Thomas Jan Boltje

Human milk oligosaccharides (HMOs) are unconjugated and structurally diverse glycans synthesized in the lactating mammary gland through the stepwise action of glycosyltransferases that extend a free lactose core. Several HMOs are capped with sialic acids, including 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL), that promote early-life microbiota development and contribute to immune system and neuronal functions. These health-promoting properties make sialylated HMOs attractive biomolecules for incorporation in infant nutrition and functional food products. Mammalian cell lines lack endogenous HMO production, limiting mechanistic studies of HMO biosynthesis and constraining production strategies based on human cells. Here, we developed a human cell-based strategy for the production of the two common sialyllactose isomers 3′-SL and 6′-SL in glycoengineered human embryonic kidney (HEK293) cells. We co-expressed LALBA and B4GALT1, that together form the lactose synthase complex, to introduce free lactose biosynthesis capacity into a genetically engineered human cell line without sialylation (HEK293ΔSia). Stable expression of either ST3GAL or ST6GAL isoenzymes in HEK293ΔSia cells revealed that ST3GAL3/4/5, and especially ST3GAL5, efficiently convert lactose into 3′-SL while ST6GAL1 and ST6GAL2 produce the 6′-SL isomer. These results provide insights into the in vivo ability of sialyltransferase isoenzymes to use lactose as substrate. Establishing HMOs biosynthesis pathways into controllable human cell systems offers an alternative strategy for production of HMOs and provides a starting point to unlock biosynthesis of more complex HMOs in human cells.

Metabolic adaptations are key in the virulence of the pathogen Mycobacterium tuberculosis (Mtb). However, our current understanding of these adaptations is limited to common pathways in central carbon metabolism. Here we apply an untargeted bottom-up approach to discover unknown stress-responsive metabolites and their biosynthetic enzyme. We show that upon exposure to hypoxia, nitric oxide and activated macrophages, Mtb rapidly produces high levels of an unknown metabolite that we identify as 6′-γ-amino-butyric acid-trehalose (GABA-trehalose). Formation of millimolar GABA-trehalose levels under these stresses is driven by a rapid rise in GABA, which is also excreted. We demonstrate that GABA-trehalose is produced from GABA and trehalose by the uncharacterized ATP-grasp enzyme Rv1722, involving a carboxylate-hydroxyl ligation that is non-canonical for ATP-grasp enzymes. Phylogenetic analyses demonstrate that the gene rv1722 is present in most slow-growing mycobacteria but absent in most rapid-growing mycobacteria. While the role of GABA-trehalose in Mtb metabolism remains unclear, we postulate that the increased NADH/NAD+ ratio under hypoxia and nitric oxide exposure promotes GABA formation and inhibits its breakdown, leading to GABA accumulation and excretion. Rv1722-driven coupling of GABA and trehalose constitutes an alternative to excretion that conserves carbon and nitrogen. Taken together, our bottom-up approach reveals a new stress-response pathway in Mtb that rapidly produces large quantities of GABA-trehalose. These findings extend our knowledge of the metabolic adaptations that a major human pathogen utilizes in response to immune system-imposed stresses.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are key immune receptors that bind to cell surface sialic acids, leading to modulation of the immune system. Interrupting the Siglec–sialoglycan binding in cancer has been proposed as a potential antitumor response strategy. We previously showed that colon-cancer-associated Fusobacterium nucleatum (Fn) ATCC 51191 interacts with Siglec-7 via its lipopolysaccharide (LPS), revealing Fn LPS as a new ligand for Siglec-7. Here, we used glycoengineered cells carrying sialic acid variants to investigate the capacity of LPS isolated from F. nucleatum strains to disrupt the interaction between sialic acid expressed on mammalian cells and Siglec-7. We first showed that LPS extracted from Fusobacterium polymorphum ATCC 10953, F. nucleatum ssp. animalis ATCC 51191, and F. nucleatum ssp. nucleatum ATCC 25586 strains bound to recombinant Siglec-7 in vitro, while no binding was observed with the Siglec-7R124A mutant, suggesting that the binding occurred through the carbohydrate binding V-set domain. Using glycoengineered Jurkat cells and HEK293T cells carrying modified sialic acid forms, we demonstrated that F. nucleatum LPS could significantly disrupt the binding of Siglec-7 to these cells and that this inhibition was decreased following neuraminidase treatment, confirming that the interaction between Fn LPS and Siglec-7 is carbohydrate-mediated. We further validated these data using Jurkat cells and HEK293T cells expressing high-affinity sialic acid ligands. We showed that F. nucleatum LPS significantly disrupted the binding of Siglec-7 to these cells in a specific manner. These findings offer novel insights into the development of glycomimetic approaches for limiting colon cancer progression.

The stereoselective synthesis of 1,2‐ cis glycosidic bonds is a significant challenge in carbohydrate chemistry. 1,2‐ Cis galactosides can be obtained by using C‐4 acyl protection groups as a stereodirecting group, yet the underlying mechanism that steers selective galactosylation has remained elusive. Herein, we investigate the stereodirecting effect of C‐4 acyl groups in galactosides using glycosylation reactions, exchange NMR spectroscopy and DFT‐calculations. We found no experimental evidence for C‐4 dioxepanium ion formation through C‐4 acyl neighboring group participation. Instead, β‐glycosyl triflates were detected using exchange NMR which could afford α‐galactosides via S _N 2‐like displacement. Computational studies of the product‐forming transition state geometries reveal that the C‐4 benzoate group shields the β‐face of the galactosyl donor, thereby disfavoring reactions that proceed via the α‐glycosyl triflate pathway. These findings can explain the stereodirecting effect of C‐4 acyl groups on galactosyl donors, and provides fundamental insights for the development of stereoselective glycosylation methods in the future.

The stereoselective synthesis of 1,2‐ cis glycosidic bonds is a significant challenge in carbohydrate chemistry. 1,2‐ Cis galactosides can be obtained by using C‐4 acyl protection groups as a stereodirecting group, yet the underlying mechanism that steers selective galactosylation has remained elusive. Herein, we investigate the stereodirecting effect of C‐4 acyl groups in galactosides using glycosylation reactions, exchange NMR spectroscopy and DFT‐calculations. We found no experimental evidence for C‐4 dioxepanium ion formation through C‐4 acyl neighboring group participation. Instead, β‐glycosyl triflates were detected using exchange NMR which could afford α‐galactosides via S _N 2‐like displacement. Computational studies of the product‐forming transition state geometries reveal that the C‐4 benzoate group shields the β‐face of the galactosyl donor, thereby disfavoring reactions that proceed via the α‐glycosyl triflate pathway. These findings can explain the stereodirecting effect of C‐4 acyl groups on galactosyl donors, and provides fundamental insights for the development of stereoselective glycosylation methods in the future.

The stereoselective synthesis of glycosidic bonds can be promoted by the addition of stereodirecting solvents. In particular, nitrile and ether based solvents are known to promote the selective production of β- or α-glycosides, respectively. Even though the application of co-solvents in glycosylation protocols is well-established, the understanding of their impact on the glycosylation mechanism as well as the reaction intermediates formed upon their addition, remains incomplete. As the exact mechanism of solvent stereo direction remains debated, this limits the ability to develop stereoselective glycosylation methods. Herein, we report the application of relaxation and exchange NMR techniques to establish that acetonitrile and tetrahydrofuran form covalent glycosyl-nitrilium and glycosyl-oxonium ions, respectively. In contrast, other ethereal solvents such as diethyl ether and 1,4-dioxane were not able to do so. This work highlights the potential of exchange NMR to study reaction mechanisms, provides evidence for the formation of glycosyl-solvent adducts despite their very low populations, and improves our ability to develop better stereoselective glycosylation protocols.

Sialic acid mimetics (SAMs) are chemically modified sialic acid derivatives that can act as metabolic inhibitors or as sugar donors for sialyltransferases.

Glioblastoma (GBM) is a highly aggressive brain tumor marked by invasive growth and therapy resistance. Tumor cells adapt to hostile conditions, such as hypoxia and nutrient deprivation, by activating survival mechanisms including autophagy and metabolic reprogramming. Among GBM-associated changes, hypersialylation, particularly, the aberrant expression of polysialic acid (PSA), has been linked to increased plasticity, motility, and immune evasion. PSA, a long α2,8-linked sialic acid polymer typically attached to the NCAM, is abundant in the embryonic brain and re-expressed in cancers, correlating with poor prognosis. Here, we investigated how PSA expression was regulated in GBM cells under nutrient-limiting conditions. Serum starvation induced a marked increase in PSA-NCAM, driven by upregulation of the polysialyltransferase ST8SiaIV and an autophagy-dependent recycling of sialic acids from degraded glycoproteins. Inhibition of autophagy or sialidases impaired PSA induction, and PSA regulation appeared dependent on p53 function. Immunohistochemical analysis of GBM tissues revealed co-localization of PSA and LC3, particularly around necrotic regions. In conclusion, we identified a novel mechanism by which GBM cells sustain PSA-NCAM expression via autophagy-mediated sialic acid recycling under nutrient stress. This pathway may enhance cell migration, immune escape, and stem-like properties, offering a potential therapeutic target in GBM.

Achieving skin regeneration following destruction of the epidermis and dermis (e.g. full-thickness wounds) has remained an unachieved goal. The wound healing response is complex and consists of multiple overlapping phases which are tightly choreographed by the ebb and flow of effector molecules. Mimicking this spatiotemporal aspect in pro-regenerative biomaterials may enhance their efficacy and eventually lead to skin regeneration. However, robust spatiotemporal signalling has remained difficult to achieve. The field of bioorthogonal click and click-to-release chemistry may be key to creating spatiotemporal signalling biomaterials. The ability to safely and effectively conjugate or release molecules in complex biological environments has transformed many areas of research. In this review, we aim to highlight the complex nature of wound healing and address how click and click-to-release chemistry approaches could contribute to the development of biomaterials with spatiotemporal control over effector molecules.