Photocrosslinking approaches can be used to map interactome networks within the context of living cells. Photocrosslinking methods rely on use of metabolic engineering or genetic code expansion to incorporate photocrosslinking analogs of amino acids or sugars into cellular biomolecules. Immunological and mass spectrometry techniques are used to analyze crosslinked complexes, thereby defining specific interactomes. Because photocrosslinking can be conducted in native, cellular settings, it can be used to define context-dependent interactions. Photocrosslinking methods are also ideally suited for determining interactome dynamics, mapping interaction interfaces, and identifying transient interactions in which intrinsically disordered proteins and glycoproteins engage. Here we discuss the application of cell-based photocrosslinking to the study of specific problems in immune cell signaling, transcription, membrane protein dynamics, nucleocytoplasmic transport, and chaperone-assisted protein folding.

The plasma membrane of eukaryotic cells is coated with carbohydrates. By virtue of their extracellular position and recognizable chemical features, cell surface glycans mediate many receptor-ligand interactions. Recently, mammalian extracellular hydrolytic enzymes have been shown to modify the structure of cell surface glycans and consequently, alter their binding properties. These cell surface glycan remodeling events can cause rapid changes in critical signal transduction phenomena. This review highlights recent studies on the roles of eukaryotic extracellular sialidases, sulfatases, and a deacetylase in regulation of intracellular signaling. We also describe possible therapies that target extracellular glycan remodeling processes and discuss the potential for new discoveries in this area.The glycocalyx stands between extracellular signals and intracellular responses. But the glycoproteins, glycolipids and proteoglycans that comprise the glycocalyx are not simply a barrier; rather, they serve essential roles in the transduction of signals from the outside to the inside of cells. Indeed, most cell surface receptors are glycosylated and many specifically recognize glycans that are attached to their ligands. Cell surface and extracellular glycans have been demonstrated to play critical roles in the control and modulation of a variety of signal transduction pathways (1-4). For example, in embryonic development, signaling through the Notch receptor requires that the receptor be modified with O-fucose glycans (5). If these glycans are absent, gestational death occurs. In the adult nervous system, the myelin-associated glycoprotein (MAG) binds to glycoproteins and glycolipids on axons, forming signaling complexes that inhibit axonal outgrowth (6). And in the immune system, specific cell surface glycans are essential components of the signal transduction pathways that lead to processes such as B-cell receptor activation and T-cell apoptosis (7,8). Glycosylation also regulates intracellular signal transduction events: for example, the addition of O-linked Nacetylglucosamine (O-GlcNAc) to histone lysine methyl transferase MLL5 activates this enzyme, causing it to methylate histone H3 and thereby leading to cell lineage determination (9).As more signaling roles for glycans are identified, an essential task is to understand the mechanisms that regulate the glycosylation state of a cell or molecule (Figure 1). Most glycans are assembled in the endoplasmic reticulum (ER) and Golgi through the coordinate action of many membrane-associated glycosyltransferases and other glycan-modifying enzymes, such as sulfotransferases and epimerases. ER-and Golgi-resident glycosyltransferases require nucleotide-sugar donors, such as uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), guanidine diphosphate-fucose (GDP-fucose), and cytidine monophosphate-sialic acids (CMPsialic acids), that are synthesized in the cytoplasm or nucleus. Once assembled in the secretory *To whom correspondence should be addressed (Jennifer.Kohler@UTSou...

Sialidases hydrolytically remove sialic acids from sialylated glycoproteins and glycolipids. Sialidases are widely distributed in nature and sialidase-mediated desialylation is implicated in normal and pathological processes. However, mechanisms by which sialidases exert their biological effects remain obscure, in part because sialidase substrate preferences are poorly defined. Here we report the design and implementation of a sialidase substrate specificity assay based on chemoselective labeling of sialosides. We show that this assay identifies components of glycosylated substrates that contribute to sialidase specificity. We demonstrate that specificity of sialidases can depend on structure of the underlying glycan, a characteristic difficult to discern using typical sialidase assays. Moreover, we discovered that S. pneumoniae sialidase NanC strongly prefers sialosides containing the Neu5Ac form of sialic acid, versus those that contain Neu5Gc. We propose using this approach to evaluate sialidase preferences for diverse potential substrates.

Sialic acid-mediated interactions play critical roles on the cell surface, providing impetus for the development of methods to study this important monosaccharide. In particular, photocrosslinking sialic acids incorporated onto cell surfaces have allowed covalent capture of transient interactions between sialic acids and sialic acid-recognizing proteins via crosslinking. However, natural sialic acids also present on the cell surface compete with photocrosslinking sialic acids in binding events, limiting crosslinking yields. In order to improve the utility of one such photocrosslinking sialic acid, SiaDAz, we examined a number of sialidases, enzymes that remove sialic acids from glycoconjugates, to find one that would cleave natural sialic acids but remain inactive toward SiaDAz. Using this sialidase, we improved SiaDAz-mediated crosslinking of an anti-sialyl Lewis X antibody and of endoglin. This protocol can be applied generally to sialic acid-mediated interactions and will facilitate identification of sialic acid binding partners.