Harry J. Wichers

Crystal Structure of Agaricus Bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone.

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ∼392 residues and two L subunits of ∼150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ∼100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

OLEUROPEIN AND RELATED COMPOUNDS

In this paper, oleuropein and some other related phenolic compounds are reviewed. Their occurrence, distribution, biosynthesis and transformation during maturation and during industrial processing (preparation of table olives and oil production) are described. Their role in human health is proposed based on current human, animal and in vitro studies as molecules with antioxidant and antimicrobial properties.

Antiinflammatory and Immunomodulating Properties of Fungal Metabolites

We discuss current information on the ability of extracts and isolated metabolites from mushrooms to modulate immune responses. This can result in a more enhanced innate and acquired disease resistance. The major immunomodulating effects of these active substances derived from mushrooms include mitogenicity and activation of immune effector cells, such as lymphocytes, macrophages, and natural killer cells, resulting in the production of cytokines, including interleukins (ILs), tumor necrosis factor alpha (TNF)-α, and interferon gamma (INF)-γ. In particular, the ability of selective mushroom extracts to modulate the differentiation capacity of CD4+ T cells to mature into TH1 and/or TH2 subsets will be discussed. As a consequence these extracts will have profound effects in particular diseases, like chronic autoimmune TH1-mediated or allergic TH2-mediated diseases. Immunosuppressive effects by mushroom components have also been observed. The therapeutic effects of mushrooms, such as anticancer activity, suppression of autoimmune diseases, and allergy have been associated with their immunomodulating effects. However, further studies are needed to determine the molecular mechanisms of the immunomodulating effects of mushrooms metabolites both individually and in complex mixtures, for example, extracts.

An easy and fast test to compare total free radical scavenger capacity of foodstuffs

An easy and fast test has been designed to compare the total free radical scavenging capacity (RSC) of various food samples. Black and green teas from different countries, and wines of different brands were studied and compared as examples of coloured liquids (water- and methanol-soluble); oils from different sources were used as examples of lipidic foodstuffs; apples of different varieties and spinach were analysed as solid foods. Dilutions of extracts of the described foodstuffs were prepared and aliquots of each dilution were spotted onto TLC silica gel layers in the form of a dot-blot test: layers were stained with a methanolic solution of the 2,2-diphenyl-1-picrylhydrazyl radical. Dots of extracts of foodstuffs with RSC turned yellow, with a colour intensity depending on the RSC compounds present in the dilutions. After 1 h staining, the intensity of the yellow colour was measured with a chromameter (b* parameter) at one of the dilutions at which the colour value was linearly correlated to the concentration of the sample. According to these readings the different samples were organised in decreasing order of b* values, an order which corresponded to the decreasing order of RSC as determined by spectrophotometric methods. The dot-blot test was sensitive enough to detect differences of RSC between varieties and brands of water- or methanolsoluble products, but was not adequate for lipid-based compounds. The test was also able to follow the variation of RSC during food processing as in, for example, the heat-treatment of spinach. Copyright (C) 2000 John Wiley and Sons, Ltd.

THP-1 cell line: An in vitro cell model for immune modulation approach

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions.

Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds

An assay was developed to study inflammation-related immune responses of food compounds on monocytes and macrophages derived from THP-1 cell line. First strategy focused on the effects after stimulation with either lipopolysaccharide (LPS) or Concanavalin A (ConA). Gene expression kinetics of inflammation-related cytokines (IL-1β, IL-6, IL-8, IL-10 and TNF-α), inflammation-related enzymes (iNOS and COX-2), and transcription factors (NF-κB, AP-1 and SP-1) were analyzed using RT-PCR. Time dependent cytokine secretion was investigated to study the inflammation-related responses at protein level. LPS stimulation induced inflammation-related cytokine, COX-2 and NF-κB genes of THP-1 monocytes and THP-1 macrophages with the maximum up-regulation at 3 and 6 h, respectively. These time points, were subsequently selected to investigate inflammation modulating activity of three well known immuno-modulating food-derived compounds; quercetin, citrus pectin and barley glucan. Co-stimulation of LPS with either quercetin, citrus pectin, or barley glucan in THP-1 monocytes and macrophages showed different immuno-modulatory activity of these compounds. Therefore, we propose that simultaneously exposing THP-1 cells to LPS and food compounds, combined with gene expression response analysis are a promising in vitro screening tool to select, in a limited time frame, food compounds for inflammation modulating effects.

Food allergens of plant origin: their molecular and evolutionary relationships

Abstract Along with other forms of allergic disease, food allergies appear to be on the increase, with childhood allergies to foods such as peanuts being of particular concern. Around 7–10 foods are responsible for the majority of allergies, including several of plant origin, notably peanut. Allergies are usually triggered by the protein components in a food, which are also known as allergens. However, not all the proteins in an allergenic food like peanut are allergens. Why should this be? This question has been addressed by an EU-funded inter-disciplinary network of clinicians, food chemists and plant biochemists called Protall. From the groups considerations it is clear that, whilst the abundance of a protein in a food is one factor involved in determining its allergenic potential, this is not sufficient on its own to predict its allergenicity. Through an analysis of common properties of plant food allergens that trigger a reaction via the gastrointestinal tract it has become evident that they belong almost exclusively to three structurally related protein superfamilies—the prolamin superfamily (which includes the prolamin storage proteins of cereals, non specific lipid transfer proteins, α-amylase inhibitors, and 2S albumins), the cupin superfamily (specifically the 11S and 7S globulin storage proteins) and the cysteine proteases. Whilst we cannot as yet predict the allergenicity of a given protein, such an understanding of the structural attributes of proteins that predispose them to becoming allergens is important if we are to understand what makes some foods more allergenic than others. It is also highly relevant to developing more reliable means of assessing the allergenic risks posed by novel foods, developing effective processing strategies for reducing allergen loads in foods and developing novel therapeutic options as an alternative to dietary exclusion.

Novel peptides with tyrosinase inhibitory activity.

Tyrosinase inhibition by peptides may find its application in food, cosmetics or medicine. In order to identify novel tyrosinase inhibitory peptides, protein-based peptide libraries made by SPOT synthesis were used to screen for peptides that show direct interaction with tyrosinase. One of the peptide libraries studied consists of overlapping, octameric peptides derived from industrial proteins as beta-casein, alpha-lactalbumin, beta-lactoglobulin, ovalbumin, gliadin, glycinin, and beta-conglycinin. On-membrane activity staining resulted in a set of peptides that are not only able to bind to tyrosinase, but are able to inhibit tyrosinase as well. Peptides containing aspartic or glutamic acid residues usually do not bind very well to tyrosinase. Strong tyrosinase-binding peptides always contain one or more arginine residues, often in combination with phenylalanine, while lysine residues can be found equally among nonbinding peptides as well as moderate tyrosinase-binding peptides. The presence of the hydrophobic, aliphatic residues valine, alanine or leucine appears to be important for tyrosinase inhibition. Therefore, good tyrosinase inhibitory peptides preferably contain arginine and/or phenylalanine in combination with valine, alanine and/or leucine.

Effect of Heating and Glycation on the Allergenicity of 2S Albumins (Ara h 2/6) from Peanut

Background Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. Methodology/Principal Findings Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. Conclusions/Significance Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.

Immunomodulatory capacity of fungal proteins on the cytokine production of human peripheral blood mononuclear cells

Immunomodulation by fungal compounds can be determined by the capacity of the compounds to influence the cytokine production by human peripheral blood mononuclear cells (hPBMC). These activities include mitogenicity, stimulation and activation of immune effector cells. Eight mushroom strains (Agaricus blazei, Coprinus comatus, Flammulina velutipes, Ganoderma lucidum, Grifola frondosa, Volvariella volvacea, Lentinus edodes, and Pleurotus ostreatus) were tested for the immunomodulating activity of the isolated protein fractions and polysaccharides fractions present in mycelia and culture liquid. The fungal proteins and polysaccharides have been investigated for their in vitro effect on the cytokine profile (IFN-gamma, IL-4, IL-10, IL-12 and TNF-alpha) of unstimulated or hPBMC stimulated with the polyclonal stimulations PMA/Ca-I, ConA or LPS. In addition to their influence on the cytokine profile, the hemagglutination activity of the fungal proteins on rabbit red blood cells was determined. Proteins from V. volvacea and G. lucidum showed immunomodulating activity without the presence of any mitogen, however, neither of them decreased the production of IL-4 and IFN-gamma in combination with a stimulus. All used stimuli resulted in an induction of IL-12 in the presence of the protein extracts, suggesting a direct effect on monocytes. This effect might lead to the indirect immunomodulation of T cell activation and cytokine production. In addition, both protein extracts showed more hemagglutination activity after trypsin treatment of the rabbit red blood cells, indicating the presence of carbohydrate-binding proteins, like lectins and FIPs. In conclusion, the protein extracts of V. volvacea and G. lucidum contain immunomodulating activity by acting directly on monocytes and thereby modulating T cell activation. Further purification of the fungal extracts is needed to clarify whether there are FIPs or lectins present that are responsible for this immunomodulating activity.