Biology subjects

Mikolajczyk, K.

Publications and source records attributed to Mikolajczyk, K..

6 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↗

bioRxiv2024

B4GALT5-deficient CHO-Lec2 cells expressing human alpha1,4-galactosyltransferase: a glycoengineered cell model for studying Shiga toxin receptors

Human 1,4-galactosyltransferase (A4galt) is a glycosyltransferase existing in humans as two isoforms, widespread A4galt (named A4G) and its rare variant with p.Q211E substitution (A4Gmut). Both isoforms produce Gb3 (Gal1[->]4Gal{beta}1[->]4Glc-Cer) on glycosphingolipids and P1 glycotope (Gal1[->]4Gal{beta}1[->]4GlcNAc-R) on glycoproteins, which serve as receptors for Shiga toxin types 1 and 2 (Stx1 and Stx2). Stx1 is bound by Gb3 and P1 glycotope, while Stx2 is recognized solely by Gb3. To elucidate the role of these receptors, CHO-Lec2 cells expressing human A4G and A4Gmut were modified by disrupting the hamster B4GALT5 gene using CRISPR/Cas9 technology. The B4GALT5 gene encodes {beta}1,4-galactosyltransferase 5 (B4galt5), synthesizing lactosylceramide, the key substrate for Gb3 synthesis. Consequently, B4GALT5-deficient CHO-Lec2-expressing A4G and A4Gmut cells lacked Gb3 glycosphingolipid but retained the ability to synthesize glycoprotein-based P1 glycotope. Both B4GALT5-deficient CHO-Lec2 cells expressing A4G and A4Gmut demonstrated no binding of Stx1B and Stx2B. The cytotoxicity assay showed that B4GALT5-deficient CHO-Lec2 cells expressing A4G were completely resistant to Stx1 holotoxin while A4Gmut-expressing cells revealed reduced sensitivity to Stx2. The glycoengineered CHO-Lec2 cells obtained in this study provide a valuable model for studying receptors for Stxs, enabling a detailed assessment of their roles in toxin binding and cytotoxicity.

biochemistry↗

bioRxiv2024

Gb3 trisaccharide-bearing exosomes as a novel neutralizer for Shiga toxin type 1

Shiga toxin types 1 (Stx1) and 2 (Stx2), produced by Shiga toxin-producing Escherichia coli (STEC) and Shigella dysenteriae, are key virulence factors responsible for severe foodborne diseases, such as hemorrhagic colitis and hemolytic uremic syndrome (HUS). The receptors for Stxs are Gb3 and P1 glycotope, which contain the Gal1[->]4Gal epitope and are synthesized by human 1,4-galactosyltransferase (A4galt). Stx-related infections pose a global public health challenge, owing to the limited therapeutic options due to the restricted use of antibiotics. Therefore, there is an urgent need to develop novel therapeutic strategies. This study proposes an innovative strategy utilizing exosomes derived from CHO-Lec2 cells, which were modified with Functional-Spacer-Lipid (FSL) conjugates bearing the Gb3 carbohydrate epitope (exo-Gb3-FSL). Flow cytometry analysis confirmed the presence of Gal1[->]4Gal disaccharides on exo-Gb3-FSL constructs, enabling them to bind Stx1. Moreover, using CHO-Lec2 cells evaluated the ability of exo-Gb3-FSL agents to bind Stx1 and protect these cells from Stx1-mediated cytotoxicity. For Stx1-treated CHO-Lec2 cells, increased cell survival was observed when using 25 {micro}M exo-Gb3-FSL constructs, compared to control cells. These findings highlight the potential of exosome-based anti-Stx1 agents as promising alternatives to conventional therapies. This innovative strategy may provide novel directions for studies on Stx1 neutralization, offering a valuable strategy for the treatment of Stx-related diseases. Highlights- Exo-Gb3-FSL constructs hold Stx1 binding Gal1[->]4Gal epitope. - Stx1 was effectively bound by exo-Gb3-FSL constructs. - Exo-Gb3-FSL protected CHO-Lec2 cells from Stx1. - Exo-Gb3-FSL is a promising anti-Stx1 agent.

microbiology↗

bioRxiv2024

Delving into human α1,4-galactosyltransferase acceptor specificity: the role of enzyme dimerization

Human 1,4-galactosyltransferase (A4galt), a Golgi apparatus-resident GT, synthesizes Gb3 glycosphingolipid (GSL) and P1 glycotope on glycoproteins (GPs), which are receptors for Shiga toxin types 1 and 2. Despite the significant role of A4galt in glycosylation processes, the molecular mechanisms underlying its varied acceptor specificities remain poorly understood. Here, we attempted to elucidate A4galt specificity towards GSLs and GPs by exploring its interaction with GTs with various acceptor specificities, GP-specific {beta}1,4-galactosyltransferase 1 (B4galt1) and GSL-specific {beta}1,4-galactosyltransferase isoenzymes 5 and 6 (B4galt5 and B4galt6). Using a novel NanoBiT assay, we found that A4galt can form homodimers and heterodimers with B4galt1 and B4galt5 in two cell lines, human embryonic kidney cells (HEK293) and Chinese hamster ovary cells (CHO-Lec2). We found that A4galt-B4galts heterodimers preferred N-terminally tagged interactions, while in A4galt homodimers, the favored localization of the fused tag depended on the cell line used. Furthermore, by employing AlphaFold for state-of-the-art structural prediction, we analyzed the interactions and structures of these enzyme complexes. Our analysis highlighted that the A4galt-B4galt5 heterodimer exhibited the highest prediction confidence, indicating a significant role of A4galt heterodimerization in determining enzyme specificity toward GSLs and GPs. These findings enhance our knowledge of A4galt acceptor specificity and may contribute to a better comprehension of pathomechanisms of the Shiga toxin-related diseases.

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↗