Gdp mannose

Enzyme activity was assayed as previously described [51 (link)], using 200 ng α-1,6-mannobiose (Dextra Laboratories, Reading, UK) as the rhamnose acceptor and 500 µM UDP-L-rhamnose (Chemily Glycoscience; Peachtree Corners, GA, USA). The reaction products were analyzed by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) with a Dionex system (Thermo Fisher Scientific), using a CarboPac PA-100 column (4.6 × 250 mm) and a linear gradient of 10–100 mM sodium acetate in 100 mM NaOH at a flow rate of 0.8 mL min⁻¹ for 30 min. [52 (link)]. For assays including treatment with α-L-rhamnosidase, the reaction product of rhamnosyltransferase activity was mixed with 1 U enzyme (Megazyme, Bre, Ireland) and incubated for 60 min at 50 °C. The reactions were stopped by boiling for 10 min and subjected to monosaccharide separation by HPAEC-PAD using the following conditions: a CarboPac PA-200 analytical column (3 × 250 mm) with a CarboPac PA-200 guard column (3 × 50 mm) and an isocratic gradient of 3.2 mM NaOH with a flux rate of 0.15 mL min⁻¹ for 25 min [53 (link)]. Fluorescence-assisted carbohydrate analysis (FACE) was performed essentially as previously described [54 (link)]. UDP-glucose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-galactose were from Sigma-Aldrich.

For intact mass spectrometry analysis, 10 μg of purified MBP-RIPK1_DD was incubated with 1 μg of purified GST-NleB1, GST-NleB2 or mutants for 3 hours at 37°C in TBS supplemented with 10 mM MgCl₂ and 10 mM MnCl₂ in the presence of sugar donors. UDP-GlcNAc, UDP-glucose, UDP-GalNAc, UDP-galactose, UDP-GlcA or GDP-mannose (Sigma) were used at 10 mM, 50 μM or 0.5 μM where indicated and when incubated individually, or at 25 μM concentrations when co-incubated. For immunoblot analysis, 3 μg of purified MBP-RIPK1_DD or His-FADD were incubated with 1 μg of purified GST-NleB1, GST-NleB2 or mutants for 3 hours at 37°C in TBS supplemented with 10 mM MgCl₂ and 10 mM MnCl₂ in the presence of sugar donors at 25 μM.