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Entamoeba histolytica (E. histolytica) is a protozoan responsible for intestinal amebiasis in at least 500 million people per year, although only 10% of those infected show severe symptoms. It is known that E. histolytica captures molecules released during the host immune response through membrane receptors that favor its pathogenetic mechanisms for the establishment of amebic invasion. It has been suggested that E. histolytica interacts with acetylcholine (ACh) through its membrane. This promotes the increase of virulence factors and diverse mechanisms carried out by the amoeba to produce damage. The aim of this study is to identify a membrane receptor in E. histolytica trophozoites for ACh. Methods included identification by colocalization for the ACh and Gal/GalNAc lectin binding site by immunofluorescence, western blot, bioinformatic analysis, and quantification of the relative expression of Ras 5 and Rab 7 GTPases by RT-qPCR. Results show that the Gal/GalNAc lectin acts as a possible binding site for ACh and this binding may occur through the 150 kDa intermediate subunit. At the same time, this interaction activates the GTPases, Ras, and Rab, which are involved in the proliferation, and reorganization of the amoebic cytoskeleton and vesicular trafficking. In conclusion, ACh is captured by the parasite, and the interaction promotes the activation of signaling pathways involved in pathogenicity mechanisms, contributing to disease and the establishment of invasive amebiasis.

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Changes in protein glycosylation are a hallmark of transformed cells and modulate numerous phenomena associated with cancer progression, such as the acquisition of multidrug resistance (MDR) phenotype. Different families of glycosyltransferases and their products have already been described as possible modulators of the MDR phenotype. Among the glycosyltransferases intensively studied in cancer research, UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (pp-GalNAc-T6), which is widely expressed in many organs and tissues, stands out. Its influence in several events associated with kidney, oral, pancreatic, renal, lung, gastric and breast cancer progression has already been described. However, its participation in the MDR phenotype has never been studied. Here, we demonstrate that human breast adenocarcinoma MCF-7 MDR cell lines, generated by chronic exposure to doxorubicin, in addition to exhibiting increased expression of proteins belonging to the ABC superfamily (ABCC1 and ABCG2), and anti-apoptotic proteins (Blcl-2 and Bcl-xL), also present high expression of pp-GalNAc-T6, the enzyme currently proposed as the main responsible for the biosynthesis of oncofetal fibronectin (onf-FN), a major extracellular matrix component expressed by cancer cells and embryonic tissues, but absent in healthy cells. Our results show that onf-FN, which is generated by the addition of a GalNAc unit at a specific threonine residue inside the type III homology connective segment (IIICS) domain of FN, is strongly upregulated during the acquisition of the MDR phenotype. Also, the silencing of pp-GalNAc-T6, not only compromises the expression of the oncofetal glycoprotein, but also made the MDR cells more sensitive to all anticancer drugs tested, partially reversing the MDR phenotype. Taken together, our results demonstrate for the first time the upregulation of the O-glycosylated oncofetal fibronectin, as well as the direct participation of pp-GalNAc-T6 during the acquisition of a MDR phenotype in a breast cancer model, giving credence to the hypothesis that in transformed cells, glycosyltransferases and/or their products, such as unusual extracellular matrix glycoproteins can be used as potential therapeutic targets for the treatment of cancer.

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BACKGROUND: Post-translational modifications are key factors in the modulation of nuclear protein functions controlling cell physiology and an individual's health. OBJECTIVES: This study examined the influence of protein restriction during the perinatal period on the nuclear O-N-acetylgalactosamine (O-GalNAc) glycosylation of cells from the liver and parts of the brain in the rat. METHODS: Pregnant Wistar rats were divided into 2 groups on day 14 of pregnancy and fed ad libitum 1 of 2 isocaloric diets containing 24% (well-fed) or 8% (protein-restricted diet) casein until the end of the experiment. Male pups were studied after weaning at 30 d of life. Animals and their organ/tissues (liver, cerebral cortex, cerebellum and hippocampus) were weighed. Cell nuclei were purified, and the presence in nucleus and cytoplasm of all factors required for the initiation of O-GalNAc glycan biosynthesis, i.e., the sugar donor (UDP-GalNAc), enzyme activity (ppGalNAc-transferase) and the glycosylation product (O-GalNAc glycans), were evaluated by western blotting, fluorescent microscopy, enzyme activity, enzyme-lectin sorbent assay and mass spectrometry. RESULTS: The perinatal protein deficit reduced progeny weight, as well as the cerebral cortex and cerebellum weight. UDP-GalNAc levels in the cytoplasm and nuclei of the liver, the cerebral cortex, cerebellum, or hippocampus were not affected by the perinatal dietary protein deficits. However, this deficiency affected the ppGalNAc-transferase activity localized in the cerebral cortex and hippocampus cytoplasm as well as in the liver nucleus, thus reducing the "writing" ppGalNAc-transferase activity of O-GalNAc glycans. In addition, liver nucleoplasm from protein-restricted offspring revealed a significant reduction in the expression of O-GalNAc glycans on important nuclear proteins. CONCLUSIONS: Our results report an association between the consumption of a protein-restricted diet by the dam and her progeny with the modulation in the offspring' liver nuclei O-GalNAc glycosylation, which may ultimately regulate nuclear protein functions.

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Breast cancer is a public health concern and is currently the fifth cause of mortality worldwide. Identification of different biological subtypes is essential for clinical management; therefore, the role of pathologists is essential and useful tools for immunohistochemistry diagnosis are needed. Polypeptide-GalNAc-transferases are emerging novel biomarkers related to cancer behavior and GalNAc-T13, correlated with aggressiveness in some tumors, is an interesting candidate. Few monoclonal antibodies reacting with native proteins, and not affected by fixation and paraffin embedding, have been reported. The aim of this work was to develop a useful monoclonal antibody anti-GalNAc-T13 and to assess its potential significance in breast cancer diagnosis. We evaluated 6 human breast cancer cell lines, 338 primary breast tumors and 48 metastatic lymph nodes and looked for clinical significance correlating GalNAc-T13 expression with patients' clinical features and survival. We found high GalNAc-T13 expression in 43.8% of the cases and observed a significant higher expression in metastatic lymph nodes, correlating with worse overall survival. We hypothesized several possible molecular mechanisms and their implications. We conclude that GalNAc-T13 may be a novel biomarker in breast cancer, useful for routine pathological diagnosis. Elucidation of molecular mechanisms related to aggressiveness should contribute to understand the role of GalNAc-T13 in breast cancer biology.

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Amoebiasis in humans is caused by the protozoan parasite Entamoeba histolytica, which cytotoxic activity has been demonstrated on a wide variety of target cells. The process involves the adherence of the parasite to the cell, and such adherence is mediated by an amoebic surface lectin, known as Gal/GalNAc lectin. It is composed of heavy, intermediate, and light subunits. The carbohydrate recognition domain (CRD) has been identified within a cysteine-rich region in the lectin heavy subunit and has an amino acid sequence identity to the receptor-binding domain of hepatocyte growth factor (HGF). Recombinant CRD has been previously shown to compete with HGF for binding to the c-Met receptor IgG fusion protein. In the present study, we searched for evidence of interaction between the Gal/GalNAc lectin at the surface of trophozoites with the c-Met receptor expressed at the surface of HepG2 in coculture assays. Immunoprecipitation of the coculture lysate indicated interaction of the c-Met with a 60 kDa peptide recognized by antiamoebic lectin antibody. Colocalization of both molecules was detected by fluorescence confocal microscopy. Incubation of HepG2 cells with HGF before coculture with trophozoites prevents the cytotoxic effect caused by the parasites but not their adherence to the cells. Our results point to Gal/GalNAc lectin as a ligand of the c-Met receptor at the surface of HepG2 cells.

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BACKGROUND: Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation. METHODS: Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry. RESULTS: Clostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles. CONCLUSIONS: Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes. GENERAL SIGNIFICANCE: Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.

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Altered expression of miR-182 has been observed in various types of human cancer. The purpose of this study was to investigate the expression of miR-182 and its role in prostate cancer (PCa). Expression of miR-182 and ST6GALNAC5 in tumor tissues and the Du145 PCa cell line was analyzed. Cell proliferation assay, colony formation assay, transwell assay, and wound healing assay were performed. The impact of miR-182 on tumor growth was investigated using a xenograft model. The results indicated that expression of miR-182 was higher in PCa tissues and cell lines, while ST6GALNAC5 was decreased. Downregulating miR-182 significantly inhibited the capacities of proliferation and invasion of PC3 and Du145 cells. ST6GALNAC5 was demonstrated to be a target of miR-182 by luciferase assay, and western blot results indicated PI3K/Akt pathway was involved in miR-182 associated effects on PC3 and Du145 cells. The animal experiment suggested that knockdown of miR-182 inhibited tumor growth. Our study proved that miR-182 participated in the proliferation and invasion of PCa cells via mediating expression of ST6GALNAC5 and established a miR-182/ST6GALNAC5/PI3K/AKT axis in regulation of tumor progression. Our investigation provided a basis for further exploration of the application of miR-182 or ST6GALNAC5-associated therapies for PCa patients.

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In many ways, cancer cells are different from healthy cells. A lot of tactical nano-based drug delivery systems are based on the difference between cancer and healthy cells. Currently, nanotechnology-based delivery systems are the most promising tool to deliver DNA-based products to cancer cells. This review aims to highlight the latest development in the lipids and polymeric nanocarrier for siRNA delivery to the cancer cells. It also provides the necessary information about siRNA development and its mechanism of action. Overall, this review gives us a clear picture of lipid and polymer-based drug delivery systems, which in the future could form the base to translate the basic siRNA biology into siRNA-based cancer therapies.

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Biological functions of nuclear proteins are regulated by post-translational modifications (PTMs) that modulate gene expression and cellular physiology. However, the role of O-linked glycosylation (O-GalNAc) as a PTM of nuclear proteins in the human cell has not been previously reported. Here, we examined in detail the initiation of O-GalNAc glycan biosynthesis, representing a novel PTM of nuclear proteins in the nucleus of human cells, with an emphasis on HeLa cells. Using soluble nuclear fractions from purified nuclei, enzymatic assays, fluorescence microscopy, affinity chromatography, MS, and FRET analyses, we identified all factors required for biosynthesis of O-GalNAc glycans in nuclei: the donor substrate (UDP-GalNAc), nuclear polypeptide GalNAc -transferase activity, and a GalNAc transferase (polypeptide GalNAc-T3). Moreover, we identified O-GalNAc glycosylated proteins in the nucleus and present solid evidence for O-GalNAc glycan synthesis in this organelle. The demonstration of O-GalNAc glycosylation of nuclear proteins in mammalian cells reported here has important implications for cell and chemical biology.

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Colorectal carcinoma (CRC) is the second leading cause of cancer mortality worldwide. O-glycosylated mucins at the cell surface of colonic mucosa exhibit alterations in cancer and are involved in fundamental biological processes, including invasion and metastasis. Certain members of the GalNAc-transferase family may be responsible for these changes and are being investigated as novel biomarkers of cancer. In the present study the prognostic significance of GalNAc-T6 was investigated in patients with CRC patients. GalNAc-T6 expression was observed in all three colon cancer cell lines analyzed by reverse transcription-polymerase chain reaction, immunofluorescence and flow cytometry. A cohort of 81 colon cancer specimens was analyzed by immunohistochemical staining using MAb T6.3. It was demonstrated that GalNAc-T6 was expressed in 35/81 (43%) cases of colon cancer but not in the normal colonic mucosa. No association was observed with the clinical-pathologic parameters. However, patients expressing GalNAc-T6 had a significantly increased overall survival (median, 58 months; P<0.001) compared with GalNAc-T6 negative patients, especially those with advanced disease. These results suggest that GalNAc-T6 expression predicts an improved outcome in patients with CRC. The molecular mechanism underlying the less aggressive behavior of colon cancer cells expressing GalNAc-T6 remains to be elucidated.

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BACKGROUND: Studies show that insects can adapt to the toxins of Bacillus thuringiensis under field and laboratory conditions through the development of resistance to the bacterium and its formulations. This has been demonstrated in the failure to control Tuta absoluta populations in Brazil. This study evaluated membrane receptors using peroxidase-labeled lectins and the midgut histochemistry of T. absoluta populations to assess susceptibility to the insecticides Bt fomulations. The histochemistry analysis used Periodic Acid-Schiff for glycogen and Ponceau Xylidine for total proteins. The presence of glucose/mannose and N-acetylgalactosamine (GalNAc) was analyzed using specific lectins. One susceptible and one tolerant population were used in the study; insects were exposed to the insecticide concentrations recommended by the manufacturers. The midgut was collected after an interval of 20 min and analyzed using optical microscopy. RESULTS: Bt fomulation interferes with the glycogen content, whereas XenTari® interferes with the protein content, irrespective of the level of susceptibility. High expression of GalNAc residues was observed using soybean lectin labeling, indicating a direct relationship between the glycosylation pattern and susceptibility to Bt fomulation in the Pelotas population. CONCLUSION: The use of Bt fomulation caused greater alterations in the larval intestinal histophysiology compared to the use of XenTari® . © 2018 Society of Chemical Industry.

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Lectins play crucial roles for innate immune responses in invertebrates by recognizing and eliminating pathogens. In this study, a lectin from the mussel Mytilus californianus (MCL) was identified and characterized. The lectin was purified by affinity chromatography in α-lactose-agarose resin showing an experimental molecular mass of 18000 Da as determined by SDS-PAGE and MALDI-TOF mass spectrometry. It was specific for binding d-galactose and N-Acetyl-d-galactosamine that contained carbohydrate moieties that were also inhibited by melibiose and raffinose. It had the ability to agglutinate all types of human erythrocytes, as well as rabbit red blood cells. Circular dichroism analyzes have indicated that this lectin possessed an α/ß fold with a predominance of ß structures. This was consistent with the structure of the protein that was determined by the X-ray diffraction techniques. MCL was crystallized in the space group C21 and it diffracted to 1.79 Å resolution. Two monomers were found in the asymmetric unit and they formed dimers in solution. The protein has shown to be a member of the ß-trefoil family, with three sugar binding sites per monomer. In accord with fluorescence-based thermal shift assays, we observed that the MCL Tm increased about 10 °C in the presence of galactose. Furthermore, we have determined the complete amino acid sequence by cDNA sequencing. The gene had two ORF2 proteins, one resulting in a 180 residue protein with a theoretical molecular mass of 20227 Da, and another resulting in a 150 residue protein with a theoretical molecular mass of 16911 Da. The difference between the theoretical and experimental values was due to the presence of a glycosylation that was observed by the glycosylation assay. A positive microbial agglutination and a growth inhibition activity were observed against Gram-negative and Gram-positive bacteria. The M. californianus lectin is the fourth member of the recently proposed new family of lectins that have been reported to date, occurring only in mollusks belonging to the family Mytilidae. It is the first member to be glycosylated and with a strong tendency to form large oligomers.

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Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a trematode zoonosis of interest in public health and livestock production. Like other helminths, F. hepatica modulates the host immune response by inducing potent polarized Th2 and regulatory T cell immune responses and by downregulating the production of Th1 cytokines. In this work, we show that F. hepatica glycans increase Th2 immune responses by immunomodulating TLR-induced maturation and function of dendritic cells (DCs). This process was mediated by the macrophage Gal/GalNAc lectin (MGL) expressed on DCs, which recognizes the Tn antigen (GalNAc-Ser/Thr) on parasite components. More interestingly, we identified MGL-expressing CD11c+ cells in infected animals and showed that these cells are recruited both to the peritoneum and the liver upon F. hepatica infection. These cells express the regulatory cytokines IL-10, TNFα and TGFß and a variety of regulatory markers. Furthermore, MGL+ CD11c+ cells expand parasite-specific Th2/regulatory cells and suppress Th1 polarization. The results presented here suggest a potential role of MGL in the immunomodulation of DCs induced by F. hepatica and contribute to a better understanding of the molecular and immunoregulatory mechanisms induced by this parasite.

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Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B-CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [א(2)(1)=18.26; P<0.0001], lipoprotein lipase expression [א(2)(1)=13.72; P=0.0002] and disease prognosis [א(2)(1)=15.49; P<0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.

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