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. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the -3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.

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 0.1-160 ng/ml. Serum/plasma DES/IDS level was stable at room temperature or 4°C for 20 h, and for three freeze-thaw cycles. Interferences from endogenous compounds and ion suppression/enhancing effect were also evaluated. Our results suggest the absolute necessity of using an IS in the measurement. We found that serum/plasma DES/IDS levels from patients with chronic obstructive pulmonary disease and cystic fibrosis were significantly higher compared with healthy smokers. CONCLUSION: These results demonstrate that the LC-MS/MS method provides sensitive, reproducible and accurate quantification of total serum/plasma DES/IDS.

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. fumigatus possesses an active AfAGM1 (A. fumigatus N-acetylphosphoglucosamine mutase), a key enzyme in the biosynthesis of UDP (uridine diphosphate)-GlcNAc (N-acetylglucosamine), the nucleotide sugar donor for chitin synthesis. A conditional agm1 mutant revealed the gene to be essential. Reduced expression of agm1 resulted in retarded cell growth and altered cell wall ultrastructure and composition. The crystal structure of AfAGM1 revealed an amino acid change in the active site compared with the human enzyme, which could be exploitable in the design of selective inhibitors. AfAGM1 inhibitors were discovered by high-throughput screening, inhibiting the enzyme with IC50s in the low µM range. Together, these data provide a platform for the future development of AfAGM1 inhibitors with antifungal activity.

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 ß-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)-ß-activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro-inflammatory cytokines such as transforming growth factor-ß, 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. With the help of a novel O-GlcNAc site-specific antibody, we demonstrate that O-GlcNAcylation of TAB1 is induced by IL-1 and osmotic stress, known inducers of the TAK1 signalling cascade. By reintroducing wild-type or an O-GlcNAc-deficient mutant TAB1 (S395A) into Tab1(-/-) mouse embryonic fibroblasts, we determined that O-GlcNAcylation of TAB1 is required for full TAK1 activation upon stimulation with IL-1/osmotic stress, for downstream activation of nuclear factor κB and finally production of IL-6 and TNFα. This is one of the first examples of a single O-GlcNAc site on a signalling protein modulating a key innate immunity signalling pathway.

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. RESULTS: 390 study participants were recruited from the following groups: healthy volunteers, stable asthma, stable and 'during an exacerbation' COPD. Compared with healthy non-smokers, we found increased urinary or blood desmosine levels in patients with COPD, but no differences in patients with asthma or healthy smokers. The elevation of urinary desmosine levels was associated with the exacerbation status in patients with COPD. Approximately 40% of patients with stable and 'during an exacerbation' COPD showed elevated blood desmosine levels. Blood desmosine levels were strongly associated with age and were negatively correlated with lung diffusing capacity for carbon monoxide. CONCLUSION: The results suggest that urinary desmosine levels are raised by exacerbations of COPD whereas blood desmosine levels are elevated in a subgroup of patients with stable COPD and reduced lung diffusing capacity. The authors speculate that a raised blood desmosine level may identify patients with increased elastin degradation suitable for targeted therapy. Future prospective studies are required to investigate this hypothesis.

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 1.0 and 480.0 ng/mL. The intra- and interassay imprecision, accuracy, and recovery for quality control samples were within acceptable range (<25%). Urinary total DES/IDS level was stable at room temperature or 4 degrees C for 20 h, and for three freeze/thaw cycles. The assay was employed to measure urine samples from COPD patients and demographically matched healthy volunteers. The total urinary DES/IDS levels were approximately 3-fold higher in COPD patients compared to healthy volunteers. The suitability of using urinary free DES to estimate elastin degradation was also evaluated in a second cohort. Despite urinary free and total DES/IDS levels being highly correlated, our data suggest that urinary total DES/IDS level is a preferred biomarker for elastin degradation. These results demonstrate that the LC-MS/MS method provides sensitive, reproducible and accurate quantification of urinary total DES/IDS as a biomarker for monitoring elastin degradation in diseases such as COPD.