Journal: Biomolecules

Article Title: Mouse WIF1 Is Only Modified with O -Fucose in Its EGF-like Domain III Despite Two Evolutionarily Conserved Consensus Sites

doi: 10.3390/biom10091250

Figure Lengend Snippet: In vitro O -fucosylation assay with WIF1 EGF-LDs III and V followed either by click chemistry and blotting technique or by trypsin digestion and mass spectrometry. ( A ) Isolated WT EGF-LDs III and V or T/A mutated on T 255 and T 319 respectively were first incubated with recPOFUT1. Then, azido-labeled GDP-fucose and click chemistry (CuAAC) was performed using alkynyl biotin to covalently attach biotin to fucose (red filled triangle) if transferred to an EGF-LD by recPOFUT1. After separation by SDS-PAGE and protein transfer, protein biotinylation was detected using streptavidin-HRP. Mouse NOTCH1 EGF-LD 26 (N1-EGF-LD 26), known to be modified with O -fucose in mouse NOTCH1, was used as a positive control. Positive signals resulted from successful in vitro O -linked azido-fucose transfer to EGF-LDs (upper panel), for which quantity and purity were checked by Coomassie blue-stained polyacrylamide gels (lower panel). ( B ) EGF-LD III and EGF-LD V, produced and purified from E. coli BL21 strain, were first independently incubated with recPOFUT1 and GDP-fucose to induce in vitro O -fucosylation. After reduction, alkylation and trypsin digestion, resulting peptides were analyzed by micro-LC multiple reaction monitoring-mass spectrometry (MRM-MS). Non-modified peptides were detected for EGF-LDs III and V (left panels) but the peptide modified with O -fucose was only revealed for EGF-LD III (right panels). The amino acid sequence of peptide generated by trypsin for each WIF1 EGF-LD is indicated with its O -fucosylation site in red and in brackets. Conserved cysteines (in orange) are numbered.

Article Snippet: Before click chemistry experiments, in vitro glycosyltransferase reactions were carried out with 1 μg recPOFUT1 mixed with 2 nmoles GDP-azido-fucose (R&D Systems Inc., Minneapolis, MN, USA) and 2 μg isolated EGF-LD or 1 μg recombinant WIF1 protein variant in 25 μL reaction buffer and incubated overnight at 37 °C.

Techniques: In Vitro, Mass Spectrometry, Isolation, Incubation, Labeling, SDS Page, Modification, Positive Control, Staining, Produced, Purification, Targeted Proteomics, Sequencing, Generated

Journal: Biomolecules

Article Title: Mouse WIF1 Is Only Modified with O -Fucose in Its EGF-like Domain III Despite Two Evolutionarily Conserved Consensus Sites

doi: 10.3390/biom10091250

Figure Lengend Snippet: In vitro O -fucose elongation assay with EGF-LD III or WIF1-V5-His proteins followed either by trypsin/thermolysin co-digestion and mass spectrometry ( A ) or by click chemistry and blotting technique ( B ). ( A ) EGF-LD III, produced and purified from E. coli BL21 strain, was co-incubated with recPOFUT1, recLFNG and appropriate donor nucleotide sugars (GDP-fucose, UDP-GlcNAc) to induce in vitro O -fucosylation and subsequent extension with GlcNAc. After reduction, alkylation and trypsin/thermolysin digestion, the peptide of interest FNGGT 255 C 3 of EGF-LD III, which was analyzed by micro-LC MRM-MS, was mainly modified with O -fucose ( O -Fuc) and also modified with the O -linked disaccharide GlcNAc-Fuc but at a lesser extent. ( B ) WT and mutated (T255A, T319A and T255/319A) recombinant mouse WIF1-V5-His, purified from stable Flp-In TM CHO cells, were subjected to incubation with recLFNG and UDP-Azido-GlcNAc. Then, click chemistry (CuAAC) was performed using alkynyl biotin to covalently attach biotin to GlcNAc if transferred to O -fucose carried by WIF1-V5-His. After separation by SDS-PAGE and protein transfer, protein biotinylation was detected using streptavidin-HRP. Positive signals corresponded to successful in vitro LFNG-mediated azido-GlcNAc transfer to recombinant WIF1 proteins (upper panel), for which quantity and purity were checked by silver nitrate-stained polyacrylamide gels (lower panel). RecLFNG, which remains bound to untransferred azido-GlcNAc, appeared labelled around 40 kDa after incubation with WT or T319A WIF1.

Article Snippet: Before click chemistry experiments, in vitro glycosyltransferase reactions were carried out with 1 μg recPOFUT1 mixed with 2 nmoles GDP-azido-fucose (R&D Systems Inc., Minneapolis, MN, USA) and 2 μg isolated EGF-LD or 1 μg recombinant WIF1 protein variant in 25 μL reaction buffer and incubated overnight at 37 °C.

Techniques: In Vitro, Mass Spectrometry, Produced, Purification, Incubation, Modification, Recombinant, SDS Page, Staining