Cell membrane glycoproteins are often highly fucosylated and sialylated, playing important roles in protein signaling, binding, and cellular processing functions. However, due to technical limitations, it is difficult to characterize and observe fucose-mediated protein-protein interactions.
Carlito B. Lebrilla's team from UC Davis published an article titled "Protein oxidation of fucose environments (POFE) reveals fucose–protein interactions" in Chemical Science, describing a proximity-labeling-based method to decipher the binding environment of cellular fucosylated glycoproteins in situ. The authors call this method "Protein oxidation of fucose environments (POFE)".
In previous studies, the authors used an iron-based proximity tag, FeBABE, to identify the Sialic Acid environment on the cell surface. Kizuka et al. reported a new 7-alkynyl-fucose (7AlkFuc) probe that can be metabolically incorporated into cell surface fucosylated glycoproteins. The authors wanted to use this probe to metabolically label cells, and based on the alkynyl group on fucose, they coupled the proximity labeling probe azido-FeBABE, and the catalyst motif acted as a proximity labeling probe. With the addition of hydrogen peroxide, fucose-azido-FeBABE catalyzed the formation of hydroxyl radicals, which in turn oxidized the amino acids near the labeled fucose residues, and then identified the oxidized peptides.
Fig. 1 Protein oxidation of fucose environments (POFE). (Xie, et al., 2024)
First, the authors tested three common fucose reporter probes, 6-azido-fucose (6AzFuc), 6-alkynyl-fucose (6AlkFuc), and 7AlkFuc. Among them, 7AlkFuc had the best incorporation effect, modifying up to 20% of fucose-containing Glycans on PNT2 cells. 6AzFuc had only 5% incorporation after three days of treatment, while 6AlkFuc was found to generally inhibit cellular fucosylation. Therefore, 7AlkFuc was chosen as a fucose reporter probe.
After optimizing the incorporation conditions, cells were metabolically labeled and coupled to FeBABE. Since peptide oxidation can occur during biological processes or common sample preparation workflows, a baseline of cellular background oxidation was first determined through control experiments. The experiments yielded over 150 oxidized peptides corresponding to over 80 proteins from PNT2 and Caco-2 cells. The authors compared the oxidized proteins enriched using the fucose probe with previous results based on the sialic acid probe. For PNT2 cells, comparison of oxidized proteins with the fucose probe revealed a 60% overlap with the sialic acid probe. Comparison with Caco-2 using the same Fucose probe revealed less than 30% similarity. The higher percentage of fucosylated glycans in Caco-2 cells resulted in a greater proportion of unique proteins not identified in a sialic acid environment.
To explore the spatial distribution of proteins near fucose, the authors treated with hydrogen peroxide for different times and monitored the extent of oxidation using oxidative proteomics. The total number of oxidized proteins increased with increasing incubation time. After 10 minutes of reaction, other glycoproteins and several non-glycosylated proteins were oxidized. The number of proteins involved in carbohydrate binding increased significantly after 15 minutes of incubation, indicating that proteins involved in these pathways are oxidized due to interactions with fucosylated glycoproteins.
The authors then evaluated the interaction of a specific glycoprotein, LEG3, and found a similar pattern for PNT2 and Caco-2, where carbohydrate binding, cell adhesion, and signaling proteins increased after 15 minutes of incubation time. This suggests a time-dependent transmission of oxidation based on proximity to the fucosylated source glycoprotein. Oxidized proteins were further annotated based on cellular component analysis, all of which were found in the extracellular space or plasma membrane. After 30 minutes, an additional 20 proteins were identified, but only 4 were annotated as plasma membrane proteins.
Identification of specific oxidation sites can provide direct experimental evidence for interactions between individual proteins, revealing potential interaction partners of surface glycoproteins. LEG3 interacts with LG3BP, which is fucosylated, and the Phe190 residue in the LEG3 protein is oxidized in both PNT2 and Caco-2 cells. The authors obtained the 3D structures of LEG3 and LG3BP and performed protein-protein docking using HADDOCK. To identify the specific LG3BP N-glycosylation site responsible for the interaction, the authors specified the interaction between LEG3-Phe190 (oxidized residue) and LG3BP-Asn69/125/192/398/551. After verification using HADDOCK modeling predictions and other methods, the results showed that the glycan in LG3BP-Asn551 was responsible for the oxidation of LEG3-Phe190.
Afterwards, N-glycan residues were incorporated on LG3BP-Asn551 to determine the effect of specific N-glycan residues on sugar-protein interactions. The authors simulated several tetra-antennary N-glycans on Asn551, and after equilibrium, the contact frequency and type between N-glycans and amino acid residues were monitored. First, it was observed that the frequency of hydrogen bond interactions between N-glycans and proteins increased greatly after the addition of fucose or sialic acid residues, and the effect became more obvious after the addition of sialic acid and fucose. Based on the type of interaction analysis, the results showed that the addition of core fucose to N-glycans significantly changed the conformation of N-glycans, exposing the core N-acetylglucosamine residues to more interactions, especially with LEG3-Arg183.
In summary, by combining protein oxidation information of the fucose environment with Glycoproteomic Analysis, specific fucose-mediated interactions can be studied in detail.
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