A photo-cross-linking GlcNAc analog enables covalent capture of N-linked glycoprotein-binding partners on the cell surface.

N-glycans are displayed on cell-surface proteins and can engage in direct binding interactions with membrane-bound and secreted glycan-binding proteins (GBPs). Biochemical identification and characterization of glycan-mediated interactions is often made difficult by low binding affinities. Here we describe the metabolic introduction of a diazirine photo-cross-linker onto N-acetylglucosamine (GlcNAc) residues of N-linked glycoproteins on cell surfaces. We characterize sites at which diazirine-modified GlcNAc is incorporated, as well as modest perturbations to glycan structure. We show that diazirine-modified GlcNAc can be used to covalently cross-link two extracellular GBPs, galectin-1 and cholera toxin subunit B, to cell-surface N-linked glycoproteins. The extent of cross-linking correlates with display of the preferred glycan ligands for the GBPs. In addition, covalently cross-linked complexes could be isolated, and protein components of cross-linked N-linked glycoproteins were identified by proteomics analysis. This method may be useful in the discovery and characterization of binding interactions that depend on N-glycans.

Mass Spectrometric Method for the Unambiguous Profiling of Cellular Dynamic Glycosylation.

Various biological processes at the cellular level are regulated by glycosylation which is a highly microheterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through metabolic incorporation of non-natural sugars into glycan epitopes and their detection using bio-orthogonal probes. However, this approach possesses a significant drawback due to nonspecific background reactions and ambiguity of non-natural sugar metabolism. Here, we report a probe-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry (MS). The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc) biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC (zwitterionic-hydrophilic interaction chromatography) based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N- and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this, we could deduce interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput, as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.

Cell type and receptor identity regulate cholera toxin subunit B (CTB) internalization.

Cholera toxin (CT) is a secreted bacterial toxin that binds to glycoconjugate receptors on the surface of mammalian cells, enters mammalian cells through endocytic mechanisms and intoxicates mammalian cells by activating cytosolic adenylate cyclase. CT recognizes cell surface receptors through its B subunit (CTB). While the ganglioside GM1 has been historically described as the sole receptor, CTB is also capable of binding to fucosylated glycoconjugates, and fucosylated molecules have been shown to play a functional role in host cell intoxication by CT. Here, we use colonic epithelial and respiratory epithelial cell lines to examine how two types of CT receptors-gangliosides and fucosylated glycoconjugates-contribute to CTB internalization. We show that fucosylated glycoconjugates contribute to CTB binding to and internalization into host cells, even when the ganglioside GM1 is present. The contributions of the two classes of receptors to CTB internalization depend on cell type. Additionally, in a cell line that harbours both classes of receptors, gangliosides dictate the efficiency of CTB internalization. Together, the results lend support to the idea that fucosylated glycoconjugates play a functional role in CTB internalization, and suggest that CT internalization depends on both receptor identity and cell type.

Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins.

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3ß/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.

GM1 ganglioside-independent intoxication by Cholera toxin.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.

Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells.

Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium.

Effects of altered sialic acid biosynthesis on N-linked glycan branching and cell surface interactions.

GNE (UDP-GlcNAc 2-epimerase/ManNAc kinase) myopathy is a rare muscle disorder associated with aging and is related to sporadic inclusion body myositis, the most common acquired muscle disease of aging. Although the cause of sporadic inclusion body myositis is unknown, GNE myopathy is associated with mutations in GNE. GNE harbors two enzymatic activities required for biosynthesis of sialic acid in mammalian cells. Mutations to both GNE domains are linked to GNE myopathy. However, correlation between mutation-associated reductions in sialic acid production and disease severity is imperfect. To investigate other potential effects of GNE mutations, we compared sialic acid production in cell lines expressing wild type or mutant forms of GNE. Although we did not detect any differences attributable to disease-associated mutations, lectin binding and mass spectrometry analysis revealed that GNE deficiency is associated with unanticipated effects on the structure of cell-surface glycans. In addition to exhibiting low levels of sialylation, GNE-deficient cells produced distinct N-linked glycan structures with increased branching and extended poly-N-acetyllactosamine. GNE deficiency may affect levels of UDP-GlcNAc, a key metabolite in the nutrient-sensing hexosamine biosynthetic pathway, but this modest effect did not fully account for the change in N-linked glycan structure. Furthermore, GNE deficiency and glucose supplementation acted independently and additively to increase N-linked glycan branching. Notably, N-linked glycans produced by GNE-deficient cells displayed enhanced binding to galectin-1, indicating that changes in GNE activity can alter affinity of cell-surface glycoproteins for the galectin lattice. These findings suggest an unanticipated mechanism by which GNE activity might affect signaling through cell-surface receptors.

Fucosylation and protein glycosylation create functional receptors for cholera toxin.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera.

Photoaffinity probes for studying carbohydrate biology.

Carbohydrates and carbohydrate-containing biomolecules engage in binding events that underlie many essential biological processes. Yet these carbohydrate-mediated interactions are often poorly characterized, due to their low affinities and heterogenous natures. The use of photocrosslinking functional groups offers a way to photochemically capture carbohydrate-containing complexes, which can be isolated for further analysis. Here we survey progress in the synthesis and use of carbohydrate-based photoprobes, reagents that incorporate carbohydrates or their analogs, photocrosslinking moieties, and affinity purification handles. Carbohydrate photoprobes, used in combination with modern mass spectrometry methods, can provide important new insights into the cellular roles of carbohydrates and glycosylated molecules.

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.

Transient-state kinetic analysis of transcriptional activator·DNA complexes interacting with a key coactivator.

Several lines of evidence suggest that the prototypical amphipathic transcriptional activators Gal4, Gcn4, and VP16 interact with the key coactivator Med15 (Gal11) during transcription initiation despite little sequence homology. Recent cross-linking data further reveal that at least two of the activators utilize the same binding surface within Med15 for transcriptional activation. To determine whether these three activators use a shared binding mechanism for Med15 recruitment, we characterized the thermodynamics and kinetics of Med15·activator·DNA complex formation by fluorescence titration and stopped-flow techniques. Combination of each activator·DNA complex with Med15 produced biphasic time courses. This is consistent with a minimum two-step binding mechanism composed of a bimolecular association step limited by diffusion, followed by a conformational change in the Med15·activator·DNA complex. Furthermore, the equilibrium constant for the conformational change (K(2)) correlates with the ability of an activator to stimulate transcription. VP16, the most potent of the activators, has the largest K(2) value, whereas Gcn4, the least potent, has the smallest value. This correlation is consistent with a model in which transcriptional activation is regulated at least in part by the rearrangement of the Med15·activator·DNA ternary complex. These results are the first detailed kinetic characterization of the transcriptional activation machinery and provide a framework for the future design of potent transcriptional activators.

Impact of nonnatural amino acid mutagenesis on the in vivo function and binding modes of a transcriptional activator.

Protein-protein interactions play an essential role in cellular function, and methods to discover and characterize them in their native context are of paramount importance for gaining a deeper understanding of biological networks. In this study, an enhanced nonsense suppression system was utilized to incorporate the nonnatural amino acid p-benzoyl-L-phenylalanine (pBpa) throughout the transcriptional activation domain of the prototypical eukaryotic transcriptional activator Gal4 in vivo (S. cerevisiae). Functional studies of the pBpa-containing Gal4 mutants suggest that this essential binding interface of Gal4 is minimally impacted by these substitutions, with both transcriptional activity and sensitivity to growth conditions maintained. Further supporting this are in vivo cross-linking studies, including the detection of a key binding partner of Gal4, the inhibitor protein Gal80. Cross-linking with a range of pBpa-containing mutants revealed a Gal4 x Gal80 binding interface that extends beyond that previously predicted by conventional strategies. Thus, this approach can be broadened to the discovery of novel binding partners of transcription factors, information that will be critical for the development of therapeutically useful small molecule modulators of these protein-protein interactions.