Proximity Tagging Identifies the Glycan-Mediated Glycoprotein Interactors of Galectin-1 in Muscle Stem Cells

Abstract

Myogenic differentiation, the irreversible developmental process where precursor myoblast muscle stem cells become contractile myotubes, is heavily regulated by glycosylation and glycan–protein interactions at the cell surface and the extracellular matrix. The glycan-binding protein galectin-1 has been found to be a potent activator of myogenic differentiation. While it is being explored as a potential therapeutic for muscle repair, a precise understanding of its glycoprotein interactors is lacking. These gaps are due in part to the difficulties of capturing glycan–protein interactions in live cells. Here, we demonstrate the use of a proximity tagging strategy coupled with quantitative mass-spectrometry-based proteomics to capture, enrich, and identify the glycan-mediated glycoprotein interactors of galectin-1 in cultured live mouse myoblasts. Our interactome dataset can serve as a resource to aid the determination of mechanisms through which galectin-1 promotes myogenic differentiation. Moreover, it can also facilitate the determination of the physiological glycoprotein counter-receptors of galectin-1. Indeed, we identify several known and novel glycan-mediated ligands of galectin-1 as well as validate that galectin-1 binds the native CD44 glycoprotein in a glycan-mediated manner.

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

“…With the addition of exogenous biotin (or related biotin analogues), biotinylation occurs with spatiotemporal control inside the targeted organelle. Pioneering studies have demonstrated the utility of these proximity based labeling methods in deciphering the protein interactome 39,40 , the protein composition of membraneless organelles 41,42 , and interrogation of kinase substrates 43 . Whether these methods are compatible with capturing the subcellular redoxome remains to be seen.Across all organelles, the mitochondrial cysteine redoxome is particularly intriguing.…”

GlycoID Proximity Labeling to Identify O‐GlcNAcylated Protein Interactomes in Live Cells

“…In addition to O‐GlcNAc labeling with GlycoID, other glycan types have been successfully mapped using proximity labeling. A sensitive probe for cell surface O‐linked and N‐linked glycans bearing galactose and N ‐acetylgalactosamine (GalNAc) have been reported using galectin‐APEX2 fusion constructs (Joeh et al., 2020; Vilen et al., 2021). Sialic acids on N‐linked glycans have been successfully labeled by linking sialic acid lectins (Siglecs) with metallaphotoredox MicroMap catalysts in a cell surface labeling technique called GlycoMap (Meyer et al., 2022).…”

(Glycan Binding) Activity‐Based Protein Profiling in Cells Enabled by Mass Spectrometry‐Based Proteomics

“…These biotinylated proteins can then be enriched for proteomic analysis. So far, our group has applied this technique to study galectin-3 [ 93 ] in the context of hepatic fibrosis and galectin-1 [ 94 ] in the context of skeletal muscle regeneration. Both techniques identified several known and novel interactors, which were successfully validated for direct glycan mediated binding to the GBP via ELISA.…”