Extension of N- and O-glycans with linear sialic acid polymers (polySias) was described on a limited number of mammalian glycoproteins, including the α2,8-polysialyltransferases ST8Sia II and ST8Sia IV. Previous in vitro analyses have shown that ST8Sia IV from the salmonid fish Coregonus maraena (Cma) had high polysialylation activity and broader donor substrate specificity compared to its human counterpart. In this study, we investigated whether the fish ST8Sia IV was able to polysialylate N- and O-glycans of glycoproteins with a focus on its autopolysialylation capacity. Using a combination of strategies, including plant-based glycoengineering, we found that the Cma ST8Sia IV is able to use both types of acceptors for the formation of long polySia chains with a degree of polymerization of >40 consisting of N-acetyl-neura-minic acid and N-glycolyl-neura-minic acid. Given the importance of polySias in multiple health and disease states, the Cma ST8Sia IV represents a useful biocatalyst with applications in the fields of biosafe therapeutics and glycobiology.

The interplay of the carbohydrate polysialic acid (polySia) with its interaction partners plays an important role in several physiological systems of vertebrates. The objective of this study was to develop a flexible analytical system for visualizing interaction partners of polySia using blotting and fluorescence cell staining strategies. We selected a streptavidin-biotin system due to the wide range of commercially available tools for this analytical application. After chemical polysialylation of streptavidin, the resulting bioconjugate was used to target polySia interaction partners after they had been separated via native agarose gel electrophoresis and transferred to a PVDF membrane. Furthermore, neutrophil extracellular traps (NETs) were employed to visualize polySia interaction partners within a cellular system using fluorescence-labeled biotin. The obtained results demonstrate that polysialylated streptavidin is a flexible bioconjugate that can be applied to investigate polySia interaction partners using various blotting and fluorescence imaging techniques.

BackgroundExposure to cadmium, a trace metallic element, is a major health concern. Cadmium is associated with a higher risk and predisposition to cardiovascular disease. Identifying molecular targets involved in such an effect is complexified by in utero embryonic and fetal development.MethodsTo overcome those difficulties, we used the established vertebrate heart model of Xenopus laevis to analyze the neural cell adhesion molecules NCAM and FGF receptors involved in early cardiac development under cadmium treatment. Cadmium exposure is performed from fertilization until the completion of mature heart development at the end of stage 45. Additional molecular modifications occurring within the heart are detected in the expressing signaling system of Xenopus oocytes.ResultsExposure to cadmium results in the absence of heart ventricular myocardial trabeculae and disrupts the regulation of NCAM adhesion molecules and FGF receptor signaling in Xenopus. An increase in polysialylation (PSA) of NCAM is observed, accompanied by the deregulation in the expression of Golgi effectors Rab11 GTPase and Golph3. The sialyltransferases ST8Sia2 and ST8Sia4 are not increased at the transcriptional level but are accumulated in the Golgi apparatus. The highly sialylated NCAM interacts with the FGF receptor, prevents the formation of a complex with Integrin, FAK is O-GlcNAcylated, and the receptor translocation to the nucleus is impaired. Furthermore, the polysialylated-NCAM/FGF receptor signaling recruits higher amounts of Shp2 and leads to Erk2 hyperphosphorylation. Additionally, blocking FAK with a specific antibody in the normal polysialylated-NCAM/FGF receptor signaling causes the deregulated molecular phenotype.ConclusionsThese results represent a significant advancement for future studies in environmental toxicology and cardiac developmental dysfunctions resulting from cadmium exposure.

Sialyltransferases catalyze the transfer of sialic acid to glycoconjugates within the Golgi apparatus, generating cell-surface sialoglycans that regulate cell-cell and immune communication. This process can be modulated by inhibiting α-2,6-sialyltransferase 1 (ST6GAL1), one of the most important sialyltransferases. Most β-galactoside ST6GAL1 inhibitors are polar CMP-Neu5Ac derivatives with limited membrane permeability, a limitation shared by the noncarbohydrate inhibitor JFD 00458. Here, we designed JFD 00458 analogs to improve permeability while maintaining ST6GAL1 inhibition and direct target engagement. Incorporation of sulfonic-acid bioisosteres, particularly sulfonylureas, improved passive permeability, as measured by parallel artificial membrane permeability assay, with the best analog reaching log(P_app [cm/s]) = -4.3. Potency was also improved in an expanded series of sulfonylureas, with the best inhibitor displaying an IC50 of 1.9 µM. Microscale thermophoresis confirmed direct ST6GAL1 binding, with similar apparent Kd values of approximately 22-25 µM for JFD 00458 and the optimized sulfonylureas. Saturation transfer difference NMR identified the substituted phenoxy phenyl core as a common ST6GAL1-contacting epitope, while closer contacts from the sulfonylurea-linked aromatic substituents in 6d and 6e were associated with the highest inhibitory potency across the series. These results establish sulfonylureas as membrane-permeable sulfonic-acid bioisosteres and provide a structure-activity framework for further ST6GAL1 inhibitor optimization.

Polysialic acid (polySia) is a glycan polymer composed of a linear chain of sialic acid residues detected on a small set of proteins in mammalian cells. This negatively charged glycan modulates various cellular functions and has potential biomedical applications. However, the targeted synthesis of polySia remains challenging due to biosynthetic and structural complexity. In this study, we investigated three polysialyltransferases from the salmonid Coregonus maraena (Cma-ST8Sia) for their application in glycoengineering. For this, the corresponding genes Cmast8Sia2-R1, Cmast8Sia2-R2, and Cmast8Sia4 were transiently expressed in Nicotiana benthamiana, and confocal laser scanning microscopy of GFP-fused Cma-ST8Sia exhibited trans-Golgi localization. Co-expression of Cma-ST8Sia with genes from the mammalian sialic acid biosynthesis pathway resulted in autopolysialylation of CmaST8SiaII-R1 and CmaST8SiaIV, but not of CmaST8SiaII-R2. Furthermore, co-expression with glycoproteins demonstrated polysialylation of recombinant proteins with distinct efficiencies and chain lengths. Inactive EndoN-coupled resin was used to enrich polysialylated proteins, and by the application of HPLC/DMB labeling, a degree of polymerization up to 45 was determined. Finally, a microglia inhibition assay demonstrated the biological activity of the engineered polySia. Collectively, these findings advance the capacity to engineer high-quality polySia in an eco-friendly, sustainable system, facilitating in-depth studies and the rational design of complex glycans.

Most major organs, like muscles, bones, vessels and kidneys, develop from the mesoderm, one of three germ cell layers in triploblastic organisms. Sialic acids significantly affect embryonic development by regulating cell division, migration and death through signaling pathways and cell adhesion, which support morphogenesis. Loss of early biosynthetic enzymes reduces embryonic viability and leads to complex phenotypes, while the loss of terminal enzymes primarily results in tissue-specific defects in mesoderm-derived organs. Key sialylated glycoproteins involved in the developmental processes of mesoderm and mesoderm-derived organs have been identified across various species as major effectors. These enzymes and glycoproteins are of significant interest and are discussed in the present review.

Mothers experiencing depressive symptoms or gestational stress are at risk of developing maternal distress, which, in its most severe form, can progress to postpartum depression, profoundly affecting maternal well-being and infant care. Using a late-gestation restraint stress model, we investigated whether postpartum treatment of lactating dams with probiotic Limosilactobacillus reuteri (L. reuteri) could reverse stress-induced disruptions in maternal behavior and neurobiology. L. reuteri effectively improved maternal care, and restored reactivity to pup separation in stressed dams. Postpartum L. reuteri also corrected corticosterone-oxytocin imbalances in plasma, normalized hypothalamic oxytocin levels, the unglycosylated and glycosylated oxytocin receptor isoforms and it regulated the ratio of precursor to mature brain-derived neurotrophic factor (BDNF). Additionally, L. reuteri restored gut microbiota balance, increasing Lactobacillus spp. while reducing pathogenic strains in fecal pellets of stressed dams during lactation and post-weaning, correlating with both maternal oxytocin and corticosterone plasma levels. This study is the first to demonstrate the therapeutic efficacy of a probiotic and its molecular support in counteracting the adverse effects of gestational stress on maternal care through brain-gut axis mechanisms. These findings support probiotic-based interventions as safe, complementary preventive strategy for improving maternal health in conditions involving impaired maternal care and microbiota dysbiosis.

Following fertilization, there is an initial period of rapid cell division that leads to the formation of a multicellular structure known as the blastula, or blastocyst. Within this structure, sialic acids play a key role in influencing cellular processes such as signaling, cell-to-cell contact, and adhesion. In species that develop internally, the blastocyst undergoes implantation and placentation, which depend on maternal immunomodulation facilitated by sialylated proteins and enzymes involved in the biosynthesis of sialic acids. Although research has shown that the elimination of certain initial enzymes in the sialic acid synthetic pathway can lead to reduced embryonic viability, the precise role of these enzymes remains to be further investigated, particularly in the blastula of externally developing species, which have received limited attention. Recently developed blastoid models present promising prospects for future research in this field.

N-acetylglucosaminyltransferases involved in branched N-glycans synthesis, a major post-translational modification, are gathered in the CAZy glycosyltransferase family 54. To date, the origin and evolution of this biosynthetic pathway are unknown, and the functional organization of the Golgi enzymes remains elusive. Over 230 metazoan GT54-related genes were identified, and sequence-based analysis of vertebrate MGAT4 proteins shed light on evolutionary conserved peptide motifs and structural features like the lectin domain (CBM94). Molecular phylogeny analyses disentangled their evolutionary relationships, revealing the deep ancestry of two metazoan clusters, and unveiled the existence of seven vertebrate MGAT4 subfamilies. Comparative genomics and sequence-based analyses identified an evolutionarily conserved subgroup of GT54 gathering MGAT4A, MGAT4B, and MGAT4D, whereas the other subgroup comprised of MGAT4C, MGAT4E, MGAT4F, and MGAT4G evolved faster. Biochemical analyses conducted with representatives of each subgroup revealed the existence of two acceptor substrate specificities and suggested their intricate functional organization with the other Golgi branching enzymes.

N-acetylglucosaminyltransferases involved in branched N-glycans synthesis, a major post-translational modification, are gathered in the CAZy glycosyltransferase family 54. To date, the origin and evolution of this biosynthetic pathway are unknown, and the functional organization of the Golgi enzymes remains elusive. Over 230 metazoan GT54-related genes were identified, and sequence-based analysis of vertebrate MGAT4 proteins shed light on evolutionary conserved peptide motifs and structural features like the lectin domain (CBM94). Molecular phylogeny analyses disentangled their evolutionary relationships, revealing the deep ancestry of two metazoan clusters, and unveiled the existence of seven vertebrate MGAT4 subfamilies. Comparative genomics and sequence-based analyses identified an evolutionarily conserved subgroup of GT54 gathering MGAT4A, MGAT4B, and MGAT4D, whereas the other subgroup comprised of MGAT4C, MGAT4E, MGAT4F, and MGAT4G evolved faster. Biochemical analyses conducted with representatives of each subgroup revealed the existence of two acceptor substrate specificities and suggested their intricate functional organization with the other Golgi branching enzymes.

The β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) which synthesizes the histo-blood group antigen Sda is highly expressed by normal colon, but it is dramatically down-regulated in colorectal cancer (CRC). High B4GALNT2 expression in CRC tissues is a marker of longer survival. The molecular bases of B4GALNT2 inhibition in CRC are largely obscure. A key role may be played by transcription factors and miRNA. Through an in silico analysis of The Cancer Genome Atlas and of the Cancer Cell Line Encyclopedia, we identified the transcription factors FOXD1, FOXF2 and PGR as well as mir-204-5p as potential inhibitory agents. Their transient transfection in the cell line GP2d, whose B4GALNT2 is closer to that of a normal mucosa, confirmed their inhibitory activity with a crucial role for FOXD1. The latter inhibited B4GALNT2 also in the middle B4GALNT2 expresser cell line Caco2. Deletion experiments of the putative FOXD1 binding sites in the ~ 2800 bp sequence upstream of the B4GALNT2 transcriptional start site cloned in frame with the luciferase reporter gene, confirmed the regulatory role of FOXD1. Finally, FOXD1 knock down in the non-B4GALNT2 expresser cell line SW948 stimulated B4GALNT2. Thus, FOXD1 and miR-204-5p emerged as crucial new player of B4GALNT2 down-regulation in CRC.

Heavy metals are released into the environment in increasing amounts from different natural and anthropogenic sources. Among them, cadmium contaminates aquatic habitats and represents a threat to Amphibians. To assess the risks of exposure to cadmium in the aquatic environment, we studied the survival rate of early tadpoles of Xenopus laevis under exposure to CdCl2 for 6 days in the concentration range between 0.15 and 150 µM of Cd2+. Tadpoles survived and reached stage 45 before feeding at all concentrations tested except 150 µM Cd2+, which significantly induced death. With an exposure of 15 µM Cd2+, tadpoles' mean body length decreased, heart rate increased, fastest swimming speed decreased, and distance traveled was greater compared to unexposed controls. Additionally, a witness of neuronal normal development, the neural cell adhesion molecules (NCAM) expression, was decreased. Moreover, this cell-surface glycoprotein exhibited higher polysialylation, a post-translational modification capable to reduce cell adhesion properties and to affect organ development. Our study highlights the effects of Cd2+ on a series of parameters including morphology, physiology, and behavior. They emphasize the deregulation of molecular NCAM suggesting this effector is an interesting biomarker to detect cadmic toxicity in early tadpoles.

Structural variation of N-glycans is essential for the regulation of glycoprotein functions. GalNAcb1-4GlcNAc (LacdiNAc or LDN), a unique subterminal glycan structure synthesized by B4GALNT3 or B4GALNT4, is involved in the clearance of N- glycoproteins from the blood and maintenance of cell stemness. Such regulation of glycoprotein functions by LDN is largely different from that by the dominant subterminal structure, N-acetyllactosamine (Galb1-4GlcNAc, LacNAc). However, the mechanisms by which B4GALNT activity is regulated and how LDN plays different roles from LacNAc remain unclear. Here, we found that B4GALNT3 and four have unique domain organization containing a noncatalytic PA14 domain, which is a putative glycan-binding module. A mutant lacking this domain dramatically decreases the activity toward various substrates, such as N-glycan, O-GalNAc glycan, and glycoproteins, indicating that this domain is essential for enzyme activity and forms part of the catalytic region. In addition, to clarify the mechanism underlying the functional differences between LDN and LacNAc, we examined the effects of LDN on the maturation of N-glycans, focusing on the related glycosyltransferases upstream and downstream of B4GALNT. We revealed that, unlike LacNAc synthesis, prior formation of bisecting GlcNAc in N-glycan almost completely inhibits LDN synthesis by B4GALNT3. Moreover, the presence of LDN negatively impacted the actions of many glycosyltransferases for terminal modifications, including sialylation, fucosylation, and human natural killer-1 synthesis. These fi ndings demonstrate that LDN has significant impacts on N-glycan maturation in a completely different way from LacNAc, which could contribute to obtaining a comprehensive overview of the system regulating complex N-glycan biosynthesis.

N-Glycan branching critically regulates glycoprotein functions and is involved in various diseases. Among the glycosyltransferases involved in N-glycan branching is the human N-acetylglucosaminyltransferase-IV (GnT-IV) family, which has four members: GnT-IVa, GnT-IVb, GnT-IVc, and GnT-IVd. GnT-IVa and GnT-IVb have glycosyltransferase activity that generates the type-2 diabetes-related β1,4-GlcNAc branch on the α1,3-Man arm of N-glycans, whereas GnT-IVc and GnT-IVd do not. Recently, this enzyme family was found to have a unique lectin domain in the C-terminal region, which is essential for enzyme activity toward glycoprotein substrates but not toward free N-glycans. Furthermore, interaction between the lectin domain of GnT-IV and N-glycan attached to GnT-IV enables self-regulation of GnT-IV activity, indicating that the lectin domain plays a unique and pivotal role in the regulation of GnT-IV activity. In this review, we summarize the GnT-IV family's biological functions, selectivity for glycoprotein substrates, and regulation of enzymatic activity, with a focus on its unique C-terminal lectin domain.

Human sialyltransferases primarily utilize CMP-Sias, especially transferring Neu5Ac from CMP-Neu5Ac to various acceptors. Advances in chemical biology have led to the synthesis of novel CMP-Sia donors suitable for bioorthogonal reactions in cell-based assays. However, the compatibility of these donors with all human enzymes remains uncertain. We synthesized a non-natural CMP-Sia donor with an alkyne modification on the N-acyl group of Neu5Ac, which was effectively used by human ST6Gal I and ST3Gal I. A sensitive MicroPlate Sialyltransferase Assay (MPSA) was developed and expanded to a panel of 13 human STs acting on glycoproteins. All assayed enzymes tolerated CMP-SiaNAl, allowing for the determination of kinetic parameters and turnover numbers. This study enhances the biochemical characterization of human sialyltransferases and opens new avenues for developing sialyltransferase inhibitors.

BackgroundSialic acids are essential monosaccharides influencing several biological processes and disease states. The sialyltransferases catalyze the transfer of Sia residues to glycoconjugates playing critical roles in cellular recognition and signaling. Despite their importance, the molecular mechanisms underlying their substrate specificity, especially between different organisms, remain poorly understood. Recently, the human ST8Sia IV, a key enzyme in the synthesis of polysialic acids, was found to accept only CMP-Neu5Ac as a sugar-donor, whereas the whitefish Coregonus maraena enzyme showed a wider donor substrate specificity, accepting CMP-Neu5Ac, CMP-Neu5Gc, and CMP-Kdn. However, what causes these differences in donor substrate specificity is unknown.MethodsComputational approaches were used to investigate the structural and biochemical determinants of the donor substrate specificity in ST8Sia IV. Accurate structural models of the human and fish ST8Sia IV catalytic domains and their complexes with three sialic acid donors (CMP-Neu5Ac, CMP-Neu5Gc, and CMP-Kdn) were generated. Subsequently, molecular dynamics simulations were conducted to analyze the stability and interactions within these complexes and identify differences in complex stability and substrate binding sites between the two ST8Sia IV.ResultsOur MD simulations revealed that the human enzyme effectively stabilizes CMP-Neu5Ac, whereas CMP-Neu5Gc and CMP-Kdn are unstable and explore different conformations. In contrast, the fish ST8Sia IV stabilizes all three donor substrates. Based on these data, we identified the key interacting residues for the different Sias parts of the substrate donors.General significanceThis work advances our knowledge of the enzymatic mechanisms governing sialic acid transfer, shedding light on the evolutionary adaptations of sialyltransferases.

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

The human polysialyltransferases ST8Sia II and ST8Sia IV catalyze the transfer of several Neu5Ac residues onto glycoproteins forming homopolymers with essential roles during different physiological processes. In salmonids, heterogeneous set of sialic acids polymers have been described in ovary and on eggs cell surface and three genes st8sia4, st8sia2-r1 and st8sia2-r2 were identified that could be implicated in these heteropolymers. The three polysialyltransferases from the salmonid Coregonus maraena were cloned, recombinantly expressed in HEK293 cells and the ST8Sia IV was biochemically characterized. The MicroPlate Sialyltransferase Assay and the non-natural donor substrate CMP-SiaNAl were used to demonstrate enzyme activity and optimize polysialylation reactions. Polysialylation was also carried out with natural donor substrates CMP-Neu5Ac, CMP-Neu5Gc and CMP-Kdn in cell-free and cell-based assays and structural analyses of polysialylated products using the anti-polySia monoclonal antibody 735 and endoneuraminidase N and HPLC approaches. Our data highlighted distinct specificities of human and salmonid polysialyltransferases with notable differences in donor substrates use and the capacity of fish enzymes to generate heteropolymers. This study further suggested an evolution of the biological functions of polySia. C. maraena ST8Sia IV of particular interest to modify glycoproteins with a variety of polySia chains.

Every eukaryotic cell is covered with a thick layer of complex carbohydrates with essential roles in their social life. In Deuterostoma, sialic acids present at the outermost positions of glycans of glycoconjugates are known to be key players in cellular interactions including host-pathogen interactions. Their negative charge and hydrophilic properties enable their roles in various normal and pathological states and their expression is altered in many diseases including cancers. Sialylation of glycoproteins and glycolipids is orchestrated by the regulated expression of twenty sialyltransferases in human tissues with distinct enzymatic characteristics and preferences for substrates and linkages formed. However, still very little is known on the functional organization of sialyltransferases in the Golgi apparatus and how the sialylation machinery is finely regulated to provide the ad hoc sialome to the cell. This review summarizes current knowledge on sialyltransferases, their structure–function relationships, molecular evolution, and their implications in human biology.

Sialic acids are a family of 9-carbon monosaccharides with particular physicochemical properties. They modulate the biological functions of the molecules that carry them and are involved in several steps of the reproductive process. Sialoglycoproteins participate in the balance between species recognition and specificity, and the mechanisms of these aspects remain an issue in gametes formation and binding in metazoan reproduction. Sialoglycoproteins form a specific coat at the gametes surface and specific polysialylated chains are present on marine species oocytes. Spermatozoa are submitted to critical sialic acid changes in the female reproductive tract facilitating their migration, their survival through the modulation of the female innate immune response, and the final oocyte-binding event. To decipher the role of sialic acids in gametes and at fertilization, the dynamical changes of enzymes involved in their synthesis and removal have to be further considered.