Characterisation of a recombinant β-xylosidase (xylA) from Aspergillus oryzae expressed in Pichia pastoris

β-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae β-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with Km and Vmax values of 1.0 mM and 250 μmol min−1 mg−1 respectively against 4-nitrophenyl β-xylopyranoside. Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4). Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length

Galectin-9 Controls CD40 Signaling through a Tim-3 Independent Mechanism and Redirects the Cytokine Profile of Pathogenic T Cells in Autoimmunity

While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4loCD40+ effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4loCD40+ T cells. Understanding the dynamics of the CD40 receptor(s) and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones

Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(−9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(−9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA

Fungal enzyme sets for plant polysaccharide degradation

Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families

P048 Gene expression profiling of transplant formalin-fixed paraffin-embedded biopsies: comparison of a custom TaqMan® low density array and quantitative nuclease-protection assay

We developed a custom TLDA to monitor changes of gene expression in FFPE renal and small bowel allografts. Here we carried out a parallel comparison of expression profiles of marker genes obtained with TaqMan® technology and with the automated qNPA system. Kidney, small bowel and heart transplant FFPE biopsies with different levels of rejection were selected. 5–6 curls of 10μm were cut from each biopsy for analysis in TLDA (47 markers including immune, inflammatory and apoptosis genes), and in HTG Edge System using the Immuno-Oncology Expression assay (HTGIOE, 26 markers), or HTG EdgeSeq Oncology Biomarker assay (HTGESOB, 2558 genes, format for NGS). Data was presented as differential expression. There are 10 overlapping markers between our TLDA and the HTGIOE assay. Analysis of 30 kidney biopsies with both technologies gave similar results for all 10 markers. Several markers were not detected in some samples analyzed with the HTG Edge System, mostly in normal donor and non-rejection samples. 11 unique markers of HTGIOE assay showed potential value for monitoring gene expression in kidney. 55 samples of heart biopsies were analyzed in TLDA and HTGIOE. Similar results were obtained with both technologies for all overlapping markers. 15 useful markers for monitoring gene expression in heart were found in HTGIOE assay. HTGIOE markers often did not express in non-rejection samples. We also analyzed 17 small bowel biopsies in HTGESOB, which contains 39 of the 47 markers in our TLDA (8 for SB, 17 for both kidney and small bowel, and 14 for kidney). 17.3% of the 2558 genes in the panel showed 2-fold increase expression relative to non-rejection. 15 markers in our TLDA (6 small bowel specific, 7 for both kidney and small bowel, and 2 kidney specific) were in the 5% of markers with the highest fold change. We showed that expression analysis carried out with TLDA and qNPA are comparable. The qNPA technology developed by HTG Molecular Diagnostics, Inc. is a good alternative to assess expression in biopsies. We found that the HTGIOE could be an excellent automated tool to evaluate panels with low number of markers in a clinical lab. The HTGESOB allows evaluation of a large number of genes, but it includes a NGS step that makes it costlier and time consuming. It is a good exploratory tool for searching new markers