Expression data from Congenital disorders of Glycosylation type-1 patients (CDG-I)
ABSTRACT: Disruption of N-linked glycosylation has a broad impact on proper glycosylation of nascent glycoproteins in the endoplasmic reticulum, which affect multiple signalling pathways( by changing the stability of membrane proteins or the signalling ability of membrane receptors) and may be responsible of the fibrotic stage associated to CDG type-I. We used microarrays to characterize the global changes in gene expression in three distinct groups of CDG-I patients and we identified a common perturbation in the expression of genes encoding for proteins involved in the stress as well as in the fibrotic responses. Keywords: disease state analysis Overall design: Primary fibroblasts, obtained from forearm skin biopsies of healthy control subjects and three distinct groups of CDG-I patients, were cultured in F12 DMEM supplemented with 4.5 g/l glucose and 10% fetal calf serum prior to RNA extraction and Hybridization on human HG 133 A Affymetrix Microarrays. To avoid bias related to the analysis of a specific CDG-I type or to a specific individual expression pattern, we have considered three patients for the CDG-Ic and -Ie group and two patients for the CDG-Ig group. Moreover the total RNA was extracted from three different independent biological replicates.
INSTRUMENT(S): [HG-U133A] Affymetrix Human Genome U133A Array
Project description:Disruption of N-linked glycosylation has a broad impact on proper glycosylation of nascent glycoproteins in the endoplasmic reticulum, which affect multiple signalling pathways( by changing the stability of membrane proteins or the signalling ability of membrane receptors) and may be responsible of the fibrotic stage associated to CDG type-I. We used microarrays to characterize the global changes in gene expression in three distinct groups of CDG-I patients and we identified a common perturbation in the expression of genes encoding for proteins involved in the stress as well as in the fibrotic responses. Experiment Overall Design: Primary fibroblasts, obtained from forearm skin biopsies of healthy control subjects and three distinct groups of CDG-I patients, were cultured in F12 DMEM supplemented with 4.5 g/l glucose and 10% fetal calf serum prior to RNA extraction and Hybridization on human HG 133 A Affymetrix Microarrays. To avoid bias related to the analysis of a specific CDG-I type or to a specific individual expression pattern, we have considered three patients for the CDG-Ic and -Ie group and two patients for the CDG-Ig group. Moreover the total RNA was extracted from three different independent biological replicates.
Project description:Irani2015 - Genome-scale metabolic model of
This model is described in the article:
Genome-scale metabolic model
of Pichia pastoris with native and humanized glycosylation of
Irani ZA, Kerkhoven E, Shojaosadati
SA, Nielsen J.
Biotechnol. Bioeng. 2015 Oct;
Pichia pastoris is used for commercial production of human
therapeutic proteins, and genome-scale models of P. pastoris
metabolism have been generated in the past to study the
metabolism and associated protein production by this yeast. A
major challenge with clinical usage of recombinant proteins
produced by P. pastoris is the difference in N-glycosylation of
proteins produced by humans and this yeast. However, through
metabolic engineering a P. pastoris strain capable of producing
humanized N-glycosylated proteins was constructed. The current
genome-scale models of P. pastoris do not address native nor
humanized N-glycosylation, and we therefore developed
ihGlycopastoris, an extension to the iLC915 model with both
native and humanized N-glycosylation for recombinant protein
production, but also an estimation of N-glycosylation of P.
pastoris native proteins. This new model gives a better
predictions of protein yield, demonstrates the effect of the
different types of N-glycosylation of protein yield, and can be
used to predict potential targets for strain improvement. The
model represents a step towards a more complete description of
protein production in P. pastoris, which is required for using
these models to understand and optimize protein production
processes. This article is protected by copyright. All rights
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Project description:Renal cell carcinoma (RCC) accounts for around 2-3% of all adult neoplasms and its incidence is increasing. RCC is largely resistant to standard cancer treatment regimens including chemotherapy and radiation therapy, whilst immunotherapy modestly provides benefit to only a small subset of patients. As a result, prognosis for RCC patients with advanced disease is very poor, with a 5-year survival rate of less than 10%. An early event in the development of familial and up to 90% of sporadic RCC is the loss and/or mutation/methylation of the von Hippel-Lindau (VHL) tumour suppressor gene. The tumor suppressor role of VHL relies primarily on its ability to target members of the hypoxia-inducible factors (HIF) for proteosomal degradation, however it has also been implicated in other biological processes linked to oncogenesis, such as the extracellular matrix assembly and the regulation of actin cytoskeleton, that are less clearly defined. Targeting the VHL/HIF pathway may dramatically improve RCC treatment, as demonstrated by an increasing number of novel agents that have shown promising activity in recent clinical trials6. Identification of other possible proteins regulated by VHL that may be involved in tumour suppression will improve our understanding of RCC pathobiology and also provide novel disease biomarkers or potential targets. A number of our studies have identified changes in glycosylation in RCC, at least some of which appear to alter as a result of changes in the VHL gene. Lectin binding immunohistochemistry shows altered reactivity with a number of lectins in the malignant tissue compared with the normal tissue. Comparison of plasma membrane protein enriched fractions from human RCC cells stably transfected with control vector or with wild type VHL led to the identification of VHL-associated changes in the forms of several plasma membrane proteins which we beelive to be due to differential glycosylation. For one of these we have now shown that it is differentially O-glycosylated and this has been confimed in cell lysates and patient tissue by Western blotting. A parallel approach using 2D-PAGE analysis of RCC cell line conditioned medium (CM) has also identified a tumour-specific glycoform of a secretory protein that appears to undergo differential N-glycosylation in RCC and this has been confirmed in analysis of some matched primary tumour/normal pairs. These findings are potentially very interesting, as these proteins could serve as prognostic or diagnostic markers, or even as targets for improving RCC therapy. We are therefore keen to explore the underlying mechanism of differential glycosylation that may be associated to VHL dependent gene expression. As there are an immense number of enzymes involved in the conjugation of N- and particularly O-glycans to proteins, the glyco-microarray is an ideal tool to elucidate this further. For chip analyses we have three cell lines generated from human RCC cells (RCC4, 786-0 and UMRC2) that have been stably transfected with control vector or with wild type VHL i.e. 6 cell lines in total. Whole RNA was extracted from each cell line in 3 replicates per line.
Project description:To define a genetic syndrome of severe atopy, elevated serum IgE, immune deficiency, autoimmunity, and motor and neurocognitive impairment, eight patients from two families who had similar syndromic features were studied. Whole exome sequencing was performed to identify disease-causing mutations. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). The result defines a new Congenital Disorder of Glycosylation.
Project description:Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide characterized by aberrant O-glycosylation in the hinge region of IgA1. The basis for the abnormal glycosylation in IgAN is still unknown, but an important involvement of the enzyme core 1, beta 1,3-galactosyltransferase 1 (C1GALT1) is known. However, the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in the IgAN pathogenesis has not yet been reported. In this study, by high-throughput microRNA profiling, we identified 37 miRNAs differentially expressed in peripheral blood mononuclear cells (PBMCs) from IgAN patients compared to healthy subjects. Among them, upregulated miR-148b potentially targeted C1GALT1, INVS and PTEN, three genes notably downregulated in IgAN patients. C1GALT1 expression levels in IgAN patients were reduced and negatively correlated with the upregulated miR-148b expression. We demonstrated the biological relationship between miR-148b and C1GALT1 by transient transfection experiments ex vivo. When we reduced the upregulated miR-148b function in PBMCs of IgAN patients an increase of the C1GALT1 mRNA and protein levels was observed. We validated biologically also the miR-148b targeting of INVS , involved in the altered modulation of the WNT–β-catenin and PI3K/Akt pathways in IgAN patients. All together our data evidence an important role of miR-148b in the pathogenesis of IgAN, which could explain the aberrant glycosylation of IgA1 in the pathogenesis and should light on a potential target for the theraphy of the disease. Overall design: The role of miRNAs expression in the pathogenesis of IgAN has not been well explored. To identify miRNAs differentially expressed in IgAN respect to healthy subjects (HS), we analyzed the global miRNA expression profile in PBMCs from IgAN patients and HS using the miRNA microarray approach. Among 723 human miRNAs represented on the microarrays, 147 were expressed in each sample. Unsupervised hierarchical clustering analysis generated a tree with IgAN and HS clearly separated into two group (Fig. 1). This separation was further confirmed by displaying the relationships among miRNAs expression patterns using principal component analysis (PCA) (Fig. S1). Applying a fold change threshold > 2 (false discovery rate < 0.01), 35 miRNAs were found to be significantly up-regulated and 2 were significantly down-regulated in IgAN. Complete list of differentially expressed miRNAs is shown in Table S1. To validate the microarray results we performed real-time PCR (q-RT-PCR) for miR-148b, miR-188-5p, miR-361-3p, miR-886-3p, let-7b, let-7d on miRNAs isolated from PBMCs of an independent set of 10 IgAN patients and 10 HS with the same clinical and demographic characteristics of the population used for microarray experiments. The expression of all analyzed miRNAs was significantly higher in IgAN patients confirming microarray results
Project description:These research areas concentrate on stress induced proteases in recombinant Escherichia coli, glycosylation heterogeneity due to bioprocess conditions produced in mammalian cells, and metabolic engineering of E. coli. The hypothesis of this project is that recombinant protein glycosylation is inefficient under normal bioreactor conditions since the additional glycosylation reactions necessary for the recombinant protein exceed the metabolic capacity of the cells. Normal bioreactor conditions have been optimized for cell growth, and sometimes for protein productivity. Only recently has it been accepted that optimal glycosylation may not occur under optimal growth or protein productivity conditions. Specific Aim: Determine the relationship between bioreactor conditions and glycosylation gene expression in NS0 cells. EXPERIMENT: Mouse NS0 myeloma cells were grown in culture, stressed with 5 mM NaCl, 10 mM proline plus 5 mM ammonia, or 5 mM ammonia, along with an unstressed control group. The growth of the cultures were followed until the late exponential phase (90 hours), at which time two 50 mLs of cells were harvested and RNA extracted. Samples were prepared in triplicate, for a total of 12 samples. The RNA was amplified and labeled by Microarray Core (E) and hybridized to the GLYCOv3 microarrays.
Project description:Dr. Esko's laboratory focuses on the structure, function, and biosynthesis of glycoproteins and proteoglycans. This laboratory also works on the design and synthesis of small molecule inhibitors of glycosylation. We analyzed the expression patterns of glycosyltransferase, growth factors, and receptors in endothelial cells derived from NDST1 deficient and wild-type mice using cancer cell line types U937 and LS180 .
Project description:Dr. Esko's laboratory focuses on the structure, function, and biosynthesis of glycoproteins and proteoglycans. This laboratory also works on the design and synthesis of small molecule inhibitors of glycosylation. Overall design: We analyzed the expression patterns of glycosyltransferase, growth factors, and receptors in endothelial cells derived from NDST1 deficient and wild-type mice using cancer cell line types U937 and LS180 .
Project description:Ovarian cancer is one of the most common cancer among women in the world, and chemotherapy remains the principal treatment for patients. However, drug resistance is a major obstacle to the effective treatment of ovarian cancers and the underlying mechanism is not clear. An increased understanding of the mechanisms that underline the pathogenesis of drug resistance is therefore needed to develop novel therapeutics and diagnostic. Herein, we report the comparative analysis of the doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI/ADR-RES cells using integrated global proteomics and N-glycoproteomics. A total of 1525 unique N-glycosite-containing peptides from 740 N-glycoproteins were identified and quantified, of which 253 N-glycosite-containing peptides showed significant change in the NCI/ADR-RES cells. Meanwhile, stable isotope labeling by amino acids in cell culture (SILAC) based comparative proteomic analysis of the two ovarian cancer cells led to the quantification of 5509 proteins. As about 50% of the identified N-glycoproteins are low-abundance membrane proteins, only 44% of quantified unique N-glycosite-containing peptides had corresponding protein expression ratios. The comparison and calibration of the N-glycoproteome versus the proteome classified 14 change patterns of N-glycosite-containing peptides, including 8 up-regulated N-glycosite-containing peptides with the increased glycosylation sites occupancy, 35 up-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy, 2 down-regulated N-glycosite-containing peptides with the decreased glycosylation sites occupancy, 46 down-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy. Integrated proteomic and N-glycoproteomic analyses provide new insights, which can help to unravel the relationship of N-glycosylation and multidrug resistance (MDR), understand the mechanism of MDR, and discover the new diagnostic and therapeutic targets.
Project description:Dr. Jameson's research focus is on development and regulation of lymphocytes, especially T cells. Recent work has suggested that differential glycosylation effects the sensitivity of the T cell receptors and its coreceptors, suggesting that regulation of glycosylation may be a critical element in controlling T cell development, survival and functional activity. Overall design: Determination of how glycosylation enzymes/substrates change in gene expression during development of mouse CD8 T cells. Highly purified pre-selection CD4+8+ thymocytes (using transgenic/knockout mice in our colony) are compared to mature CD8 T cells from the lymph node. The goal is to build on data suggesting that this developmental step involves regulated expression of sialyltransferases (and/or neuraminidases). Highly purified pre-selection CD4+8+ thymocytes (using transgenic/knockout mice in our colony) are compared to mature CD8 T cells from the lymph node. Pre-selection CD4+8+ (DP) thymocytes were sorted from TCRa-/- thymi. Post-selection (post-positive selection) DP thymocytes came from an OT-I TCR transgenic mouse. Naïve (CD44lo) CD8 T cells from lymph node of OT-I mice were used as naïve CD8 T cells. For activated cells, naïve OT-I T cells were activated for 48 hours in vitro with cognate antigen (SIINFEKL peptide/Kb) (displayed on cell sized latex beads) in the presence of IL-2 and IL-12.