The active site of O-GlcNAc transferase imposes constraints on substrate sequence.

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides.

Characterization and validation of an isotope-dilution LC-MS/MS method for quantification of total desmosine and isodesmosine in plasma and serum.

Background: Desmosine/isodesmosine (DES/IDS) is a promising biomarker for estimating activity of elastin degradation. RESULTS/METHODOLOGY: A stable isotope dilution LC-MS/MS method for measuring serum/plasma DES/IDS was developed and validated. The reportable range of this assay was 0.

Genetic and structural validation of Aspergillus fumigatus N-acetylphosphoglucosamine mutase as an antifungal target.

Aspergillus fumigatus is the causative agent of IA (invasive aspergillosis) in immunocompromised patients. It possesses a cell wall composed of chitin, glucan and galactomannan, polymeric carbohydrates synthesized by processive glycosyltransferases from intracellular sugar nucleotide donors. Here we demonstrate that A.

O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis.

Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human OGT recognizes the sugar donor and acceptor peptide and uses a new catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base as well as an essential lysine. This mechanism seems to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate and explains the unexpected specificity of a recently reported metabolic OGT inhibitor.

O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release.

Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro-inflammatory cytokines such as transforming growth factor-β, tumour necrosis factor (TNF), interleukin-1 (IL-1) and wnt family ligands. TAK1 is found in complex with binding partners TAB1-3, phosphorylation and ubiquitination of which has been found to regulate TAK1 activity. In this study, we show that TAB1 is modified with N-acetylglucosamine (O-GlcNAc) on a single site, Ser395.

Clinical validity of plasma and urinary desmosine as biomarkers for chronic obstructive pulmonary disease.

Background: Although an increased concentration of degraded elastin products in patients with chronic obstructive pulmonary disease (COPD) has been reported for many years, its clinical validity and utility remain uncertain due to technical difficulties, small study groups and the unknown relationship between exacerbation and elastin degradation. The objectives of this study were to determine the validity of urinary and blood total desmosine/isodesmosine in patients with COPD and asthma and to evaluate their relationship to exacerbation status and lung function.

Methods: Urinary and blood desmosine levels were measured using validated isotopic dilution liquid chromatography-tandem mass spectrometry methods.

Measurement of urinary total desmosine and isodesmosine using isotope-dilution liquid chromatography-tandem mass spectrometry.

The current LC-MS based desmosine/isodesmosine (DES/IDS) assays may be unsatisfactory for clinical use due to lack of an appropriate internal standard or low throughput. A fast and reliable LC-MS method using a D(5)-DES as an internal standard for measuring urinary total DES/IDS was developed and validated in this study. The reportable range of this assay was 1.

Determination of oxolinic acid in cow's milk and human urine by means of a single-use phosphorimetric sensor.

A single-use phosphorimetric drop plane sensor for the determination of oxolinic acid (OXA) is proposed. The sensor was formed by a 30x16 mm(2) rectangular strip of Mylar type polyester as solid support that contained a circular sensing zone, 6 mm in diameter and 20 mum in thickness, formed by PVC plasticized with tributylphosphate adhered to its surface. When the strip was introduced for 1 hour into a sample solution, the analyte was retained in the sensing zone, making it possible to directly measure the phosphorescence intensity emitted by the OXA in the solid phase, at lambda(exc)=330 nm and lambda(em)=449 nm.