Publications by authors named "Jennifer J Kohler"

50 Publications

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

ACS Chem Biol 2020 10 4;15(10):2692-2701. Epub 2020 Sep 4.

Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States.

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, -acetyl-d-galactosamine (GalNAc) and -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.
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http://dx.doi.org/10.1021/acschembio.0c00453DOI Listing
October 2020

Human UDP-galactose 4'-epimerase (GALE) is required for cell-surface glycome structure and function.

J Biol Chem 2020 01 9;295(5):1225-1239. Epub 2019 Dec 9.

Department of Biochemistry, Duke University, Durham, North Carolina 27710

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. , signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4'-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.
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http://dx.doi.org/10.1074/jbc.RA119.009271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996900PMC
January 2020

Not All Quiet on the Sugar Front: Glycan Combatants in Host-Pathogen Interactions.

Biochemistry 2020 09 7;59(34):3061-3063. Epub 2019 Oct 7.

Department of Biochemistry, UT Southwestern Medical Center, Dallas, Texas 75390, United States.

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http://dx.doi.org/10.1021/acs.biochem.9b00524DOI Listing
September 2020

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

Interface Focus 2019 Apr 15;9(2):20180076. Epub 2019 Feb 15.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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.
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http://dx.doi.org/10.1098/rsfs.2018.0076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388018PMC
April 2019

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

Proc Natl Acad Sci U S A 2018 06 21;115(23):5956-5961. Epub 2018 May 21.

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710;

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 14-3-3β/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.
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http://dx.doi.org/10.1073/pnas.1722437115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003352PMC
June 2018

GM1 ganglioside-independent intoxication by Cholera toxin.

PLoS Pathog 2018 02 12;14(2):e1006862. Epub 2018 Feb 12.

Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.

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.
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http://dx.doi.org/10.1371/journal.ppat.1006862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825173PMC
February 2018

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

ACS Infect Dis 2018 05 22;4(5):758-770. Epub 2018 Feb 22.

Department of Microbiology and Immunology, Institute of Biomedicine , University of Gothenburg , SE-40530 Gothenburg , Sweden.

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.
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http://dx.doi.org/10.1021/acsinfecdis.7b00085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948155PMC
May 2018

Hyposialylated IgG activates endothelial IgG receptor FcγRIIB to promote obesity-induced insulin resistance.

J Clin Invest 2018 01 27;128(1):309-322. Epub 2017 Nov 27.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Type 2 diabetes mellitus (T2DM) is a common complication of obesity. Here, we have shown that activation of the IgG receptor FcγRIIB in endothelium by hyposialylated IgG plays an important role in obesity-induced insulin resistance. Despite becoming obese on a high-fat diet (HFD), mice lacking FcγRIIB globally or selectively in endothelium were protected from insulin resistance as a result of the preservation of insulin delivery to skeletal muscle and resulting maintenance of muscle glucose disposal. IgG transfer in IgG-deficient mice implicated IgG as the pathogenetic ligand for endothelial FcγRIIB in obesity-induced insulin resistance. Moreover, IgG transferred from patients with T2DM but not from metabolically healthy subjects caused insulin resistance in IgG-deficient mice via FcγRIIB, indicating that similar processes may be operative in T2DM in humans. Mechanistically, the activation of FcγRIIB by IgG from obese mice impaired endothelial cell insulin transcytosis in culture and in vivo. These effects were attributed to hyposialylation of the Fc glycan, and IgG from T2DM patients was also hyposialylated. In HFD-fed mice, supplementation with the sialic acid precursor N-acetyl-D-mannosamine restored IgG sialylation and preserved insulin sensitivity without affecting weight gain. Thus, IgG sialylation and endothelial FcγRIIB may represent promising therapeutic targets to sever the link between obesity and T2DM.
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http://dx.doi.org/10.1172/JCI89333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749535PMC
January 2018

A Conserved Splicing Silencer Dynamically Regulates O-GlcNAc Transferase Intron Retention and O-GlcNAc Homeostasis.

Cell Rep 2017 08;20(5):1088-1099

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Modification of nucleocytoplasmic proteins with O-GlcNAc regulates a wide variety of cellular processes and has been linked to human diseases. The enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) add and remove O-GlcNAc, but the mechanisms regulating their expression remain unclear. Here, we demonstrate that retention of the fourth intron of OGT is regulated in response to O-GlcNAc levels. We further define a conserved intronic splicing silencer (ISS) that is necessary for OGT intron retention. Deletion of the ISS in colon cancer cells leads to increases in OGT, but O-GlcNAc homeostasis is maintained by concomitant increases in OGA protein. However, the ISS-deleted cells are hypersensitive to OGA inhibition in culture and in soft agar. Moreover, growth of xenograft tumors from ISS-deleted cells is compromised in mice treated with an OGA inhibitor. Thus, ISS-mediated regulation of OGT intron retention is a key component in OGT expression and maintaining O-GlcNAc homeostasis.
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http://dx.doi.org/10.1016/j.celrep.2017.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5588854PMC
August 2017

Chemical Modulation of Protein O-GlcNAcylation via OGT Inhibition Promotes Human Neural Cell Differentiation.

ACS Chem Biol 2017 08 19;12(8):2030-2039. Epub 2017 Jun 19.

Department of Chemistry, Stanford University , Stanford, California 94305, United States.

The enzymes that determine protein O-GlcNAcylation, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), act on key transcriptional and epigenetic regulators, and both are abundantly expressed in the brain. However, little is known about how alterations in O-GlcNAc cycling affect human embryonic stem cell (hESC) neural differentiation. Here, we studied the effects of perturbing O-GlcNAcylation during neural induction of hESCs using the metabolic inhibitor of OGT, peracetylated 5-thio-N-acetylglucosamine (Ac-5SGlcNAc). Treatment of hESCs with Ac-5SGlcNAc during induction limited protein O-GlcNAcylation and also caused a dramatic decrease in global levels of UDP-GlcNAc. Concomitantly, a subpopulation of neural progenitor cells (NPCs) acquired an immature neuronal morphology and expressed early neuronal markers such as β-III tubulin (TUJ1) and microtubule associated protein 2 (MAP2), phenotypes that took longer to manifest in the absence of OGT inhibition. These data suggest that chemical inhibition of OGT and perturbation of protein O-GlcNAcylation accelerate the differentiation of hESCs along the neuronal lineage, thus providing further insight into the dynamic molecular mechanisms involved in neuronal development.
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http://dx.doi.org/10.1021/acschembio.7b00232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850955PMC
August 2017

Carb cutting works better with a partner.

Nat Struct Mol Biol 2017 05;24(5):433-435

Department of Biochemistry, UT Southwestern Medical Center, Dallas, Texas, USA.

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http://dx.doi.org/10.1038/nsmb.3405DOI Listing
May 2017

Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

FASEB J 2017 08 25;31(8):3574-3586. Epub 2017 Apr 25.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Texas, USA;

Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.
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http://dx.doi.org/10.1096/fj.201700043RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503716PMC
August 2017

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

J Biol Chem 2017 06 19;292(23):9637-9651. Epub 2017 Apr 19.

From the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038 and

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 -linked glycan structures with increased branching and extended poly--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 -linked glycan structure. Furthermore, GNE deficiency and glucose supplementation acted independently and additively to increase -linked glycan branching. Notably, -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.
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http://dx.doi.org/10.1074/jbc.M116.764597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465488PMC
June 2017

Soluble klotho binds monosialoganglioside to regulate membrane microdomains and growth factor signaling.

Proc Natl Acad Sci U S A 2017 01 9;114(4):752-757. Epub 2017 Jan 9.

Department of Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390;

Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.
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http://dx.doi.org/10.1073/pnas.1620301114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278494PMC
January 2017

Pyrimidine Salvage Enzymes Are Essential for De Novo Biosynthesis of Deoxypyrimidine Nucleotides in Trypanosoma brucei.

PLoS Pathog 2016 Nov 7;12(11):e1006010. Epub 2016 Nov 7.

Department of Pharmacology University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.

The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for growth. Thymidine kinase (TK) catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD), which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA), which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5'-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5'-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug.
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http://dx.doi.org/10.1371/journal.ppat.1006010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098729PMC
November 2016

Glycan specificity of neuraminidases determined in microarray format.

Carbohydr Res 2016 Jun 8;428:31-40. Epub 2016 Apr 8.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Neuraminidases hydrolytically remove sialic acids from glycoconjugates. Neuraminidases are produced by both humans and their pathogens, and function in normal physiology and in pathological events. Identification of neuraminidase substrates is needed to reveal their mechanism of action, but high-throughput methods to determine glycan specificity of neuraminidases are limited. Here we use two glycan labeling reactions to monitor neuraminidase activity toward glycan substrates. While both periodate oxidation and aniline-catalyzed oxime ligation (PAL) and galactose oxidase and aniline-catalyzed oxime ligation (GAL) can be used to monitor neuraminidase activity toward glycans in microtiter plates, only GAL accurately measured neuraminidase activity toward glycans displayed on a commercial glass slide microarray. Using GAL, we confirm known linkage specificities of three pneumococcal neuraminidases and obtain new information about underlying glycan specificity.
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http://dx.doi.org/10.1016/j.carres.2016.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4885666PMC
June 2016

Advances in cell surface glycoengineering reveal biological function.

Glycobiology 2016 08 10;26(8):789-96. Epub 2016 Apr 10.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA

Cell surface glycans are critical mediators of cell-cell, cell-ligand, and cell-pathogen interactions. By controlling the set of glycans displayed on the surface of a cell, it is possible to gain insight into the biological functions of glycans. Moreover, control of glycan expression can be used to direct cellular behavior. While genetic approaches to manipulate glycosyltransferase gene expression are available, their utility in glycan engineering has limitations due to the combinatorial nature of glycan biosynthesis and the functional redundancy of glycosyltransferase genes. Biochemical and chemical strategies offer valuable complements to these genetic approaches, notably by enabling introduction of unnatural functionalities, such as fluorophores, into cell surface glycans. Here, we describe some of the most recent developments in glycoengineering of cell surfaces, with an emphasis on strategies that employ novel chemical reagents. We highlight key examples of how these advances in cell surface glycan engineering enable study of cell surface glycans and their function. Exciting new technologies include synthetic lipid-glycans, new chemical reporters for metabolic oligosaccharide engineering to allow tandem and in vivo labeling of glycans, improved chemical and enzymatic methods for glycoproteomics, and metabolic glycosyltransferase inhibitors. Many chemical and biochemical reagents for glycan engineering are commercially available, facilitating their adoption by the biological community.
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http://dx.doi.org/10.1093/glycob/cww045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018048PMC
August 2016

Pneumococcal Neuraminidase Substrates Identified through Comparative Proteomics Enabled by Chemoselective Labeling.

Bioconjug Chem 2016 Apr 22;27(4):1013-22. Epub 2016 Mar 22.

Department of Biochemistry, The University of Texas Southwestern Medical Center , Dallas, Texas 75390-9038, United States.

Neuraminidases (sialidases) are enzymes that hydrolytically remove sialic acid from sialylated proteins and lipids. Neuraminidases are encoded by a range of human pathogens, including bacteria, viruses, fungi, and protozoa. Many pathogen neuraminidases are virulence factors, indicating that desialylation of host glycoconjugates can be a critical step in infection. Specifically, desialylation of host cell surface glycoproteins can enable these molecules to function as pathogen receptors or can alter signaling through the plasma membrane. Despite these critical effects, no unbiased approaches exist to identify glycoprotein substrates of neuraminidases. Here, we combine previously reported glycoproteomics methods with quantitative proteomics analysis to identify glycoproteins whose sialylation changes in response to neuraminidase treatment. The two glycoproteomics methods-periodate oxidation and aniline-catalyzed oxime ligation (PAL) and galactose oxidase and aniline-catalyzed oxime ligation (GAL)-rely on chemoselective labeling of sialylated and nonsialylated glycoproteins, respectively. We demonstrated the utility of the combined approaches by identifying substrates of two pneumococcal neuraminidases in a human cell line that models the blood-brain barrier. The methods deliver complementary lists of neuraminidase substrates, with GAL identifying a larger number of substrates than PAL (77 versus 17). Putative neuraminidase substrates were confirmed by other methods, establishing the validity of the approach. Among the identified substrates were host glycoproteins known to function in bacteria adherence and infection. Functional assays suggest that multiple desialylated cell surface glycoproteins may act together as pneumococcus receptors. Overall, this method will provide a powerful approach to identify glycoproteins that are desialylated by both purified neuraminidases and intact pathogens.
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838540PMC
April 2016

Enhanced Cross-Linking of Diazirine-Modified Sialylated Glycoproteins Enabled through Profiling of Sialidase Specificities.

ACS Chem Biol 2016 Jan 16;11(1):185-92. Epub 2015 Nov 16.

Department of Biochemistry, The University of Texas Southwestern Medical Center , Dallas, Texas 75390-9038, United States.

Sialic-acid-mediated interactions play critical roles on the cell surface, providing an impetus for the development of methods to study this important monosaccharide. In particular, photo-cross-linking sialic acids incorporated onto cell surfaces have allowed covalent capture of transient interactions between sialic acids and sialic-acid-recognizing proteins via cross-linking. However, natural sialic acids also present on the cell surface compete with photo-cross-linking sialic acids in binding events, limiting cross-linking yields. In order to improve the utility of one such photo-cross-linking 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 cross-linking of an antisialyl 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.
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http://dx.doi.org/10.1021/acschembio.5b00775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731091PMC
January 2016

Fucosylation and protein glycosylation create functional receptors for cholera toxin.

Elife 2015 Oct 29;4:e09545. Epub 2015 Oct 29.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States.

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.
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http://dx.doi.org/10.7554/eLife.09545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686427PMC
October 2015

Enhanced transfer of a photocross-linking N-acetylglucosamine (GlcNAc) analog by an O-GlcNAc transferase mutant with converted substrate specificity.

J Biol Chem 2015 Sep 3;290(37):22638-48. Epub 2015 Aug 3.

From the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390 and

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins in multicellular organisms. O-GlcNAc modification is catalyzed by the O-GlcNAc transferase (OGT), which transfers N-acetylglucosamine (GlcNAc) from the nucleotide sugar donor UDP-GlcNAc to serine or threonine residues of protein substrates. Recently, we reported a novel metabolic labeling method to introduce the diazirine photocross-linking functional group onto O-GlcNAc residues in mammalian cells. In this method, cells are engineered to produce diazirine-modified UDP-GlcNAc (UDP-GlcNDAz), and the diazirine-modified GlcNAc analog (GlcNDAz) is transferred to substrate proteins by endogenous OGT, producing O-GlcNDAz. O-GlcNDAz-modified proteins can be covalently cross-linked to their binding partners, providing information about O-GlcNAc-dependent interactions. The utility of the method was demonstrated by cross-linking highly O-GlcNAc-modified nucleoporins to proteins involved in nuclear transport. For practical application of this method to a broader range of O-GlcNAc-modified proteins, efficient O-GlcNDAz production is critical. Here we examined the ability of OGT to transfer GlcNDAz and found that the wild-type enzyme (wtOGT) prefers the natural substrate, UDP-GlcNAc, over the unnatural UDP-GlcNDAz. This competition limits O-GlcNDAz production in cells and the extent of O-GlcNDAz-dependent cross-linking. Here we identified an OGT mutant, OGT(C917A), that efficiently transfers GlcNDAz and, surprisingly, has altered substrate specificity, preferring to transfer GlcNDAz rather than GlcNAc to protein substrates. We confirmed the reversed substrate preference by determining the Michaelis-Menten parameters describing the activity of wtOGT and OGT(C917A) with both UDP-GlcNAc and UDP-GlcNDAz. Use of OGT(C917A) enhances O-GlcNDAz production, yielding improved cross-linking of O-GlcNDAz-modified molecules both in vitro and in cells.
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http://dx.doi.org/10.1074/jbc.M115.667006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566237PMC
September 2015

Cellular metabolism of unnatural sialic acid precursors.

Glycoconj J 2015 Oct 10;32(7):515-29. Epub 2015 May 10.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

Carbohydrates, in addition to their metabolic functions, serve important roles as receptors, ligands, and structural molecules for diverse biological processes. Insight into carbohydrate biology and mechanisms has been aided by metabolic oligosaccharide engineering (MOE). In MOE, unnatural carbohydrate analogs with novel functional groups are incorporated into cellular glycoconjugates and used to probe biological systems. While MOE has expanded knowledge of carbohydrate biology, limited metabolism of unnatural carbohydrate analogs restricts its use. Here we assess metabolism of SiaDAz, a diazirine-modified analog of sialic acid, and its cell-permeable precursor, Ac4ManNDAz. We show that the efficiency of Ac4ManNDAz and SiaDAz metabolism depends on cell type. Our results indicate that different cell lines can have different metabolic roadblocks in the synthesis of cell surface SiaDAz. These findings point to roles for promiscuous intracellular esterases, kinases, and phosphatases during unnatural sugar metabolism and provide guidance for ways to improve MOE.
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http://dx.doi.org/10.1007/s10719-015-9593-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641449PMC
October 2015

Recognition of diazirine-modified O-GlcNAc by human O-GlcNAcase.

Medchemcomm 2014 Aug;5(8):1227-1234

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038.

The mammalian O-GlcNAc hydrolase (OGA) removes O-GlcNAc from serine and threonine residues on intracellular glycoproteins. OGA activity is sensitive to N-acyl substitutions to O-GlcNAc, with alkyl diazirine-modified O-GlcNAc (O-GlcNDAz) being completely resistant to removal by OGA. Using homology modeling, we identified OGA residues proximal to the N-acyl position of O-GlcNAc substrate. Mutation of one of these residues, C215, results in mutant enzymes that are able to hydrolytically remove O-GlcNDAz from a model compound. Further, the C215A mutant is capable of removing O-GlcNDAz from a peptide substrate. These results can be used to improve metabolism of O-GlcNAc analogs in cells. In addition, the enzyme specificity studies reported here provide new insight into the active site of OGA, an important drug target.
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http://dx.doi.org/10.1039/C4MD00164HDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109824PMC
August 2014

Glycosylation of the nuclear pore.

Traffic 2014 Apr 13;15(4):347-61. Epub 2014 Feb 13.

Department of Biochemistry, University of Texas Southwestern Medical Centre, 5323 Harry Hines Boulevard, Dallas, TX, 75390, USA.

The O-linked β-N-acetylglucosamine (O-GlcNAc) posttranslational modification was first discovered 30 years ago and is highly concentrated in the nuclear pore. In the years since the discovery of this single sugar modification, substantial progress has been made in understanding the biochemistry of O-GlcNAc and its regulation. Nonetheless, O-GlcNAc modification of proteins continues to be overlooked, due in large part to the lack of reliable methods available for its detection. Recently, a new crop of immunological and chemical detection reagents has changed the research landscape. Using these tools, approximately 1000 O-GlcNAc-modified proteins have been identified. While other forms of glycosylation are typically associated with extracellular proteins, O-GlcNAc is abundant on nuclear and cytoplasmic proteins. In particular, phenylalanine-glycine nucleoporins are heavily O-GlcNAc-modified. Recent experiments are beginning to provide insight into the functional implications of O-GlcNAc modification on certain proteins, but its role in the nuclear pore has remained enigmatic. However, tantalizing new results suggest that O-GlcNAc may play roles in regulating nucleocytoplasmic transport.
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http://dx.doi.org/10.1111/tra.12150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4001855PMC
April 2014

Introduction to glycosylation and mass spectrometry.

Methods Mol Biol 2013 ;951:1-17

Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Glycosylation is increasingly recognized as a common and biologically significant post-translational modification of proteins. Modern mass spectrometry methods offer the best ways to characterize the glycosylation state of proteins. Both glycobiology and mass spectrometry rely on specialized nomenclature, techniques, and knowledge, which pose a barrier to entry by the nonspecialist. This introductory chapter provides an overview of the fundamentals of glycobiology, mass spectrometry methods, and the intersection of the two fields. Foundational material included in this chapter includes a description of the biological process of glycosylation, an overview of typical glycoproteomics workflows, a description of mass spectrometry ionization methods and instrumentation, and an introduction to bioinformatics resources. In addition to providing an orientation to the contents of the other chapters of this volume, this chapter cites other important works of potential interest to the practitioner. This overview, combined with the state-of-the-art protocols contained within this volume, provides a foundation for both glycobiologists and mass spectrometrists seeking to bridge the two fields.
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http://dx.doi.org/10.1007/978-1-62703-146-2_1DOI Listing
June 2013

Photoaffinity probes for studying carbohydrate biology.

J Carbohydr Chem 2012 2;31(4-6):325-352. Epub 2012 Jul 2.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038.

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.
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http://dx.doi.org/10.1080/07328303.2012.676118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518314PMC
July 2012

Photocrosslinking approaches to interactome mapping.

Curr Opin Chem Biol 2013 Feb 10;17(1):90-101. Epub 2012 Nov 10.

Department of Biochemistry, 5323 Harry Hines Boulevard, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA.

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.
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http://dx.doi.org/10.1016/j.cbpa.2012.10.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594551PMC
February 2013

Sialidase specificity determined by chemoselective modification of complex sialylated glycans.

ACS Chem Biol 2012 Sep 26;7(9):1509-14. Epub 2012 Jun 26.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9185, USA.

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 Streptococcus 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.
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http://dx.doi.org/10.1021/cb300241vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448839PMC
September 2012

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

Proc Natl Acad Sci U S A 2012 Mar 12;109(13):4834-9. Epub 2012 Mar 12.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA.

O-linked β-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.
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http://dx.doi.org/10.1073/pnas.1114356109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323966PMC
March 2012

Metabolism of diazirine-modified N-acetylmannosamine analogues to photo-cross-linking sialosides.

Bioconjug Chem 2011 Sep 25;22(9):1811-23. Epub 2011 Aug 25.

Department of Chemistry, Stanford University , Stanford, CA 94305, United States.

Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photo-cross-linking approach in which a diazirine photo-cross-linker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogues in which the length of the linker between the pyranose ring and the diazirine was varied. These analogues were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogues could be incorporated into both α2-3 and α2-6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently cross-linked to their interaction partners. We demonstrate that all three diazirine-modified analogues were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogues were less efficiently metabolized, yet could still function as effective cross-linkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogues to glycans other than sialosides, providing fidelity to selectively incorporate the cross-linker into sialylated molecules. These compounds are nontoxic and display only minimal growth inhibition at the concentrations required for cross-linking studies. This report provides essential information for the deployment of photo-cross-linking analogues to capture and study ephemeral, yet essential, sialic acid-mediated interactions.
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http://dx.doi.org/10.1021/bc2002117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178686PMC
September 2011