Fig. 4: FUT10 and FUT11 are POFUTs that are responsible for the O-fucosylation of MMRN1 EMI domains.
From: FUT10 and FUT11 are protein O-fucosyltransferases that modify protein EMI domains
a, 0.1 μM purified GFP–FUT10, GFP–FUT11 or GFP (negative control) was incubated with 0.5 μM non-fucosylated N-terminal EMI and 100 μM GDP-fucose for indicated time periods. Reaction products were reduced, alkylated, digested with trypsin and analyzed by nano LC–MS/MS. Relative abundances of O-fucosylation for each reaction were calculated from the EICs of peptides containing T216 or T265 O-fucose site and plotted as time-dependent curves. Data are presented as mean ± s.d. from biological triplicates using three batches of purified enzymes (Supplementary Fig. 8). b, Substrate concentration-dependent kinetics of GFP–FUT10 and GFP–FUT11. 50 nM purified GFP–FUT10/11 was incubated with varied concentrations of non-fucosylated N-terminal EMI, 100 μM Ultra Pure GDP-fucose and 0.3 mM MnCl2 for 15 min. Reaction products were reduced, alkylated, digested with trypsin and analyzed by nano LC–MS/MS. O-fucosylation stoichiometry on T216 site and T265 site was quantified from EICs and converted into product concentration. Kinetic analysis was performed with nonlinear Michaelis–Menten fitting in Prism 7. GDP-fucose concentration-dependent kinetics of GFP–FUT11 are in Supplementary Fig. 14. c, Data points with substrate concentrations ranging from 0 μM to 2.5 μM in b were zoomed in to show the distinct kinetic profiles of GFP–FUT10 and GFP–FUT11 with low substrate concentration. Data are presented as mean ± s.d. from biological triplicates using three batches of purified enzymes (Supplementary Fig. 13). d, Kinetic parameters calculated using the data from b with nonlinear Michaelis–Menten fitting in Prism 7.
