Polysaccharide-protein Complexes Research Articles

Current findings, future trends, and unsolved problems in studies of medicinal mushrooms

The target of the present review is to draw attention to many critically important unsolved problems in the future development of medicinal mushroom science in the twenty-first century. Special attention is paid to mushroom polysaccharides. Many, if not all, higher Basidiomycetes mushrooms contain biologically active polysaccharides in fruit bodies, cultured mycelium, and cultured broth. The data on mushroom polysaccharides are summarized for approximately 700 species of higher Hetero- and Homobasidiomycetes. The chemical structure of polysaccharides and its connection to antitumor activity, including possible ways of chemical modification, experimental testing and clinical use of antitumor or immunostimulating polysaccharides, and possible mechanisms of their biological action, are discussed. Numerous bioactive polysaccharides or polysaccharide-protein complexes from medicinal mushrooms are described that appear to enhance innate and cell-mediated immune responses and exhibit antitumor activities in animals and humans. Stimulation of host immune defense systems by bioactive polymers from medicinal mushrooms has significant effects on the maturation, differentiation, and proliferation of many kinds of immune cells in the host. Many of these mushroom polymers were reported previously to have immunotherapeutic properties by facilitating growth inhibition and destruction of tumor cells. While the mechanism of their antitumor actions is still not completely understood, stimulation and modulation of key host immune responses by these mushroom polymers appears central. Particularly and most importantly for modern medicine are polysaccharides with antitumor and immunostimulating properties. Several of the mushroom polysaccharide compounds have proceeded through phases I, II, and III clinical trials and are used extensively and successfully in Asia to treat various cancers and other diseases. A total of 126 medicinal functions are thought to be produced by medicinal mushrooms and fungi including antitumor, immunomodulating, antioxidant, radical scavenging, cardiovascular, antihypercholesterolemia, antiviral, antibacterial, antiparasitic, antifungal, detoxification, hepatoprotective, and antidiabetic effects.

Fractionation, partial characterization and bioactivity of water-soluble polysaccharides and polysaccharide–protein complexes from Pleurotus geesteranus

Fractionation and purification of mushroom polysaccharides is a critical process for mushroom clinical application. After a hot-water treatment, the crude Pleurotus geesteranus (PG) was further fractionated into four fractions (PG-1, -2, -3, -4) using gradient precipitation with water and ammonia sulphate. By controlling the initial polymer concentration and ratio of solvents, this process produced PG fractions with high chemical uniformity and narrow M w distribution without free proteins. Structurally, PG-1 and PG-2 are pure homopolysaccharide mainly composed of glucose; and PG-3 and PG-4 are heteropolysaccharide–protein complexes. PG-2, a high M w fraction mainly composed of glucose presented significant cytotoxicity at the concentration of 200 and 100 μg/ml to human breast cancer cells. Here, we report a new mushroom polysaccharides extraction and fractionation method, with which we produced four fractions of PG with PG-2 appearing effective anti-tumour activity.

Chemical modification and antitumor activities of two polysaccharide–protein complexes from Pleurotus tuber-regium

Two water-soluble polysaccharide-protein complexes, extracted from Pleurotus tuber-regium sclerotia, were modified chemically to obtain their sulfated and carboxymethylated derivatives. While C6 position of glucan was fully substituted, C2, C3, and C4 were only partially substituted by sulfate groups. C3, C4, and C6 position of glucan were partially substituted during the carboxymethylation. Chain conformation and antitumor activity of the native samples and their derivatives were studied. The native samples and derivatives existed in sphere conformation, and showed potent in vitro antitumor activities. Water-solubility and introduction of ionic groups played important roles in enhancing the antitumor activities of the polysaccharide–protein complexes.

Extracellular polysaccharide-protein complexes of a harmful alga mediate the allelopathic control it exerts within the phytoplankton community

The goal of this study was to examine the significance of allelopathy by the raphidophyte Heterosigma akashiwo in a multispecies phytoplankton community in the field. Towards this aim, we sought allelochemicals of H. akashiwo, which had allelopathic effect both in laboratory experiments and in the field. As an initial step, we showed that the allelopathic effects of H. akashiwo filtrate were both species-specific and dependent upon the cell density of the target species. Secondly, we found for the first time that extracellular, high-molecular weight allelochemicals [that is, polysaccharide-protein complexes (APPCs)] were produced by a marine phytoplankton species, H. akashiwo. Thirdly, we indicated that the purified APPCs selectively inhibited the growth of the diatom Skeletonema costatum that is a major competitor of H. akashiwo, and thereby tended to promote the formation of monospecific H. akashiwo blooms. Furthermore, we demonstrated that the inhibitory effect of APPCs on the growth of the diatoms was determined by binding to the cell surface of the target species. Finally, we succeeded in the detection of APPCs in the field samples at concentrations exceeding their experimentally determined action threshold during the H. akashiwo bloom. Strategies for ecosystem control, including mitigation of harmful algal blooms (HABs), should take into account that red-tide organisms like H. akashiwo are already part of complex webs involving inter-specific allelopathic inhibition and ecosystem control during their dense blooms.

Chemical properties and antioxidant activity of exopolysaccharides from mycelial culture of Cordyceps sinensis fungus Cs-HK1

Exopolysaccharide (EPS) was produced in a mycelial liquid culture of the Cordyceps sinensis fungus, Cs-HK1, at a rate proportional to mycelial growth. The crude EPS isolated from the culture medium by ethanol precipitation contained 65–70% sugar and about 25% protein, suggesting that the EPS was composed of polysaccharide–protein complexes. It exhibited a wide molecular weight (MW) range from about 5 kDa to more than 200 kDa. The intrinsic viscosity as an index of average MW of EPS, increased during the period of rapid mycelial growth to 11.0 dL/g on day 5, and remained relatively stable between day 5 to day 8 (stationary growth period). Both infrared spectrometry of EPS and gas chromatography of acetylated EPS suggested that the EPS had a β- d-glucan backbone. The EPS showed moderate antioxidant activities with a Trolox equivalent antioxidant capacity of 35–40 μmol Trolox/g and a ferric reducing ability of plasma of 50–52 μmol Fe(II)/g.

Characterization of polysaccharide–protein complexes by size-exclusion chromatography combined with three detectors

Two water-soluble samples (TM1 and TM2) were extracted from Pleurotus tuber-regium using 0.9% aqueous NaCl at 20 and 80 °C to obtain relatively low molecular mass fractions. The chemical structure of TM1 was analyzed to be a branched heteropolysaccharide–protein complex, and the sugar moiety was mainly β-(1→6)-, β-(1→4)-, and β-(1→3)-linked glucan containing galactose and mannose. TM2 was a branched polysaccharide–protein complex, and the sugar moiety was mainly β-(1→6)-, β-(1→4)-, and β-(1→3)-linked glucan. Preparative size-exclusion chromatography (SEC) and analytical SEC combined with three detectors were used to detect the TM1 and TM2 samples, confirming that the proteins were bonded to the polysaccharides. Furthermore, analytical SEC combined with online laser light scattering, differential refractive index detector, and UV to determine the components, weight-average molecular mass ( M w) and chain conformation of the samples. The relatively low exponent values ( ν) of 〈 S 2 〉 z 1 / 2 = k ν M w ν for the samples in 0.15 M aqueous NaCl at 25 °C suggested that TM1 and TM2 existed in compact sphere conformation in the aqueous solution. This work provided valuable information on the structure and chain conformation characterization of the polysaccharide–protein complex having relatively low M w.

Effects of Lycium barbarum extract on production and immunomodulatory activity of the extracellular polysaccharopeptides from submerged fermentation culture of Coriolus versicolor

Polysaccharopeptides (PSPs) from Coriolus versicolor have been used as immunomodulatory and anticancer agents. However, most studies have concentrated on the mycelial PSPs and not those in the fermented broth. On the other hand, Lycium barbarum fruit has been used as a traditional Chinese herbal medicine for two millennia. Its extract contains various nutrients, minerals, and also polysaccharide–protein complexes, which are proven to be bioactive. Herein we report the effects of L. barbarum fruit extract on the mycelial growth and extracellular PSP (ePSP) production of C. versicolor LH1 by using a submerged fermentation process in 20l fermenters. Fermentation production of C. versicolor biomass and its ePSP were augmented in the presence of L. barbarum extract. The ePSP such obtained differs from those obtained with normal culture medium in terms of simple sugar composition and protein content but shows similar overall chemical structures as analyzed by Fourier transformed infrared spectroscopy. Moreover, the ePSP from C. versicolor cultured with supplementary L. barbarum extract exhibits significant immunomodulatory activity as judged by its effects on the production of nitric oxide and several cytokines by murine RAW264.7 macrophages.

Atomic force microscopy of tomato and sugar beet pectin molecules

Atomic force microscopy has been used to image the structure of pectin molecules isolated from unripe tomato and sugar beet tissue. The tomato pectin molecules were found to be extended stiff chains with a weight average contour length of L W = 174 nm and a number average contour length of L N = 132 nm ( L W/ L N = 1.32). A proportion of the pectin molecules (∼30%) were found to be branched structures. Chemical analysis of the sugar beet pectin extracts showed that the samples contained protein (8.6%). This protein proved difficult to remove and is believed to be covalently attached to the polysaccharide. Imaging of the extracted pectin revealed largely un-aggregated chains: a small fraction (33%) of which were extended stiff polysaccharide chains and a major fraction (67%) of which were of polysaccharide–protein complexes containing a single protein molecule attached to one end of the polysaccharide chains (‘tadpoles’). In addition the sample contained a small number of aggregated structures. The un-aggregated pectin molecules were found to be predominately linear structures with a small fraction (∼17%) of branched structures. The branched structures were all in the free polysaccharide fraction and no branched pectin chains were observed in the protein–polysaccharide complexes. Alkali treatment was found to remove the protein. For the alkali-treated, un-aggregated structures the average contour lengths were found to be L W = 137 nm, L N = 108 nm with L W/ L N = 1.27. It is proposed that the ‘tadpole’ structures contribute to the unusual emulsifying properties of sugar beet pectin.

Antitumor and immunomodulatory activities of medicinal mushroom polysaccharides and polysaccharide-protein complexes in animals and humans (Review)

Antitumor and immunomodulatory activities of medicinal mushroom polysaccharides and polysaccharide-protein complexes in animals and humans (Review)

Cell wall proteins of Kluyveromyces fragilis: Surface and emulsifying properties

Yeast cell wall proteins were extracted from homogenized suspensions with 0.75 mol/l NaOH, yielding after precipitation at isoelectric pH a pale-brown sediment. Lyophilized sample was fractionated on Sephadex G-50 to yield three fractions (Fr 1, Fr 2 and Fr 3). Fr 1, which had the highest yields and protein content, showed the highest molecular weight and best surface properties. Fr 2 and Fr 3 were mainly composed by polysaccharide-protein complexes. Fr 1 was further subfractionated on Sephacryl S-300 to produce three fractions (Fr A, Fr B and Fr C). All subfractions, turned out to be highly foamy during evaporation. The highest yields were obtained for Fr A, which also showed the highest molecular weight. Fractions Fr 1 and their subfractions Fr B and Fr C exhibited good surface activity and high emulsifying activity. Emulsions prepared with these fractions were the most stable against creaming and coalescence. Fr 2 cream phase presented a gel-like behavior as a consequence of polysaccharides acting as thickening agents.

Medicinal mushroom modulators of molecular targets as cancer therapeutics

Empirical approaches to discover anticancer drugs and cancer treatments have made limited progress in the past several decades in finding a cure for cancer. The expanded knowledge of the molecular basis of tumorigenesis and metastasis, together with the inherently vast structural diversity of natural compounds found in mushrooms, provided unique opportunities for discovering new drugs that rationally target the abnormal molecular and biochemical signals leading to cancer. This review focuses on mushroom low-molecular-weight secondary metabolites targeting processes such as apoptosis, angiogenesis, metastasis, cell cycle regulation, and signal transduction cascades. Also discussed in this review are high-molecular-weight polysaccharides or polysaccharide-protein complexes from mushrooms that appear to enhance innate and cell-mediated immune responses, exhibit antitumor activities in animals and humans, and demonstrate the anticancer properties of selenium compounds accumulated in mushrooms.

Immunomodulatory Activities of Mushroom Glucans and Polysaccharide-Protein Complexes in Animals and Humans (A Review)

Immunomodulatory Activities of Mushroom Glucans and Polysaccharide-Protein Complexes in Animals and Humans (A Review)

Presentation of a major histocompatibility complex class 1–binding peptide by monocyte-derived dendritic cells incorporating hydrophobized polysaccharide–truncated HER2 protein complex: implications for a polyvalent immuno-cell therapy

Recognition of the essential role of dendritic cells (DCs) as professional antigen-presenting cells has prompted investigators to search for methods to use DCs as natural adjuvants in immunotherapy. A number of antigenic oligopeptides, recognized by CD8(+) cytotoxic T lymphocytes (CTLs) specific for cancer cells, have been applied in clinical trials using DCs. Such a monovalent vaccine with a single epitope for a particular type of HLA class 1 molecule would be effective. However, a polyvalent vaccine might be more potent. We designed a novel protein delivery system consisting of hydrophobized polysaccharides complexed with target proteins. The truncated HER2 protein encompassing 147 N-terminal amino acids, including the 9-mer HER2p63-71 peptide (HER2p63), TYLPTNASL, the human homologue of an antigenic murine tumor rejection peptide, was prepared. We report here that HLA-A2402(+) DCs could incorporate hydrophobized polysaccharide-truncated HER2 protein complexes and process the protein to present major histocompatibility complex class 1-binding HER2p63 peptide. The complexes enter DCs by phagocytosis, and then the truncated protein is processed through a pathway similar to that for endogenous proteins. DCs sensitized by these complexes primed and boosted HER2p63-specific CD8(+) T cells in the context of HLA-A2402. Vaccination with DCs incorporating these complexes completely suppressed lung metastases in a HER2-expressing murine tumor model. We also generated 3 CD4(+) clones reactive with different HER2- derived 25-mer peptides from lymph node cells in mice treated with CHP/HER2-147. Thus, hydrophobized polysaccharide-protein complexes are promising candidates for the construction of polyvalent vaccines.

An Improved Assay for the N-Acetyl-[formula omitted]-glucosamine Reducing Ends of Polysaccharides in the Presence of Proteins

Hyaluronan concentration and hyaluronidase activity can be assayed by using different techniques including turbidimetry, viscosimetry, ELISA, chromatography, and colorimetry. The most popular colorimetric method is that of J. Reissig et al. (1955, J. Biol. Chem. 217, 959–966), in which the color results from a reaction between the Ehrlich's reagent (DMAB) and the N-acetyl-d-glucosamine reducing end of each hyaluronan chain. Nevertheless, there are problems with this method when proteins are present in the medium. Here we propose a new interpretation of the Reissig signal for estimating such reducing ends in media containing enzymes or other proteins. This interpretation is based on the fact that the absorbance obtained by using the Reissig method results from two factors: a turbidity due to the formation of polysaccharide–protein complexes and a color resulting from the action of DMAB on the reducing end of the polysaccharide chains. The turbidity at 585 nm, the wavelength at which the color intensity is maximal, may be estimated by curve fitting the spectrum between 450 and 650 nm. Subtracting the turbidity from the absorbance gives the colorimetric intensity which represents the concentration of polysaccharide chains. Moreover, the turbidity may give additional information about the existence of polysaccharide–protein complexes and their nature.

Immunomodulation and anti-cancer activity of polysaccharide-protein complexes.

In the last three decades, numerous polysaccharides and polysaccharide-protein complexes have been isolated from mushrooms and used as a source of therapeutic agents. The most promising biopharmacological activities of these biopolymers are their immunomodulation and anti-cancer effects. They are mainly present as glucans with different types of glycosidic linkages such as (1-->3), (1-->6)-beta-glucans and (1-->3)-alpha-glucans, and as true herteroglycans, while others mostly bind to protein residues as polysaccharide-protein complexes. Three antitumor mushroom polysaccharides, i.e. lentinan, schizophyllan and protein-bound polysaccharide (PSK, Krestin), isolated respectively, from Lentinus edodes, Schizophyllum commune and Coriolus versicolor, have become large market items in Japan. Lentinan and schizophyllan are pure beta-glucans, whereas PSK is a protein-bound beta-glucan. A polysaccharide peptide (PSP), isolated from a strain of Coriolus versicolor in China, has also been widely used as an anti-cancer and immunomodulatory agent. Although the mechansim of their antitumor action is still not completely clear, these polysaccharides and polysaccharide-protein complexes are suggested to enhance cell-mediated immune responses in vivo and in vitro and act as biological response modifiers. Potentiation of the host defense system may result in the activation of many kinds of immune cells that are vitally important for the maintenance of homeostasis. Polysaccharides or polysaccharide-protein complexes are considered as multi-cytokine inducers that are able to induce gene expression of vaious immunomodulatory cytokines and cytokine receptors. Some interesting studies focus on investigation of the relationship between their structure and antitumor activity, elucidation of their antitumor mechanism at the molecular level, and improvement of their various biological activities by chemical modifications.

Development, properties and clinical use of fungous allergens

The diagnostic allergens from yeast-like and mold fungi of 18 names are developed and studied. The drugs are polysaccharide protein complexes, they do not contain toxic components and conservants. They are used for the diagnosis of sensibilization in vivo and in vitro as well as for immunotherapy.

Development, properties and clinical use of fungous allergens

The diagnostic allergens from yeast-like and mold fungi of 18 names are developed and studied. The drugs are polysaccharide protein complexes, they do not contain toxic components and conservants. They are used for the diagnosis of sensibilization in vivo and in vitro as well as for immunotherapy.

Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide-protein complexes

Two antitumor polysaccharide-protein complexes, PSPC and PSK from mushrooms, were compared for their modulating effect on cytokine and cytokine receptor gene expression. RNA samples were isolated from the splenocytes and peritoneal exudate cells of the untreated or treated mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the cytokine gene expression. Nine out of 17 cytokine mRNAs and 5 out of 6 cytokine receptor mRNAs were detected in the splenocytes and peritoneal exudate cells from both untreated and treated mice. However, IL-4 was only detected in the splenocytes while IL-7 and IL-1R(typeI) were only detected in the peritoneal exudate cells. Among the 9 cytokine genes, the expression level of M-CSF was up-regulated in splenocytes and peritoneal exudate cells of the mice by PSPC and PSK. The expression level of TNF-ga was only up-regulated in the peritoneal exudate cells by PSK, but not by PSPC.

Efficiency of crude and purified fungal antigens in serodiagnosis to discriminate mycotic from other respiratory diseases

Mycotic immunodiagnosis was performed in 186 hospitalized patients with different respiratory diseases, mostly considered as tuberculosis and others with a doubtful diagnosis. Crude histoplasmin, coccidioidin, paracoccidioidin, blastomycin, candidin, aspergillin, and sporotrichin, as well as purified polysaccharide-protein complexes (PPC) of Histoplasma capsulatum, Coccidioides immitis, and Paracoccidioides brasiliensis were used as antigens. Immune tests used included skin test (ST), gel immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF), and ELISA. A possible association with candidosis was observed in 17% of patients with tuberculosis and diabetes; one presumptive paracoccidioidomycosis, one confirmed aspergillosis, and six cases of active histoplasmosis were determined. Candidin ST showed 29% of positive reactions with an increased frequency in patients between 31 and 55 years of age. CF test showed the highest positivity percentages with crude antigens, specially for Candida antigen (26.3%) and histoplasmin (18.2%). Cross reactions were evident with crude antigens but decreased when PPC's were used in ELISA.

Cinephotomicrographic and Histochemical Observations on Cercarial Penetration and Encystment by Plagiorchis sp. in Larvae of Chaoborus sp.

Plagiorchis sp. cercariae were exposed to Chaoborus sp. larvae and filmed using 16 mm photocinemicrography. After contacting the host, cercariae crawled leechlike, constantly probing with their stylet (from this point until encystment the tail may be lost). Upon contacting an intersegmental membrane, stylet activity increased. The cercarial stage pushed inward on this membrane until it was entirely surrounded by it. The stylet moved generally in a 90? arc about every two seconds and cut through the membrane in approximately 2.45-10 minutes. Passage of the cercariae through the membrane averaged 66 seconds. Once within the hemocoel, the cercariae moved about until encystment. Initially, the cyst wall was elastic, flexible, and easily distorted by the rotation of the metacercaria. The initial 11 mean rotations per minute slowed over a 6-10 minutes period and then became irregular. At this time, a two-layered cyst wall of parasite origin was observed. Histochemically, polysaccharides, polysaccharide-protein complexes, and acid mucosubstances were observed in the ventral, dorsal, cystogenous, and penetration glands. The inner cyst wall originated from ventral and dorsal gland secretions of the cercariae and contained polysaccharides and proteins. The outer layer, from cystogenous glands, consisted mainly of acid mucosubstances and proteins. The third, host-produced layer, was formed from humoral elements and disintegrating hemocytes, and stained positive mainly for proteins. The tail contained high concentrations of glycogen. Numerous specimens Lymnaea stagnalis L. collected from the Stevens Point Area High School (SPASH) marsh, Stevens Point, Wisconsin harbored large numbers of xiphidiocercariae belonging to the genus Plagiorchis as described by McMullen (1937). These cercariae readily penetrated and encysted in Chaoborus sp. larvae in the laboratory. No cinematography but some histochemical studies of Plagiorchis (see Bock, 1988; LeFlore, 1979; Zdarska', 1969) have been published. Additional information relating to host penetration, encystment, and histochemical studies are presented here. MATERIALS AND METHODS Larvae of Chaoborus sp. were collected from Escanaba Lake, Vilas Co., Wisconsin June 1987 and 1988. Individuals of Lymnaea stagnalis were collected from a marsh on the SPASH grounds, Stevens Point, Wisconsin 1 July-15 August in 1987 and 1988. Both were refrigerated at 4?C until needed. Plagiorchis sp. cercariae dissected from snails placed with Chaoborus sp. larvae on microscope slides in a small amount of water and covered with a no. I thank Drs. William LeGrande and Stanley Szczytko for critically reading the manuscript, S. Szczytko for collecting the Chaoborus larvae, Tammy Oatman for printing some of the photomicrographs, and Judy Ratkowski for typing the manuscript. This research was supported in part by a grant from the University of Wisconsin-Stevens Point Personnel Development Committee. TRANS. AM. MICROSC. SOC., 109(2): 160-167. 1990. ? Copyright, 1990, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.17 on Fri, 02 Sep 2016 04:54:16 UTC All use subject to http://about.jstor.org/terms VOL. 109, NO. 2, APRIL 1990 1.5, 24 x 40-mm coverglass. A 16 mm Bolex movie camera mounted on a Zeiss Universal microscope was used to photograph cercarial penetration and encystment at 10 frames/sec. For histochemical studies, Chaoborus sp. larvae were exposed to Plagiorchis sp. cercariae and fixed 30-90 min postexposure either in Gendres fixative or 10% buffered formalin, depending upon the subsequent staining procedures. Gendre-fixed specimens were dehydrated, embedded in Paraplast and sectioned at 5-8 um (Humason, 1979). Five-,um sections were stained with mercuric bromphenol blue (HgBPB) (Mazia et al., 1953), a general protein stain. Eightujm sections were stained with PAS and counterstained with fast green for polysaccharides and polysaccharide-protein complexes; alcian blue pH 2.5 (AB2.5) and AB-PAS were used to stain for mucosubstances (Humason, 1979; Pearse, 1960). Sections, both treated and untreated with salivary amylase, were stained with PAS to check for glycogen. Aldehyde-fuchsin (Cameron & Steele, 1959) was used as a general morphological stain and to determine if hemocytes were present. Isolated cercariae fixed with 10% neutral formalin were frozen and sectioned at 16 um on a cryostat. Sections were stained with oil red 0 (ORO) and mounted with Aquamount (Humason, 1979). All measurements are given in micrometers.