Diluka Peiris

Metabolite profiles of interacting mycelial fronts differ for pairings of the wood decay basidiomycete fungus, Stereum hirsutum with its competitors Coprinus micaceus and Coprinus disseminatus

The paper presents the first proof-of principle study of metabolite profiles of the interacting mycelial fronts of a wood decomposer basidiomycete, Stereum hirsutum, paired with two competitor basidiomycetes, Coprinus disseminatus and C. micaceus, using TLC and GC-TOF-MS profiling. GC-TOF-MS profiles were information rich, with a total of 190 metabolite peaks detected and more than 120 metabolite peaks detected per sample. The metabolite profiles were able to discriminate between the interactions of S. hirsutum with the two species of Coprinus. In confrontation with C. micaceus, where S. hirsutum mycelial fronts always overgrew those of C. micaceus, there were down-regulations of metabolites in the interaction zone, compared to monocultures of both S. hirsutum and C. micaceus. In contrast, in pairings with C. disseminatus, whose mycelia overgrew those of S. hirsutum, there were some up-regulations compared with monoculture controls, the majority of the metabolites being characteristic of the S. hirsutum monoculture profile. These differences indicate that up-regulation of metabolites in the mycelia of S. hirsutum may be connected to a defensive role or to stress. The results also show proof of principle for the employment of metabolic profiling for biological discovery studies of metabolites produced by fungi that could be applied to natural product screening programmes.

A novel approach to determining the affinity of protein-carbohydrate interactions employing adherent cancer cells grown on a biosensor surface

The development of biological agents for the treatment of solid tumours is an area of considerable activity. We are pursuing carbohydrate-binding proteins (lectins) in a strategy aimed at targeting cancer-associated changes in glycosylation. To evaluate lectin-cancer cell interactions we developed a novel cell biosensor in which binding events take place at the cell surface, more closely mimicking an in vivo system. Metastatic, SW620, and non-metastatic, SW480, colorectal cancer cells were grown on the surface of a tissue-culture compatible polystyrene coated biosensor chip and housed in a quartz crystal microbalance (QCM) apparatus, the kinetics of binding of a diverse range of lectins was evaluated. The lectin Helix pomatia agglutinin (HPA) has been shown to bind aggressive metastatic cancer and was produced in recombinant form (His- and RFP-tagged). The affinity of HPA was in the nanomolar range to the metastatic SW620 cells but was only in the micromolar range to the non-metastatic SW480. Overall, the dissociation constant (K(D)) of the lectins tested in the new cell biosensor system was an order of magnitude lower (nanomolar range) than has generally been reported with systems such as QCM/SPR. This new cell-biosensor enables molecular interactions to be studied in a more relevant environment. An intrinsic problem with developing new biological therapies is the difficulty in determining the affinity with which proteins will interact with intact cell surfaces. This methodology will be of interest to researchers developing new biological approaches for targeting cell surfaces in a wide range of diseases, including cancer.

Identification, Cloning, and Characterization of Two N-Acetylgalactosamine-binding Lectins from the Albumen Gland of Helix pomatia

Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant production of HPA, is required. A combination of isoelectric focusing, amino acid sequence analysis, and cloning revealed two polypeptides in native HPA preparations (HPAI and HPAII), both consistent with GalNAc-binding lectins of the H-type family. Pairwise sequence alignment showed that HPAI and HPAII share 54% sequence identity whereas molecular modeling using SWISS-MODEL suggests they are likely to adopt similar tertiary structure. The inherent heterogeneity of native HPA highlighted the need for production of functional recombinant protein; this was addressed by preparing His-thioredoxin-tagged fusion products in Escherichia coli Rosetta-gami B (DE3) cells. The recombinant lectins agglutinated human blood group A erythrocytes whereas their oligosaccharide specificity, evaluated using glycan microarrays, showed that they predominantly bind glycans with terminal α-GalNAc residues. Surface plasmon resonance with immobilized GalNAc-BSA confirmed that recombinant HPAI and HPAII bind strongly with this ligand (Kd = 0.60 nm and 2.00 nm, respectively) with a somewhat higher affinity to native HPA (Kd = 7.67 nm). Recombinant HPAII also bound the breast cancer cells of breast cancer tissue specimens in a manner similar to native lectin. The recombinant HPA described here shows important potential for future studies of cancer cell glycosylation and as a reagent for cancer prognostication.

Arabidopsis AtPARK13, Which Confers Thermotolerance, Targets Misfolded Proteins

Background: Mutations in PARK13 have been implicated in Parkinson disease. Results: AtPARK13 confers thermotolerance in Arabidopsis and degrades misfolded proteins, including α-synuclein and DJ-1. Conclusion: AtPARK13 confers thermotolerance through degradation of misfolded proteins. Significance: Arabidopsis is a complementary model to investigate mechanisms associated with Parkinson disease. Mutations in HTRA2/Omi/PARK13 have been implicated in Parkinson disease (PD). PARK13 is a neuroprotective serine protease; however, little is known about how PARK13 confers stress protection and which protein targets are directly affected by PARK13. We have reported that Arabidopsis thaliana represents a complementary PD model, and here we demonstrate that AtPARK13, similar to human PARK13 (hPARK13), is a mitochondrial protease. We show that the expression/accumulation of AtPARK13 transcripts are induced by heat stress but not by other stress conditions, including oxidative stress and metals. Our data show that elevated levels of AtPARK13 confer thermotolerance in A. thaliana. Increased temperatures accelerate protein unfolding, and we demonstrate that although AtPARK13 can act on native protein substrates, unfolded proteins represent better AtPARK13 substrates. The results further show that AtPARK13 and hPARK13 can degrade the PD proteins α-synuclein (SNCA) and DJ-1/PARK7 directly, without autophagy involvement, and that misfolded SNCA and DJ-1 represent better substrates than their native counterparts. Comparative proteomic profiling revealed AtPARK13-mediated proteome changes, and we identified four proteins that show altered abundance in response to AtPARK13 overexpression and elevated temperatures. Our study not only suggests that AtPARK13 confers thermotolerance by degrading misfolded protein targets, but it also provides new insight into possible roles of this protease in neurodegeneration.

Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.

The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride.

In this study, agar plate interaction between Schizophyllum commune and Trichoderma viride was investigated to characterise the physiological responses occurring during interspecific mycelial combat. The metabolite profiles and morphological changes in both fungi paired on agar were studied relative to the modulation of phenoloxidase activity in S. commune. The calcium ionophore A23187 was incorporated in self-paired cultures of S. commune to explore possible involvement of calcium influx in the response of S. commune to T. viride. The levels of lipid peroxides and protein carbonyls in the confronted mycelia of S. commune were also measured. Contact with T. viride induced pigmentation and cell wall hydrolysis in S. commune with concomitant increase in phenoloxidase activity, rise in the levels of oxidative stress indicators and increased levels of phenolic compounds, antioxidant γ-amino butyric acid, and pyridoxine and osmo-protective sugar alcohols. Calcium ionophore mimicked the pigmentation in the T. viride-confronted mycelia of S. commune, implicating calcium influx in the response to T. viride. The changes in S. commune are indicative of targeted responses to osmotic and oxidative stresses and phenoloxidase-mediated detoxification of noxious compounds in the contact interface with T. viride, which may confer resistance in natural environments.

Quantitative proteomic analysis of the response of the wood-rot fungus, Schizophyllum commune, to the biocontrol fungus, Trichoderma viride

Aims:  Investigation of changes in the protein profile of the wood‐rot fungus, Schizophyllum commune, when paired against the biocontrol fungus, Trichoderma viride, for 48 h.

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

Background Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. Methodology In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. Results Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). Conclusion Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer.

2-DE-based proteomics for the analysis of metastasis-associated proteins.

Two-dimensional electrophoresis (2-DE) is a high-resolution technique for analysis and comparison of complex protein mixtures. With the advent of recent technical developments, its application has become significant in a wide range of fields. This chapter describes a proteomic approach for the analysis of metastasis-associated proteins using pre-fractionation of glycosylated proteins via lectin (HPA) affinity chromatography prior to separation by 2-DE. Guidelines for the preparation and storage of buffers, experimental conditions and protocols of affinity chromatography, isoelectric focussing, and SDS-PAGE conditions are provided. Critical parameters associated with the different steps of 2-DE are discussed.