Surfaceome Profiling Identifies Basigin-Chaperoned Protein Clients.

The surface proteome or "surfaceome" is a critical mediator of cellular biology, facilitating cell-to-cell interactions and communication with extracellular biomolecules. Constituents of the surfaceome can serve as biomarkers for changing cell states and as targets for pharmacological intervention. While some pathways of cell surface trafficking are well characterized to allow prediction of surface localization, some non-canonical trafficking mechanisms do not. Basigin (Bsg), a cell surface glycoprotein, has been shown to chaperone protein clients to the cell surface. However, understanding which proteins are served by Bsg is not always straightforward. To accelerate such identification, we applied a surfaceome proximity labeling method that is integrated with quantitative mass spectrometry-based proteomics to discern changes in the surfaceome of hepatic stellate cells that occur in response to the genetic loss of Bsg. Using this strategy, we observed that the loss of Bsg leads to corresponding reductions in the cell surface expression of monocarboxylate transporters MCT1 and MCT4. We also found that these relationships were unique to Bsg and not found in neuroplastin (Nptn), a related family member. These results establish the utility of the surfaceome proximity labeling method to determine clients of cell surface chaperone proteins.

Cell Surface Engineering Enables Surfaceome Profiling.

Cell surface proteins (CSPs) are vital molecular mediators for cells and their extracellular environment. Thus, understanding which CSPs are displayed on cells, especially in different cell states, remains an important endeavor in cell biology. Here, we describe the integration of cell surface engineering with radical-mediated protein biotinylation to profile CSPs. This method relies on the prefunctionalization of cells with cholesterol lipid groups, followed by sortase-catalyzed conjugation with an APEX2 ascorbate peroxidase enzyme. In the presence of biotin-phenol and H2O2, APEX2 catalyzes the formation of highly reactive biotinyl radicals that covalently tag electron-rich residues within CSPs for subsequent streptavidin-based enrichment and analysis by quantitative mass spectrometry. While APEX2 is traditionally used to capture proximity-based interactomes, we envisioned using it in a "baitless" manner on cell surfaces to capture CSPs. We evaluate this strategy in light of another CSP labeling method that relies on the presence of cell surface sialic acid. Using the APEX2 strategy, we describe the CSPs found in three mammalian cell lines and compare CSPs in adherent versus three-dimensional pancreatic adenocarcinoma cells.

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

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.

Mapping Interactions between Glycans and Glycan-Binding Proteins by Live Cell Proximity Tagging.

Interactions between glycans and glycan-binding proteins (GBPs) consist of weak, noncovalent, and transient binding events, making them difficult to study in live cells void of a static, isolated system. Furthermore, the glycans are often presented as protein glycoconjugates, but there are limited efforts to identify these proteins. Proximity labeling permits covalent tagging of the glycoprotein interactors to query GBP in live cells. Coupled with high-resolution mass spectrometry, it facilitates determination of the proteins bearing the interacting glycans. In this method, fusion protein constructs of a GBP of interest with a peroxidase enzyme allows for in situ spatiotemporal radical-mediated tagging of interacting glycoproteins in living cells that can be enriched for identification. Using this method, the capture and study of glycan-GBP interactions no longer relies on weak, transient interactions, and results in robust capture and identification of the interactome of a GBP while preserving the native cellular environment. This protocol focuses on (1) expression and characterization of a recombinant fusion protein consisting of a peroxidase and the GBP galectin-3, (2) corresponding in situ labeling and visualization of interactors, (3) and proteomic workflow and analysis of captured proteins for robust identification using mass spectrometry. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Expression, purification, and characterization of recombinant fusion protein Alternate Protocol 1: Manual Ni-NTA purification of recombinant fusion protein Basic Protocol 2: In situ proximity labeling and evaluation by fluorescence microscopy Alternate Protocol 2: Western blot analysis of in situ proximity labeling Basic Protocol 3: Proximity labeling of cells for quantitative MS-based proteomics with tandem mass tags.

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling.

Galectin-3 is a glycan-binding protein (GBP) that binds ß-galactoside glycan structures to orchestrate a variety of important biological events, including the activation of hepatic stellate cells and regulation of immune responses. While the requisite glycan epitopes needed to bind galectin-3 have long been elucidated, the cellular glycoproteins that bear these glycan signatures remain unknown. Given the importance of the three-dimensional (3D) arrangement of glycans in dictating GBP interactions, strategies that allow the identification of GBP receptors in live cells, where the native glycan presentation and glycoprotein expression are preserved, have significant advantages over static and artificial systems. Here we describe the integration of a proximity labeling method and quantitative mass spectrometry to map the glycan and glycoprotein interactors for galectin-3 in live human hepatic stellate cells and peripheral blood mononuclear cells. Understanding the identity of the glycoproteins and defining the structures of the glycans will empower efforts to design and develop selective therapeutics to mitigate galectin-3-mediated biological events.