Exo-Enzymatic Addition of Diazirine-Modified Sialic Acid to Cell Surfaces Enables Photocrosslinking of Glycoproteins

Abstract

Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ; however, use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here, we report an exoenzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves the chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAzylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

One-Step Selective Labeling of Native Cell Surface Sialoglycans by Exogenous α2,8-Sialylation

“…19 Furthermore, the use of photocross-linkable sialic acid probes with Cst-II could facilitate the identification of glycan-binding proteins that may use the α2,8disialyl epitope for recognition but have yet to be identified. 17,72 Appending complex synthetic ligands to the CMP-Neu5Ac donor derivative could expand applications of this SEEL strategy to cell-based glycan arrays, enabling the identification of high-affinity ligands for glycan-binding proteins or the development of cell-based therapies. 10,13,14,17,72,85 In addition, this Cst-II-SEEL approach may be useful in tandem with other glycosyltransferases for multiplex imaging of various cell surface glycan epitopes with different nucleotide sugar probes, such as through chemoenzymatic histology.…”

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

“…The Kohler and Capicciotti groups have used neuraminidases to cleave sialic acids from cell surfaces followed by the addition of diazirine-modified sialic acids using exogenous sialyltransferases. [89] Upon UV irradiation, GBPs and their glycoprotein ligands are crosslinked. While this strategy has not yet been coupled with MS-based identification, it represents a promising future approach to systems where metabolic incorporated of unnatural monosaccharides is inaccessible.…”

Exo-Enzymatic Cell-Surface Glycan Labeling for Capturing Glycan–Protein Interactions through Photo-Cross-Linking

“…In their report, they engineer different cell lines with a CMP-Neu5DAz derivative using an Arthrobacter ureafaciens sialidase and a bacterial α-2,6sialyltransferase fromPhotobacterium damsela and demonstrate photo-cross-linking using Daudi B-cells, highlighting the versatility of this exo-enzymatic labeling approach. 51 An exo-enzymatic approach is advantageous over an MOE strategy as the photo-cross-linking sugar can be installed on cells in a few hours, it is broadly applicable to various cell types, and cross-linking was enhanced compared to MOE. Furthermore, this method offers more control over the selective installation of the cross-linking sugar as well-defined glycan linkages, epitopes, and selective glycan subclass labeling can be achieved by harnessing the substrate specificity of the glycosyltransferase used.…”