL−1glucose with 0.05mgL−1-500mgL−1Na2SO4, at 27℃ for 10d (130rmin−1). The mycelia and culture medium were rapidly concentrated by rotary evaporation to 100 mL and extracted with 400mL 95% ethanol at 80℃ 3times to remove lipids. The de-lipided power was then used for polysaccharide extraction.

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2.3 Extraction of Polysaccharides from Wild Cordyceps and Cultured Mycelia

The Cordyceps was divided into larva and sporophore. The larva and sporophore were dried at 40℃ and then pulverized. The pulverized powders were extracted three times with 95% ethanol at 80℃ to obtain defatted powders from larva and sporophore, respectively. The enzymatic extraction method added a step of pronase K digestion of de-lipided starting materials. Briefly, the de-lipided powders from wild Cordyceps and cultured mycelia were digested by pronase K solution overnight at 50℃ and then heated at 100℃ to inactivate the enzyme. The polysaccharide-containing supernatants were collected after centrifugation at 4500rmin−1for 4min and then con- centrated by rotary-evaporation. The concentrated solutions were precipitated by adding 4 volumes of ethanol. The polysaccharide-containing precipitates were then dried. The precipitates were re-dissolved in distilled water at 80℃ for 3h and extracted with distilled water 3 more times at 80℃ allowing all polysaccharides to thoroughly dissolve. The polysaccharide-containing supernatants were precipitated again by adding 4 volumes of ethanol. The polysaccharide-containing precipitates were soaked in 2% Na2CO3or 1% NaOH solution to remove covalently linked protein and then extracted three times at 60℃ for 3h with distilled water. The collected polysaccharide-containing supernatants were neutralized, dialyzed, and precipitated with 4 volumes of ethanol. The precipitated polysaccharides were then dissolved in water and dialyzed against water using dialysis bags with 3000 Da molecular weight cut off. After thorough dialysis, each sample was dried, weighed, and used for further tests.

2.4 Fourier Transformed Infrared Spectrometry (FTIR) Analysis

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Samples (1-2mg) were dried in a P2O5desiccator for 48h, mixed with 100mg KBr, and pressed under 7kgcm−2to make transparent films, followed by placing them in an FTIR instrument (Nicolet Nexus 470, Thermo Electron, USA) and scanning from 400 to 4 000cm−1to obtain the infrared spectrum.

2.51H-NMR Analysis

The polysaccharide sample was dissolved in 500mL D2O and freeze-dried twice to replace all exchangeable protons with deuterium. The1H NMR spectrum was recorded on a Bruker DPX 400 spectrometer; acetone (the signals at 2.09ppm) was used as an external reference.

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