Biology subjects

Konada, R. S. R.

Publications and source records attributed to Konada, R. S. R..

3 recordsLinked to original sources

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↗

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↗