NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.

Sialylation of tumor cells is involved in various aspects of their malignancy (proliferation, motility, invasion, and metastasis); however, its effect on the process of immunoediting that affects tumor cell immunogenicity has not been studied. We have shown that in mice with impaired immunoediting, such as in IL-1α−/− and IFNγ−/− mice, 3-methylcholanthrene–induced fibrosarcoma cells are immunogenic and concomitantly bear low levels of surface sialylation, whereas tumor cells derived from wild type mice are nonimmunogenic and bear higher levels of surface sialylation. To study immune mechanisms whose interaction with tumor cells involves surface sialic acid residues, we used highly sialylated 3-methylcholanthrene–induced nonimmunogenic fibrosarcoma cell lines from wild type mice, which were treated with sialidase to mimic immunogenic tumor cell variants. In vivo and in vitro experiments revealed that desialylation of tumor cells reduced their growth and induced cytotoxicity by NK cells. Moreover, sialidase-treated tumor cells better activated NK cells for IFN-γ secretion. The NKG2D-activating receptor on NK cells was shown to be involved in interactions with desialylated ligands on tumor cells, the nature of which is still not known. Thus, the degree of sialylation on tumor cells, which is selected during the process of immunoediting, has possibly evolved as an important mechanism of tumor cells with low intrinsic immunogenicity or select for tumor cells that can evade the immune system or subvert its function. When immunoediting is impaired, such as in IFN-γ−/− and IL-1α−/− mice, the overt tumor consists of desialylayed tumor cells that interact better with immunosurveillance cells.

Although one typically thinks of carbohydrates as associated with cell growth and viability, glycosylation also has an integral role in many processes leading to cell death. Glycans, either alone or complexed with glycan-binding proteins, can deliver intracellular signals or control extracellular processes that promote initiation, execution and resolution of cell death programs. Herein, we review the role of glycans and glycan-binding proteins as essential components of the cell death machinery during physiologic and pathologic settings.

No abstract

A microbial fuel cell (MFC) was designed in which fuel is generated in the cell by the enzyme glucoamylase, which is displayed on the surface of yeast. The enzyme digests starch specifically into monomeric glucose units and as a consequence enables further glucose oxidation by microorganisms present in the MFC anode. The oxidative enzyme glucose oxidase was coupled to the glucoamylase digestive enzyme. When both enzymes were displayed on the surface of yeast cells in a mixed culture, superior fuel-cell performance was observed in comparison with other combinations of yeast cells, unmodified yeast, or pure enzymes. The feasibility of the use of the green macroalgae Ulva lactuca in such a genetically modified MFC was also demonstrated. Herein, we report the performance of such fuel cells as a proof of concept for the enzymatic digestion of complex organic fuels in the anode of MFCs to render the fuel more available to microorganisms.

The occurrence of N-glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N-glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N-glycans of the complex type by employing modular TFAc-protected donors for all antennae. The TFAc-protected N-glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N-glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one-pot double glycosylation. With this new method, rare N-glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.

There is increasing evidence localizes the mitochondrial chaperone heat shock protein (HSP)60, outside the cell, where it mediates interactions between immune cells and other body tissues. However, the mechanisms by which HSP60 is secreted into the extracellular environment are not fully understood. Recent studies have shown that HSP60 is actively released by a nonconventional secretion mechanism, the lipid raft-exosome pathway. In the present study, we show for the first time that HSP60, produced by 3-methylcholantrene-induced fibrosarcoma tumour cells, is secreted through the conventional endoplasmic reticulum-Golgi secretory pathway. Confocal microscopy using anti-TGN38 and anti-HSP60 antibodies together with monensin, a Golgi transport inhibitor, demonstrated the relocation of HSP60 to the Golgi of malignant cells but not primary fibroblast cells subjected to heat shock or fibroblast cell lines. Transmission electron microscopy, flow cytometry and cell fractionation of cell treated with brefeldin A, an inhibitor of endoplasmic reticulum to Golgi protein transport, further indicated that HSP60 is present both in the endoplasmic reticulum and the Golgi complex of malignant cells. We found a single mRNA with a mitochondrial targeting sequence encoding for HSP60 in the malignant cells but two HSP60 translation products, namely the native unmodified protein and a protein post-translationally modified by N-glycosylation. The N-glycans observed were composed of high-mannose structures and bi-, tri-and tetraantennary complex type structures occupying sites of the three potential glycosylation sites present on HSP60. Accordingly, we propose that HSP60 in malignant cells is transported through the endoplasmic reticulum-Golgi secretion pathway, where it acquires N-glycans, and thus can affect the immunological properties of the proteins in the tumour microenvironment. Structured digital abstractl Grp94 and Hsp60 colocalize by cosedimentation through density gradient (View interaction) Abbreviations 3-MCA, 3-methylcholantrene; BFA, brefeldin A; EGFA, epidermal growth factor receptor; ER, endoplasmic reticulum; FACS, fluorescenceactivated cell sorting; FGF-2, fibroblast growth factor-2; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; HSP, heat shock protein; MFI, mean fluorescence intensity; NP-HPLC, normal phase-HPLC; TEM, transmission electron microscopy; TLR, Toll-like receptor.

Finding new peptide biomarkers for stomach cancer in human sera that can be implemented into a clinically practicable prediction method for monitoring of stomach cancer. We studied the serum peptidome from two different biorepositories. We first employed a C8-reverse phase liquid chromatography approach for sample purification, followed by mass-spectrometry analysis. These were applied onto serum samples from cancer-free controls and stomach cancer patients at various clinical stages. We then created a bioinformatics analysis pipeline and identified peptide signature discriminating stomach adenocarcinoma patients from cancer-free controls. Matrix Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) results from 103 samples revealed 9 signature peptides; with prediction accuracy of 89% in the training set and 88% in the validation set. Three of the discriminating peptides discovered were fragments of Apolipoproteins C-I and C-III (apoC-I and C-III); we further quantified their serum levels, as well as CA19-9 and CRP, employing quantitative commercial-clinical assays in 142 samples. ApoC-I and apoC-III quantitative results correlated with the MS results. We then employed apoB-100-normalized apoC-I and apoC-III, CA19-9 and CRP levels to generate rules set for stomach cancer prediction. For training, we used sera from one repository, and for validation, we used sera from the second repository. Prediction accuracies of 88.4% and 74.4% were obtained in the training and validation sets, respectively. Serum levels of apoC-I and apoC-III combined with other clinical parameters can serve as a basis for the formulation of a diagnostic score for stomach cancer patients.