Castanospermine

The α-mannosidase and α-glucosidase activities were measured using the fluorogenic substrates 4-methylumbellyferyl-α-D-mannopyranoside (Sigma) and 4-methylumbellyferyl-α-D-glucopyranoside (Sigma), respectively. Cells were harvested by centrifugation, broken with glass beads in a FastPrep machine (Thermo Scientific), and the homogenate centrifuged at 21,000 × g and 4°C for 10 min. The supernatant was saved and used to quantify enzyme activity as reported (Mora-Montes et al., 2004 (link); Robledo-Ortiz et al., 2012 (link)). Aliquots containing 100 μg protein were resuspended in 10 mM phosphate buffer, pH 7.0, in a total volume of 200 μL. Then, 40 μM of either 4-methylumbellyferyl-α-D-mannopyranoside or 4-methylumbellyferyl-α-D-glucopyranoside were added and the reaction incubated at 37°C for 30 min. The reaction was stopped by addition of 3.3 mL 50 mM glycine-NaOH, pH 11.0, and fluorescence of the released 4-methylumbellyferone (MU) was measured in a Perkin-Elmer LS-5B luminescence spectrofluorometer, with excitation and emission set at 350 nm and 440 nm, respectively. Total enzyme activity was expressed as nmoles of MU min^-1 total protein^-1. In assays to inhibit mannosyl-oligosaccharide glucosidase, 10 μM castanospermine (Sigma) was added prior the incubation step at 37°C (Lopes-Bezerra et al., 2015 (link)).

For co-expression with different constructs, agrobacteria were mixed, infiltrated into leaves and harvested at the indicated time points. For the block of α-mannosidases, 50 μM kifunensine (Santa Cruz Biotechnology) was co-infiltrated with the agrobacteria suspension and for the block of α-glucosidases 200 μM castanospermine (Sigma-Aldrich) was co-infiltrated. Crude protein extracts or purified protein were subjected to SDS-PAGE under reducing or non-reducing (no reducing agent, no boiling of samples) conditions and after blotting the proteins were detected using anti-His (Thermo Fisher Scientific), anti-RBD (Sino Biological) anti-GFP-HRP (Miltenyi Biotec), anti-RFP (Chromotek) and anti-HA (Roche) antibodies. For deglycosylation, proteins were denatured and incubated with or without Endo H or PNGase F (both from NEB) according to the manufacturer’s instructions.

HMG-CoA reductase inhibitors were a kind gift of Bayer Pharmaceuticals PLC and AstraZeneca PLC. Deoxymannojirimycin and tunicamycin were purchased from Calbiochem. Farnesyl pyrophosphate and castanospermine were purchased from Sigma.