Biology subjects

Bereznicka, A.

Publications and source records attributed to Bereznicka, A..

3 recordsLinked to original sources

bioRxiv2026

Surface functionalization of small extracellular vesicles derived from Caco-2 and HEK293T cells in the neutralization of Shiga toxin 1 subunit B

Shiga toxins (Stx) are key virulence factors of Shiga toxin-producing Escherichia coli (STEC), which are responsible for severe foodborne infections that can progress to hemolytic-uremic syndrome (HUS). Currently, no specific anti-toxin therapies are available. In this study, we devised a glycoengineering strategy utilizing Functional-Spacer-Lipid (FSL) conjugates to create small extracellular vesicles (sEVs)-based decoy receptors for Shiga toxin type 1 (Stx1). sEVs isolated from human Caco-2 and HEK293T cells were functionalized with Gb3 trisaccharide (Gal1[->]4Gal{beta}1[->]4Glc)-containing FSL conjugates, yielding Gb3-decorated vesicles, displaying the Gal1[->]4Gal epitope. Characterization of FSL-modified sEVs confirmed that FSL incorporation did not adversely affect sEV morphology, size distribution, or surface charge. Western blotting and bead-assisted flow cytometry verified the presence of exosomal markers (CD9 and CD63) and the Gb3 epitope on modified vesicles. Gb3-tagged sEVs from both cell types exhibited high specificity in binding Stx1B, while control vesicles carrying Galili epitope (Gal1[->]3Gal{beta}1[->]4GlcNAc), lacking Stx1B binding, demonstrated negligible binding. Gb3-expressing Caco-2 cell-based assays revealed that Gb3-decorated sEVs markedly reduced Stx1B binding to Caco-2 cells, indicating effective competition with cellular receptors. Furthermore, glycoengineered sEVs did not impair Caco-2 cell viability at concentrations sufficient for Stx1B sequestration. These findings establish FSL-mediated glycoengineering as a rapid and versatile approach for generating sEV-based decoy receptors that effectively bind Stx1B. Gb3-containing human sEVs may serve as an agent for neutralizing Stx1B and potentially other glycan-binding toxins, supporting the development of promising next-generation anti-toxin therapeutics.

bioengineering↗

bioRxiv2025

Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation

Glycosylation of proteins can impact their folding, stability, trafficking and enzymatic activity. Human Gb3/CD77 synthase (1,4-galactosyltransferase, A4galt) has two occupied N-glycosylation sites. Previously, we demonstrated that the activity of recombinant enzyme relies on its N-glycosylation. In this study, we produced soluble recombinant catalytic domain of human Gb3/CD77 synthase in two expression hosts known for different glycosylation patterns: Trichoplusia ni insect cells (High Five) and human embryonic kidney cells (Expi293F). The High Five cells generate short oligomannose structures, while the Expi293F cells synthesize complex type glycans. We evaluated the activity of High Five-derived and Expi293F-derived enzymes, characterized the structures of their N-glycans and showed that High Five cells provide a higher amount and activity of the enzyme. Moreover, we used the Expi293F cells to evaluate the N- and C-terminal location of the 6xHis-tag and found that only the N-terminally tagged Expi293F-derived enzyme demonstrated activity. In contrast, the enzyme produced in High Five cells was active despite carrying a C-terminal tag. These findings highlight the role of glycosylation pattern and tag position in the activity of human recombinant glycosyltransferase produced in different hosts. Highlights- Active recombinant Gb3/CD77 synthase was obtained in High Five and Expi293F cells. - High Five cells provided higher recombinant Gb3/CD77 synthase yield than Expi293F. - Glycosylation pattern of recombinant Gb3/CD77 synthase was host specific. - Activity of Expi293F-derived enzyme depended on 6xHis-tag localization.

biochemistry↗

bioRxiv2021

Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase curtails its activity

N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Gal1[->]4Gal (Gb3 and the P1 antigen) and Gal1[->]4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

biochemistry↗