Protein oxidation of fucose environments (POFE) reveals fucose-protein interactions

Abstract

Cell membrane glycoproteins are generally highly fucosylated and sialylated, and post-translational modifications play important roles in the proteins' functions of signaling, binding and cellular processing. For these reasons, methods for measuring sialic acid-mediated protein-protein interactions have been developed. However, determining the role of fucose in these interactions has been limited by technological barriers that have thus far hindered the ability to characterize and observe fucose-mediated protein-protein interactions. Herein, we describe a method to metabolically label mammalian cells with modified fucose, which incorporates a bioorthogonal group into cell membrane glycoproteins thereby enabling the characterization of cell-surface fucose interactome. Copper-catalyzed click chemistry was used to conjugate a proximity labeling probe, azido-FeBABE. Following the addition of hydrogen peroxide (H₂O₂), the fucose-azido-FeBABE catalyzed the formation of hydroxyl radicals, which in turn oxidized the amino acids in the proximity of the labeled fucose residue. The oxidized peptides were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Variations in degree of protein oxidation were obtained with different H₂O₂ reaction times yielding the acquisition of spatial information of the fucose-interacting proteins. In addition, specific glycoprotein-protein interactions were constructed for Galectin-3 (LEG3) and Galectin-3-binding protein (LG3BP) illustrating the further utility of the method. This method identifies new fucose binding partners thereby enhancing our understanding of the cell glycocalyx.

Scheme 1. A representation of the 7-alkynyl fucose probe incorporated into fucosylated glycans on the cell surface, followed by the conjugation of the oxidative probe. Upon treatment with hydrogen peroxide, hydroxyl radicals were generated at the fucose site and oxidized the proteins in proximity.

Fig. 1. (a) N-glycomic profiles of 7-alkynyl fucose-treated and control PNT2 cells analyzed using HPLC-chip-QTOF MS. Over 200 glycans were identified in the chromatograms and were color-coded based on the glycan subtypes. Putative structures were annotated according to accurate masses and glycan compositions. (b) The numbers of oxidized proteins and peptides in PNT2 and Caco-2 cell lines generated using the POFE (protein oxidation of fucose environments) method. (c) The overlap of oxidized proteins identified from PNT2 cells using the POFE and POSE (protein oxidation of sialic acid environments) methods. (d) The overlap of oxidized proteins identified from Caco-2 cells using the POFE and POSE methods.

Fig. 2. (a) The number of oxidized proteins in both PNT2 and Caco-2 cell lines increases with longer H₂O₂ incubation times. (b) Likewise, the number of proteins involved in carbohydrate-binding, cell adhesion, signaling, and other pathways increases with longer times. This translates to increasingly larger oxidation networks in both (c) PNT2 and (d) Caco-2 cells. Proteins in the oxidation networks are categorized and color-coded into different biological pathways based on being the source protein (green) or molecular functions: carbohydrate binding (blue), signaling (yellow), cell adhesion (pink), cell adhesion and signaling (orange), and other pathways (white).

Fig. 3. In both (a) PNT2 and (b) Caco-2 cells, the oxidation network of LEG3 increases with increasing H₂O₂ incubation times. In PNT2, the proteins that are being oxidized by LGALS3 glycans are involved in carbohydrate-binding, cell adhesion, and signaling pathways while in Caco2, the proteins interacting with LGALS3 are involved in cell adhesion and other pathways. Proteins in the oxidation networks are categorized and color-coded into different biological pathways based on being the source protein (green) or molecular functions: carbohydrate binding (blue), signaling (yellow), cell adhesion (pink), cell adhesion and signaling (orange), and other pathways (white).

Fig. 4. Modeled glycan–protein interactions between LEG3 and LG3BP–ASN511 N-glycan. The LG3BP–ASN511 glycosite was modeled with glycans (a) Hex₇HexNAc₆, (b) Hex₇HexNAc₆Fuc₁, (c) Hex₇HexNAc₆NeuAc₁, and (d) Hex₇HexNAc₆Fuc₁NeuAc₁. Frequency of protein interactions with glycan residues, including mannose (Man), N-acetylglucosamine (GlcNAc), galactose (Gal), fucose (Fuc), and sialic acid (NeuAc), was counted and classified as Hbond, electrostatic, or hydrophobic.