Biology subjects

Jaroentomeechai, T.

Publications and source records attributed to Jaroentomeechai, T..

9 recordsLinked to original sources

bioRxiv2026

Topological reprogramming transforms an integral membrane oligosaccharyltransferase into a water-soluble glycosylation catalyst

Glycosyltransferases (GTs) catalyze the formation of new glycosidic bonds and thus are vital for synthesizing natures vast repertoire of glycans and glycoconjugates and for engineering glycan-related medicines and materials. However, obtaining detailed structural and functional insights for the >750,000 known GTs is limited by difficulties associated with their efficient recombinant expression. Members of the GT-C fold, in particular, pose the most significant expression challenges due to the integration and folding requirements of their multiple membrane-spanning regions. Here, we address this challenge by engineering water-soluble variants of an archetypal GT-C fold enzyme, namely the oligosaccharyltransferase PglB from Campylobacter jejuni (CjPglB), which possesses 13 hydrophobic transmembrane helices. To render CjPglB water-soluble, we leveraged two advanced protein engineering methods: one that is universal called SIMPLEx (solubilization of IMPs with high levels of expression) and the other that is custom tailored called WRAPs (water-soluble RFdiffused amphipathic proteins). Each approach was able to transform CjPglB into a water-soluble enzyme that could be readily expressed in the cytoplasm of Escherichia coli cells at yields in the 3-6 mg/L range. Importantly, solubilization was achieved without the need for detergents and with retention of catalytic function. Collectively, our findings demonstrate that both SIMPLEx and WRAPs are promising platforms for advancing the molecular characterization of even the most structurally complex GTs, while also enabling broader GT-mediated glycosylation capabilities within synthetic glycobiology applications.

synthetic biology↗

bioRxiv2026

Siglec-7 and -15 recognize repeated clustered sulfo-sialo O-glycan motifs on select O-glycoproteins

Siglecs are key immunoreceptors in immune homeostasis and cancer immunosuppression. Diverse endogenous and exogenous sialoglycan ligands induce Siglec-mediated immunoregulation, but the nature of endogenous Siglec ligands and how seemingly ubiquitous sialoglycans achieve selectivity is unclear. Here, we employed a HEK293 cell-based array and glycoprotein reporters designed from human mucins and mucin-like proteins with O-glycans in natural clusters and repeat motifs to dissect specificities of Siglec-7 and -15. We first used precomplexed Siglec-Fc chimera to demonstrate that CHST1-mediated 6-O-sulfation markedly enhanced binding to select O-glycoproteins, namely MAdCAM1, CD43, and PSGL-1. Probing binding properties of Siglec-7 expressed exogenously on CHO cells or endogenously on human monocytes by reverse assays employing fluorophore-tagged O-glycoprotein reporters revealed specific binding to the sulfo-sialyl core1 O-glycoform of the same O-glycoproteins. Our study indicates that Siglec-7 on immune cells employs multivalent interactions with repeated motifs of clustered sulfo-sialyl O-glycans to drive selectivity in ligand interactions and Siglec-induced immunosuppression.

molecular biology↗

bioRxiv2024

Biantennary N-glycans As Receptors for MARTX Toxins in Vibrio Pathogenesis

Multifunctional Autoprocessing Repeats-in-Toxin (MARTX) toxins are a diverse effector delivery platform of many Gram-negative bacteria that infect mammals, insects, and aquatic animal hosts. The mechanisms by which these toxins recognize host cell receptors for translocation of toxic effectors into the cell have remained elusive. Here, we map the first surface receptor-binding domain of a MARTX toxin from the highly lethal foodborne pathogen Vibrio vulnificus. This domain corresponds to a 273-amino acid sequence with predicted symmetrical immunoglobulin-like folds. We demonstrate that this domain binds internal N-acetylglucosamine on complex biantennary N-glycans with select preference for L1CAM and other N-glycoproteins with multiple N-glycans on host cell surfaces. This receptor binding domain is essential for V. vulnificus pathogenesis during intestinal infection. The identification of a highly conserved motif universally present as part of all N-glycans correlates with the V. vulnificus MARTX toxin boasting broad specificity and targeting nearly all cell types.

microbiology↗

bioRxiv2021

A modular platform for on-demand vaccine self-assembly enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

Engineered outer membrane vesicles (OMVs) derived from laboratory strains of bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. As mimics of the bacterial cell surface, OMVs offer a molecularly-defined architecture for programming repetitive, high-density display of heterologous antigens in conformations that elicit strong B and T cell immune responses. However, antigen display on the surface of OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. To address this shortcoming, we created a universal approach called AddVax (avidin-based dock- and-display for vaccine antigen cross (x)-linking) whereby virtually any antigen that is amenable to biotinylation can be linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen receptor (SNARE) comprised of an outer membrane scaffold protein fused to a member of the avidin family. We show that SNARE-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations were injected in wild-type BALB/c mice, strong antigen-specific antibody responses were observed that depended on the physical coupling between the antigen and SNARE-OMV delivery vehicle. Overall, these results demonstrate AddVax as a modular platform for rapid self-assembly of antigen-studded OMVs with the potential to accelerate vaccine generation, respond rapidly to pathogen threats in humans and animals, and simplify vaccine stockpiling.

synthetic biology↗

bioRxiv2020

High-resolution mapping of glycoprotein structure-activity relationships by shotgun scanning glycomutagenesis

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation at naive sites as a strategy for engineering proteins with advantageous properties is currently limited by our inability to construct large collections of glycoproteins for interrogating the structural and functional consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis (SSGM) in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. Moreover, the resulting neoglycoproteins can be readily subjected to available medium- to high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and a human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by the N-linked glycan in a manner that critically depended on the precise location of the modification. Importantly, we anticipate that our workflow for creating and characterizing large ensembles of neoglycoproteins should provide access to unexplored regions of glycoprotein structural space and to custom-made glycoproteins with desirable properties.

synthetic biology↗

bioRxiv2020

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli

We describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of the genetically engineered Escherichia coli strain, SHuffle. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called ‘cyclonals’ that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The selective power of this approach was demonstrated by facile isolation of novel complementarity-determining regions for a cyclonal that specifically recognized the basic-region leucine zipper domain of the yeast transcriptional activator protein Gcn4.Competing Interest StatementM.P.D. has ownership interest (including stock, patents, etc.) in SwiftScale Biologics, Inc. M.P.D.’s interests are reviewed and managed by Cornell University in accordance with their conflict of interest policies. M.B. is employed by NEB, which commercializes SHuffle cells. All other authors declare no competing interests.View Full Text

synthetic biology↗

bioRxiv2019

On-demand, cell-free biomanufacturing of conjugate vaccines at the point-of-care

Conjugate vaccines are among the most effective methods for preventing bacterial infections, representing a promising strategy to combat drug-resistant pathogens. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro bioconjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of bioconjugate vaccines against diverse bacterial pathogens in one hour. We show that iVAX synthesized vaccines against the highly virulent pathogen Franciscella tularensis subsp. tularensis (type A) strain Schu S4 elicited pathogen-specific antibodies in mice at significantly higher levels compared to vaccines produced using engineered bacteria. The iVAX platform promises to accelerate development of new bioconjugate vaccines with increased access through refrigeration-independent distribution and point-of-care production.

synthetic biology↗

bioRxiv2017

A pipeline for interrogating and engineering single-subunit oligosaccharyltransferases

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of post-translational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily-adaptable pipeline for virtually any ssOST of interest.\n\nAbbreviations

synthetic biology↗

bioRxiv2017

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 440 {micro}g/mL for the single-subunit OST enzyme, Protein glycosylation B (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

synthetic biology↗