Biology subjects

Kohler, J. J.

Publications and source records attributed to Kohler, J. J..

9 recordsLinked to original sources

bioRxiv2026

B3GNT7 regulates mucin glycosylation and protects against colitis and infection

Mucus covers and protects colonic epithelial cells. Mucus is mainly composed of heavily O-glycosylated proteins called mucins, and disruption of normal mucin glycosylation occurs in ulcerative colitis (UC). Mucin-2 (MUC2) is the major colonic mucin, and MUC2 O-glycans are often extended with sulfated polyLacNAc, also known as keratan sulfate (KS). The GlcNAc residues in KS are added by B3GNT family members. B3GNT7 is highly expressed in the colon, and B3GNT7 expression is dramatically reduced in UC. However, the function of B3GNT7 in colonic physiology is unexplored. Here we show that B3gnt7 is a key player in colonic physiology through its function in controlling the structure of mucus glycans. We found that B3GNT7 prefers to extend a sulfated acceptor substrate and is required for production of polyLacNAc-modified mucus in a human goblet cell model. In vivo, B3GNT7 regulates Muc2, Muc13, and Muc17 O-glycosylation. Intestinal B3GNT7 deficiency increases susceptibility to colitis and enteric infection in mice, showing that B3GNT7-dependent glycosylation confers protective properties to colonic mucus. Taken together, these results demonstrate that B3GNT7 has a function distinct from other B3GNT family members and is critical for maintaining colonic homeostasis. SIGNIFICANCE STATEMENTUlcerative colitis is a chronic inflammatory bowel disease that affects 5 million people globally. The colonic mucus layer forms a protective barrier over colonic epithelial cells and is disrupted in ulcerative colitis. Mucus is composed of mucin proteins decorated by carbohydrates, called glycans. Glycans confer protective properties to the mucus barrier, and mucin glycans change in ulcerative colitis. B3GNT7 is an enzyme that elongates glycans and is downregulated in ulcerative colitis. In this study, we use in vitro and in vivo models to demonstrate that B3GNT7 regulates colonic mucus glycans and protects mice against colitis and infection. Our findings provide molecular insight into the contributions of B3GNT7-dependent glycans to colonic homeostasis.

biochemistry↗

bioRxiv2025

Hypofucosylation promotes pertussis toxin binding to cell surface glycococonjugates and pertussis toxin-induced intracellular ERK signaling

Pertussis (whooping cough) is caused by the bacterium Bordetella pertussis. Among the virulence factors produced by B. pertussis, pertussis toxin (PT) is responsible for key disease symptoms, including impacts on the immune system. PT is an AB5 toxin, consisting of a single catalytic A subunit and five B subunits. The B pentamer recognizes cell surface glycans and facilitates intracellular delivery of the catalytic A subunit. PT also impacts host cell signaling via mechanisms that do not depend on the catalytic activity of the A subunit. In particular, PT promotes signaling through the T-cell receptor (TCR), leading to activation of extracellular signal-regulated kinase (ERK) cascades. PT prefers to bind sialylated and N-linked glycans, but other aspects of PTs glycan binding specificity remain underexplored. Here we report that the absence of fucose on mammalian cell surfaces leads to increased binding by PT. Using pharmacological inhibitors in a human bronchial epithelial cell line, we observe that sialylation and N-linked glycosylation promote PT binding while fucosylation interferes with PT binding. Similarly, CHO and Colo205 cells deficient in fucosylation exhibited enhanced PT binding as compared to the corresponding wild-type cell lines. Genetic knockout of FUT3/FUT5/FUT6 or of FUT8 led to increased PT binding, suggesting that specific fucosylated epitopes mediate protection from PT. The functional impact of altered PT binding was examined in Jurkat T cells, where removal of cell surface non-core fucose led to increased PT-dependent ERK phosphorylation. In sum, our study identifies a role for fucosylation in protecting mammalian cells from PT.

biochemistry↗

bioRxiv2025

Time-Resolved Tracking and Secretome Mapping of Intracellular, Cell Surface, and Extracellular Glycoconjugates with a Mini-Tetrazine N-Acetylglucosamine

Glycosylation is instrumental in governing essential biological processes that dictate health outcomes. Herein we describe a mini-tetrazine N-acetylglucosamine (HTz-GlcNAc) that is incorporated into N-linked glycans and through rapid live-cell labeling with trans-cyclooctene (TCO) reagents enables real-time visualization and evaluation of glycoconjugate trafficking in living cells. We utilize cell-permeable and impermeable TCO-fluorophores to perform time-resolved, multicolor live labeling of cell-surface glycoconjugates, as well as intracellular, and internalized glycoconjugates. We observe intracellular glycoconjugate trafficking throughout the secretory pathway of live cells in real-time, as well as cell surface glycan turnover and extracellular vesicle secretion across three days. In the toolkit of probes for metabolic oligosaccharide engineering, HTz-GlcNAc brings the capability of precise interrogation of intracellular and extracellular glycoconjugate trafficking, and selective tagging of intra-and-extracellular vesicles, while providing a handle for the purification and enrichment of glycans for immunoblot and proteomic analyses. TOC image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/682811v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@f65188org.highwire.dtl.DTLVardef@162c58corg.highwire.dtl.DTLVardef@1977b73org.highwire.dtl.DTLVardef@29e9ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer's Disease Susceptibility

The innate immune gene CD33, encoding a myeloid inhibitory sialic acid-binding receptor, is associated with Alzheimers disease (AD) susceptibility. The AD-associated rs3865444CC risk variant reduces splicing of the sialic acid-binding domain and increases expression of the full-length (sialic acid-binding) CD33 isoform seven-fold compared to the rs3865444AA protective genotype. Here we identify CD45 as an immune cell-specific sialic acid-dependent cis CD33 binding partner, whose phosphatase activity is inhibited by CD33. Overexpression of CD33 or loss of CD45 contributes to impaired microglial clearance of amyloid beta and amyloid beta-induced loss of dendritic spines in microglial-neuronal co-cultures, aligning with a detrimental effect of CD33-mediated inhibition of CD45. CD33-CD45 interaction frequency was increased in monocytes from individuals with the rs3865444CC risk variant compared to rs3865444AA, as well as in AD compared to controls, independent of genotype. Furthermore, an interaction between CD33 and PTPRC (encoding CD45) gene expression in human brain tissue was associated with a pathological diagnosis of AD and global burden of AD pathology. Our findings thus establish a functional interaction between CD33 and CD45 relevant to AD susceptibility and systemic myeloid dysfunction in this disease.

neuroscience↗

bioRxiv2023

Fucosylated glycoproteins and fucosylated glycolipids play opposing roles in cholera intoxication

Cholera toxin (CT) is the etiological agent of cholera. Here we report that multiple classes of fucosylated glycoconjugates function in CT binding and intoxication of intestinal epithelial cells. In Colo205 cells, knockout of B3GNT5, the enzyme required for synthesis of lacto- and neolacto-series glycosphingolipids (GSLs), reduces CT binding but sensitizes cells to intoxication. Overexpressing B3GNT5 to generate more fucosylated GSLs confers protection against intoxication, indicating that fucosylated GSLs act as decoy receptors for CT. Knockout (KO) of B3GALT5 causes increased production of fucosylated O-linked and N-linked glycoproteins, and leads to increased CT binding and intoxication. Knockout of B3GNT5 in B3GALT5 KO cells eliminates production of fucosylated GSLs but increases intoxication, identifying fucosylated glycoproteins as functional receptors for CT. These findings provide insight into molecular determinants regulating CT sensitivity of host cells.

biochemistry↗

bioRxiv2021

Exo-enzymatic addition of diazirine-modified sialic acid to cell surfaces enables photocrosslinking of glycoproteins

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 exo-enzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAz-ylation 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 2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

biochemistry↗

bioRxiv2020

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 micro-heterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through bio-orthogonal tagging of metabolically engineered non-natural sugars into glycan epitopes. However, this approach possesses a significant drawback due to non-specific background reactions and ambiguity of non-natural sugar metabolism. Here we report a tag-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry. The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, GalNAc, and GlcNAc biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC 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 some 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.

biochemistry↗

bioRxiv2019

Chemical precision glyco-mutagenesis by glycosyltransferase engineering in living cells

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-function modification on cells where the products of individual glycosyltransferases can be selectively characterized or manipulated at will.

synthetic biology↗

bioRxiv2019

The human UDP-galactose 4’-epimerase (GALE) is required for cell surface glycome structure and function

Glycan biosynthesis relies on nucleotide-sugars (NS), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease, but how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, we examined uridine diphosphate (UDP)-galactose 4-epimerase (GALE), which interconverts two pairs of essential NSs. GALE deletion in human cells triggered major imbalances in its substrate NSs and consequent dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the Fas death receptor. NS dysregulation also directly impacted cell signaling, as GALE-/- cells exhibit Fas hypoglycosylation and hypersensitivity to Fas ligand-induced apoptosis. Our results reveal a new role for GALE-mediated NS regulation in supporting death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

cell biology↗