A novel linear 3-O-methylated galactan isolated from Cantharellus cibarius activates macrophages

Highlights

• A novel linear 3-O-methylated galactan (WCCP-N-b) from C. cibarius was identified.

• WCCP-N-b increased phagocytosis and secretion of NO, TNF-α, IL-6 and IL-1β.

• WCCP-N-b activated macrophages through Akt/NF-κB, MAPKs via TLR2 receptor.

• The molecular weight of WCCP-N-b impacted the activation.

Abstract

A novel polysaccharide (WCCP-N-b) with a molecular weight of 18 kDa was isolated and purified from the fruiting bodies of Cantharellus cibarius. Monosaccharide composition, methylation analysis and NMR spectra indicated that WCCP-N-b was a linear α-1,6-galactan, partially methylated at O-3 of galactose. The molar ratio of Gal, 3-methylated-Gal, Glc and Man was 14.4:4.6:1.0:1.2. WCCP-N-b could significantly increase macrophage phagocytosis, release of NO and secretion of TNF-α, IL-6 and IL-1β. On a cellular mechanistic level, WCCP-N-b activated MAPKs and NF-κB signaling pathway via Toll-like receptor 2 (TLR2). To further elucidate the structure-function relationship, WCCP-N-b was hydrolyzed by acid. Four degraded fragments were obtained, with molecular weights of 16.1 kDa, 11.2 kDa, 5 kDa and 3.5 kDa, respectively. Their macrophage activation effects were significantly decreased along with the molecular weight decrease. Collectively, WCCP-N-b could activate RAW264.7 cells, and the activation effect was related to its molecular weight.

Introduction

Macrophages are important components of the innate immune system, playing a crucial role in host defense against infection trough immune-inflammatory responses, recognition of pathogens and phagocytosis. In addition, macrophages also exert an important role as an interface between innate and adaptive immunity (Van den Bossche, O’Neill, & Menon, 2017). Hence, macrophages are thought to be the important target cells of some antitumor and immunomodulatory drugs. Many mushroom sugars that can bind to pattern recognition receptors (PRRs) such as TLR2, TLR4, Dectin-1 (Gordon, 2002) and stimulate immune system, which present on the surface of macrophage cells, and then activate MAPKs and NF-κB signaling pathway, induce the activation of immune response (Takeuchi & Akira, 2010).

Mushrooms are used as healthy food in the world as they are rich in a great variety of bioactive components, such as proteins (Zhang et al., 2014), lipopolysaccharides (Wasser, 2011), glycoproteins (Cui et al., 2013) and polysaccharides (Meng, Cheng, Han, Chen, & Wang, 2017). Among the bioactive compounds, polysaccharides are the most abundant and extensively studied. Polysaccharides from mushrooms possess antioxidant activity (Nowacka et al., 2014), antitumor activity (Wasser, 2002) and immunoregulatory activity (Meng et al., 2018). These biologically active mushroom polysaccharides are mostly glucans (Liana Inara de Jesusa, Cordeiroa, de Freitasb, Van Griensvenc, & Iacominia, 2018) and galactans (Rosado et al., 2003) The main chains of the galactans are composed of (1→6)-linked α-d-Galp units, which are partially methylated at O-3 or substituted at C-2 by l-fucopyranosyl, d-mannopyranosyl, d-galactopyranosyl or 3-O-α-d-mannopyranosyl-α-l-fucopyranosyl residues as side chains (Rosado et al., 2003; Ruthes, Smiderle, & Iacomini, 2016) have been reported, possess immunoregulatory activity.

Cantharellus cibarius (C. cibarius, the golden chanterelle) is an edible mushroom which widely distributes in Asia, America and some European countries. It belongs to the phylum Basidiomycota, family Cantharel-laceae. It is not only consumed as health-supplementary food, but also considered as adjuvant in improving eyesight and strengthening both spleen and stomach functions in traditional Chinese medicines (Han et al., 2013; Wang, Ngai, & Ng, 2003). Polysaccharides, such as mannans (Nyman, Aachmann, Rise, Ballance, & Samuelsen, 2016) and glucans (Villares, García-Lafuente, Guillamón, & Mateo-Vivaracho, 2013; Zhao et al., 2018) have been purified from C. cibarius fruit bodies. However, few studies about galactans from C. cibarius have been reported. In this paper, a novel galactan was isolated and purified from the fruit bodies of C. cibarius. The present study is aimed to study the structure of this galactan, evaluate its immunostimulatory activity on RAW264.7 cells, investigate the molecular mechanism responsible for its immunostimulatory activity, and illuminate its structure-activity relationship.