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Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid–binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell–specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas -deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.

ispartof: GLYCOBIOLOGY vol:32 issue:6 pages:458-459 ispartof: location:England status: published

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

Application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as tissue transplants in regenerative medicine depends on cell-surface marker-based characterization and/or purification. Glycosphingolipids (GSLs) are a family of highly diverse surface-exposed biomolecules that have been neglected as potential surface markers for hiPSC-CMs due to significant analytical challenges. Here, we describe the development of a novel and high-throughput-compatible workflow for the analysis of GSL-derived glycans based on ceramide glycanase digestion, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling, and multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). GSL glycans were detected with highly reproducible migration times after repeated analysis by xCGE-LIF. We built up a migration time database comprising 38 different glycan species, and we showed exemplarily that as few as 10 pg of fucosyl lactotetra was detectable. GSL glycan profiling could be performed with 105 human induced pluripotent stem cells, and we quantitatively dissected global alterations of GSL glycosylation of human induced pluripotent stem cells (hiPSCs) and hiPSC-CMs by employing xCGE-LIF. In our study, we observed a general switch from complex GSLs with lacto- and globo-series core structures comprising the well-known human pluripotent stem cell marker stage-specific embryonic antigen 3 (SSEA3) and SSEA4 in hiPSCs toward the simple gangliosides GM3 and GD3 in hiPSC-CMs. This is the first description of GM3 and GD3 being highly abundant GSLs on the cell surface of stem cell-derived cardiomyocytes.

Polysialic acid (polySia) are α2,8- and/or α2,9-linked homopolymers with interesting properties for meningococcal vaccine development or the cure of human neurodegenerative disorders. With the goal to avoid large scale production of pathogenic bacteria, we compare in the current study the efficacy of conventional polySia production to recombinant approaches using the engineered laboratory safety strain E. coli BL21. High cell density cultivation (HCDC) experiments were performed in two different bioreactor systems. Increased cell densities of up to 11.3 (±0.4) g/L and polySia concentrations of up to 774 (±18) mg/L were reached in E. coli K1. However, cultivation of engineered E. coli BL21 strains delivered comparable cell densities but a maximum of only 133 mg/L polySia. Using established downstream procedures, host cell DNA and proteins were removed. All recombinant polySia products showed an identical degree of polymerization >90. Polymers with different glycosidic linkages could be successfully differentiated by nuclear magnetic resonance spectroscopy.

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Bibersteinia trehalosi, and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis., IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy. The new capsule polymerase family described here vastly increases the toolbox of enzymes available for biotechnology purposes. Representatives are abundantly found in human pathogens but also in animal pathogens, paving the way for the exploitation of polymerases for the development of a new generation of vaccines for animal husbandry.

Background Glycosylation is a common and complex type of protein posttranslational modification. Altered glycosylation of immunoglobulins in autoimmune diseases has led to the “altered glycan hypothesis” postulating existence of a unique glycan signature on immune cells and extracellular proteins characterized by site‐specific relative abundances of individual glycan structures and glycosylation patterns. However, it is not clear how glycosylation on leukocyte subpopulations differ between states of health or inflammation. Hypothesis Glycosphingolipid patterns on immune cells of forkhead‐box‐P3‐deficient scurfy mice differs from those on wild‐type immune cells. Methods T cells and dendritic cells were isolated from spleens of either wild‐type or age‐matched scurfy mice. Glycosphingolipids of CD4+ T cells and splenic dendritic cells from wild‐type and scurfy mice were then analyzed by multiplexed capillary gel electrophoresis coupled to laser‐induced fluorescence detection (xCGE‐LIF). In addition, flow cytometry and ChipCytometry were used to access expression patterns of various C‐type lectin receptors on antigen‐presenting cells from various organs of both wild‐type and scurfy mice. Results We, hereby report differential expression of glycosphingolipids in health and under inflammatory conditions as reflected in wild‐type and scurfy mice. Furthermore, we observed that the absence of functional regulatory T cells correlated with elevated expression of CLEC‐7A and CD205 but a reduction in levels of CLEC12A and CD206 on antigen‐presenting cells. Conclusion We hereby show that the absence of functional regulatory T cells affects expression pattern and quantities of glycosphingolipids on immune cells. Thus, glycosphingolipids could serve as biomarkers for mapping genetical and homeostatic perturbances such as those resulting from a diseased condition., (a) Attenuated levels of glycosphingolipids in splenic dendritic cells in forkhead‐box‐P3 (FOXP3)‐deficient scurfy mice lacking functional regulatory T cells. (b) Glycosphingolipids patterns on CD4+ T cells are not affected in absence of FOXP3. (c) Expression patterns and quantities of glycosphingolipids on immune cells could serve as signatures to map inflammation and homeostatic conditions in health or as biomarkers for identifying homeostatic perturbances.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO(4)-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

Biocatalysts utilize allosteric mechanisms to control selectivity, catalytic activity, and the transport of reaction components. The allosteric control of catalysis has a high potential for the development of drugs and technologies. In particular, it opens the way to specific regulation of vital enzymes with conserved active sites. Using the central metabolic enzyme UDP-glucose pyrophosphorylase from the pathogen Leishmania major (LmUGP), we demonstrate how specific allosteric inhibition sites and their links to the catalytic center can be revealed rationally, through analysis of molecular interfaces along the enzymatic reaction cycle. Two previously unknown specific allosteric inhibition sites in LmUGP were rationally identified and experimentally verified. The molecular scaffold for allosteric inhibitor targeting the pathogen’s enzyme was developed. This led to the identification of murrayamine-I as an allosteric inhibitor that selectively blocks LmUGP. The presented approach opens up the possibility of...

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Virus-neutralizing antibodies are a severe obstacle in oncolytic virotherapy. Here, we present a strategy to convert this unfavorable immune response into an anticancer immunotherapy via molecular retargeting. Application of a bifunctional adapter harboring a tumor-specific ligand and the adenovirus hexon domain DE1 for engaging antiadenoviral antibodies, attenuates tumor growth and prolongs survival in adenovirus-immunized mice. The therapeutic benefit achieved by tumor retargeting of antiviral antibodies is largely due to NK cell-mediated triggering of tumor-directed CD8 T-cells. We further demonstrate that antibody-retargeting (Ab-retargeting) is a feasible method to sensitize tumors to PD-1 immune checkpoint blockade. In therapeutic settings, Ab-retargeting greatly improves the outcome of intratumor application of an oncolytic adenovirus and facilitates long-term survival in treated animals when combined with PD-1 checkpoint inhibition. Tumor-directed retargeting of preexisting or virotherapy-induced antiviral antibodies therefore represents a promising strategy to fully exploit the immunotherapeutic potential of oncolytic virotherapy and checkpoint inhibition., The efficacy of oncolytic adenoviruses is limited by strong immune responses being induced against the oncolytic virus itself. Here, the authors generate a bispecific molecule capable of redirecting the adenovirus-specific antibodies to tumour cells and show this induces immune mediated cancer growth inhibition and enhances the therapeutic efficacy of viral oncolysis.

The negatively charged sugar sialic acid (Sia) occupies the outermost position in the bulk of cell surface glycans. Lack of sialylated glycans due to genetic ablation of the Sia-activating enzyme CMP–sialic acid synthase (CMAS) resulted in embryonic lethality around day 9.5 post coitum (E9.5) in mice. Developmental failure was caused by complement activation on trophoblasts in Cmas(–/–) implants and was accompanied by infiltration of maternal neutrophils at the fetal-maternal interface, intrauterine growth restriction, impaired placental development, and a thickened Reichert’s membrane. This phenotype, which shared features with complement receptor 1-related protein Y (Crry) depletion, was rescued in E8.5 Cmas(–/–) mice upon injection of cobra venom factor, resulting in exhaustion of the maternal complement component C3. Here we show that Sia is dispensable for early development of the embryo proper but pivotal for fetal-maternal immune homeostasis during pregnancy, i.e., for protecting the allograft implant against attack by the maternal innate immune system. Finally, embryos devoid of cell surface sialylation suffered from malnutrition due to inadequate placentation as a secondary effect.

A chemokine's sugary release As immune cells survey the body for pathogens, they circulate through the blood and migrate through the lymphatic system. The latter route allows for tissues and lymph nodes—the central hubs of the immune system—to communicate. Kiermaier et al. reveal the importance of the monosaccharide sialic acid in keeping immune cells in motion. Multiple sialic acids decorate the surface CCR7 on immune cells. CCR7 recognizes proteins called chemokines, which direct where cells move in the body. Sialic acids on CCR7 release one such chemokine present on lymph node endothelial cells from an inhibited state, allowing immune cells to enter lymph nodes. Science , this issue p. 186

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis , Actinobacillus pleuropneumoniae , Haemophilus influenzae , Bibersteinia trehalosi , and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis. IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy. The new capsule polymerase family described here vastly increases the toolbox of enzymes available for biotechnology purposes. Representatives are abundantly found in human pathogens but also in animal pathogens, paving the way for the exploitation of polymerases for the development of a new generation of vaccines for animal husbandry.

Studies on the polymerization mode of Neisseria meningitidis serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total in vitro construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates.

Aggregatibacter (A.) actinomycetemcomitans is a periodontopathogenic bacterium causing aggressive periodontitis. Here we describe a single-step transconjugation system as novel and easily applicable protocol for site-specific genetic manipulation of A. actinomycetemcomitans. Deletion of PgaC, which is involved in the synthesis of biofilm matrix, led to a reduced biofilm formation.

Crucial virulence determinants of disease causing Neisseria meningitidis species are their extracellular polysaccharide capsules. In the serogroups W and Y, these are heteropolymers of the repeating units (→6)-α-d-Gal-(1→4)-α-Neu5Ac-(2→)n in NmW and (→6)-α-d-Glc-(1→4)-α-Neu5Ac-(2→)n in NmY. The capsule polymerases, SiaDW and SiaDY, which synthesize these highly unusual polymers, are composed of two predicted GT-B fold domains separated by a large stretch of amino acids (aa 399-762). We recently showed that residues critical to the hexosyl- and sialyltransferase activity are found in the predicted N-terminal (aa 1-398) and C-terminal (aa 763-1037) GT-B fold domains, respectively. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain extends well beyond the predicted C-terminal GT-B domain and defines a new glycosyltransferase family, GT97, in CAZy (Carbohydrate-Active enZYmes Database).

ST3GAL3 encodes the Golgi enzyme beta-galactoside-alpha-2,3-sialyltransferase-III that in humans forms, among others, the sialyl Lewis a (sLe(a)) epitope on proteins. Functionally deleterious variants in this gene were previously identified in patients with either non-syndromic or syndromic intellectual disability such as West syndrome, an age-dependent epileptic encephalopathic syndrome associated with developmental arrest or regression. The aim of this study was to further elucidate the molecular and cellular mechanisms causing West syndrome by lack of ST3GAL3 function. For this purpose we generated induced pluripotent stem cell (iPSC) lines from fibroblasts obtained from a patient with West syndrome, carrying a variant in exon 12 (c.958G>C, p.(Ala320Pro)) of ST3GAL3, and a healthy sibling, using lentiviral reprogramming. iPSCs and cortical neurons derived thereof were analysed by lectin blots, mRNA sequencing, adherence assays, and FACS. While no significant difference was observed at stem cell or fibroblast level between patient and control cells, patient-derived cortical neurons displayed an altered lectin blot staining pattern, enhanced adherence to a poly-l-ornithine/laminin-coated surface and decreased levels of neurons expressing T-box transcription factor brain 1. Our results suggest that changes in the sialylation pattern on the surface of specific neuronal cell types affect adhesive interactions during development, which in turn may cause subtle changes in tissue composition that could result in the occurrence of epilepsy and might impair neural development to an extent that is detrimental to the development and maintenance of normal cognitive functions.

Neisseria meningitidis serogroups A and X are among the leading causes of bacterial meningitis in the African meningitis belt. Glycoconjugate vaccines, consisting of an antigenic carrier protein coupled to the capsular polysaccharide of the bacterial pathogen, are the most effective strategy for prevention of meningococcal disease. However, the distribution of effective glycoconjugate vaccines in this region is limited by the high cost of cultivating pathogens and purification of their capsular polysaccharides. Moreover, chemical approaches to synthesize oligosaccharide antigens have proven challenging. In the current study, we present a chemoenzymatic approach for generating tailored oligosaccharide fractions ready for activation and coupling to the carrier protein. In a first step, the elongation modes of recombinant capsular polymerases from Neisseria meningitidis serogroups A (CsaB) and X (CsxA) were characterized. We observed that CsaB is a distributive enzyme, and CsxA is a processive enzyme. Sequence comparison of these two stealth family proteins revealed a C-terminal extension in CsxA, which conferred processivity because of the existence of a second product-binding site. Deletion of the C-terminal domain converted CsxA into a distributive enzyme, allowing facile control of product length by adjusting the ratio of donor to acceptor sugars. Solid-phase fixation of the engineered capsular polymerases enabled rapid production of capsular polysaccharides with high yield and purity. In summary, the tools developed here provide critical steps toward reducing the cost of conjugate vaccine production, which will increase access in regions with the greatest need. Our work also facilitates efforts to study the relationship between oligosaccharide size and antigenicity.

Genetic replacement of adenoviral fiber knobs by ligands that enable tumor specific targeting of oncolytic adenoviruses is challenging because the fiber knob contributes to virus assembly. Here, we present a novel concept by describing stable recombinant adenoviruses with tumor specific infection mode. The fiber knob was replaced by endosialidaseNF (endoNF), the tailspike protein of bacteriophage K1F. EndoNF recognizes polysialic acid, an oncofetal antigen characteristic for high malignant tumors of neuroendocrine origin. An intramolecular chaperone contained in endoNF warrants folding and compensates for the knob function in virus assembly. Obtained recombinant viruses demonstrated polysialic acid dependent infection modes, strong oncolytic capacity with polysialic acid positive cells in culture and a high potential to inhibit tumor growth in a therapeutic mouse model of subcutaneous neuroblastoma. With a single genetic manipulation we achieved ablation of the fiber knob, introduction of a tumor specific ligand, and folding control over the chimeric fiber construct.

Polysialic acid is a glycan modification of the neural cell adhesion molecule (NCAM) produced by the polysialyltransferases ST8SIA2 and ST8SIA4. Polysialic acid has been detected in multiple sclerosis plaques, but its beneficial or adverse role in remyelination is elusive. Here, we show that, despite a developmental delay, myelination at the onset and during cuprizone-induced demyelination was unaffected in male Ncam1(−/−) or St8sia2(−/−) mice. However, remyelination, restoration of oligodendrocyte densities, and motor recovery after the cessation of cuprizone treatment were compromised. Impaired differentiation of NCAM- or ST8SIA2-negative oligodendrocyte precursors suggested an underlying cell-autonomous mechanism. In contrast, premature differentiation in ST8SIA4-negative cultures explained the accelerated remyelination previously observed in St8sia4(−/−) mice. mRNA profiling during differentiation of human stem cell-derived and primary murine oligodendrocytes indicated that the opposing roles of ST8SIA2 and ST8SIA4 arise from sequential expression. We also provide evidence that potentiation of ST8SIA2 by 9-cis-retinoic acid and artificial polysialylation of oligodendrocyte precursors by a bacterial polysialyltransferase are mechanisms to promote oligodendrocytic differentiation. Thus, differential targeting of polysialyltransferases and polysialic acid engineering are promising strategies to advance the treatment of demyelinating diseases. SIGNIFICANCE STATEMENT The beneficial or adverse role of polysialic acid (polySia) in myelin repair is a long-standing question. As a modification of the neural cell adhesion molecule (NCAM), polySia is produced by the polysialyltransferases ST8SIA2 and ST8SIA4. Here we demonstrate that NCAM and ST8SIA2 promote oligodendrocyte differentiation and myelin repair as well as motor recovery after cuprizone-induced demyelination. In contrast, ST8SIA4 delays oligodendrocyte differentiation, explaining its adverse role in remyelination. These opposing roles of the polysialyltransferases are based on different expression profiles. 9-cis-retinoic acid enhances ST8SIA2 expression, providing a mechanism for understanding how it supports oligodendrocyte differentiation and remyelination. Furthermore, artificial polysialylation of the cell surface promotes oligodendrocyte differentiation. Thus, boosting ST8SIA2 and engineering of polySia are promising strategies for improving myelin repair.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.

In severe liver injury, ductular reactions (DRs) containing bipotential hepatic progenitor cells (HPCs) branch from the portal tract. Neural cell adhesion molecule (NCAM) marks bile ducts and DRs, but not mature hepatocytes. NCAM mediates interactions between cells and surrounding matrix; however, its role in liver development and regeneration is undefined. Polysialic acid (polySia), a unique posttranslational modifier of NCAM, is produced by the enzymes, ST8SiaII and ST8SiaIV, and weakens NCAM interactions. The role of polySia with NCAM synthesizing enzymes ST8SiaII and ST8SiaIV were examined in HPCs in vivo using the choline-deficient ethionine-supplemented and 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet models of liver injury and regeneration, in vitro using models of proliferation, differentiation, and migration, and by use of mouse models with gene defects in the polysialyltransferases (St8sia 2+/−4+/−, and St8sia2−/−4−/−). We show that, during liver development, polySia is required for the correct formation of bile ducts because gene defects in both the polysialyltransferases (St8sia2+/−4+/− and St8sia2−/−4−/− mice) caused abnormal bile duct development. In normal liver, there is minimal polySia production and few ductular NCAM+ cells. Subsequent to injury, NCAM+ cells expand and polySia is produced by DRs/HPCs through ST8SiaIV. PolySia weakens cell-cell and cell-matrix interactions, facilitating HGF-induced migration. Differentiation of HPCs to hepatocytes in vitro results in both transcriptional down-regulation of polySia and cleavage of polySia-NCAM. Cleavage of polySia by endosialidase (endoN) during liver regeneration reduces migration of DRs into parenchyma. Conclusion: PolySia modification of NCAM+ ductules weakens cell-cell and cell-matrix interactions, allowing DRs/HPCs to migrate for normal development and regeneration. Modulation of polySia levels may provide a therapeutic option in liver regeneration. (Hepatology 2014;60:1727–1740)

Polysialic acid (polySia) is a unique glycan modification of the neural cell adhesion molecule NCAM and a major determinant of brain development. Polysialylation of NCAM is implemented by the two polysialyltransferases (polySTs) ST8SIA2 and ST8SIA4. Dysregulation of the polySia-NCAM system and variation in ST8SIA2 has been linked to schizophrenia and other psychiatric disorders. Here, we show reduced interneuron densities in the medial prefrontal cortex (mPFC) of mice with either partial or complete loss of polySia synthesizing capacity by ablation of St8sia2, St8sia4, or both. Cells positive for parvalbumin and perineuronal nets as well as somatostatin-positive cells were reduced in the mPFC of all polyST-deficient lines, whereas calretinin-positive cells and the parvalbumin-negative fraction of calbindin-positive cells were unaffected. Reduced interneuron numbers were corroborated by analyzing polyST-deficient GAD67-GFP knock-in mice. The accumulation of precursors in the ganglionic eminences and reduced numbers of tangentially migrating interneurons in the pallium were observed in polyST-deficient embryos. Removal of polySia by endosialidase treatment of organotypic slice cultures led to decreased entry of GAD67-GFP-positive interneurons from the ganglionic eminences into the pallium. Moreover, the acute loss of polySia caused significant reductions in interneuron velocity and leading process length. Thus, attenuation of polySia interferes with the developmental migration of cortical interneurons and causes pathological changes in specific interneuron subtypes. This provides a possible link between genetic variation in polyST genes, neurodevelopmental alterations and interneuron dysfunction in neuropsychiatric disease.

The human pathogen Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis globally. A major virulence factor of Nm is the capsular polysaccharide (CPS), which in Nm serogroup A consists of N-acetyl-mannosamine-1-phosphate units linked together by phosphodiester linkages [→6)-α-d-ManNAc-(1→OPO3−→]n. Acetylation in O-3 (to a minor extent in O-4) position results in immunologically active polymer. In the capsule gene cluster (cps) of Nm, region A contains the genetic information for CPSA biosynthesis. Thereby the open reading frames csaA, -B, and -C are thought to encode the UDP-N-acetyl-d-glucosamine-2-epimerase, poly-ManNAc-1-phosphate-transferase, and O-acetyltransferase, respectively. With the aim to use a minimal number of recombinant enzymes to produce immunologically active CPSA, we cloned the genes csaA, csaB, and csaC and functionally characterized the purified recombinant proteins. If recombinant CsaA and CsaB were combined in one reaction tube, priming CPSA-oligosaccharides were efficiently elongated with UDP-GlcNAc as the donor substrate, confirming that CsaA is the functional UDP-N-acetyl-d-glucosamine-2-epimerase and CsaB the functional poly-ManNAc-1-phosphate-transferase. Subsequently, CsaB was shown to transfer ManNAc-1P onto O-6 of the non-reducing end sugar of priming oligosaccharides, to prefer non-O-acetylated over O-acetylated primers, and to efficiently elongate the dimer of ManNAc-1-phosphate. The in vitro synthesized CPSA was purified, O-acetylated with recombinant CsaC, and proven to be identical to the natural CPSA by 1H NMR, 31P NMR, and immunoblotting. If all three enzymes and their substrates were combined in a one-pot reaction, nature identical CPSA was obtained. These data provide the basis for the development of novel vaccine production protocols.

Polysaccharides are abundant extracellular matrix components and ideal materials for next-generation bioprostheses. Because the chain length critically impacts polymer properties, biotechnological production requires enzymes that allow polymer length control. For the capsule polymerase from Neisseria meningitidis serogroup B, we describe here the switching from processive to distributive polymer elongation. Our methodology delivered a finely tuned enzyme with narrow product distribution even in the absence of advanced high throughput screening platforms.

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is crucial for nervous system development and brain plasticity. PolySia attachment is catalyzed by the polysialyltransferases (polySTs) ST8SIA2 and ST8SIA4, two enzymes with distinct but also common functions during neurodevelopment and in the adult brain. A growing body of evidence links aberrant levels of NCAM and polySia as well as variation in the ST8SIA2 gene to neuropsychiatric disorders, including schizophrenia. To investigate whether polyST deficiency might cause a schizophrenia-like phenotype, St8sia2 (-/-) mice, St8sia4 (-/-) mice and their wildtype littermates were assessed neuroanatomically and subjected to tests of cognition and sensorimotor functions. St8sia2 (-/-) but not St8sia4 (-/-) mice displayed enlarged lateral ventricles and a size reduction of the thalamus accompanied by a smaller internal capsule and a highly disorganized pattern of fibers connecting thalamus and cortex. Reduced levels of the vesicular glutamate transporter VGLUT2 pointed towards compromised glutamatergic thalamocortical input into the frontal cortex of St8sia2 (-/-) mice. Both polyST-deficient lines were impaired in short- and long-term recognition memory, but only St8sia2 (-/-) mice displayed impaired working memory and deficits in prepulse inhibition. Furthermore, only the St8sia2 (-/-) mice exhibited anhedonic behavior and increased sensitivity to amphetamine-induced hyperlocomotion. These results reveal that reduced polysialylation in St8sia2 (-/-) mice leads to pathological brain development and schizophrenia-like behavior. We therefore propose that genetic variation in ST8SIA2 has the potential to confer a neurodevelopmental predisposition to schizophrenia.

Contains fulltext : 119120.pdf (Publisher’s version ) (Closed access) OBJECTIVE: To identify the underlying genetic defect in a patient with intellectual disability, seizures, ataxia, macrothrombocytopenia, renal and cardiac involvement, and abnormal protein glycosylation. METHODS: Genetic studies involved homozygosity mapping by 250K single nucleotide polymorphism array and SLC35A1 sequencing. Functional studies included biochemical assays for N-glycosylation and mucin-type O-glycosylation and SLC35A1-encoded cytidine 5'-monophosphosialic acid (CMP-sialic acid) transport after heterologous expression in yeast. RESULTS: We performed biochemical analysis and found combined N- and O-glycosylation abnormalities and specific reduction in sialylation in this patient. Homozygosity mapping revealed homozygosity for the CMP-sialic acid transporter SLC35A1. Mutation analysis identified a homozygous c.303G>C (p.Gln101His) missense mutation that was heterozygous in both parents. Functional analysis of mutant SLC35A1 showed normal Golgi localization but 50% reduction in transport activity of CMP-sialic acid in vitro. CONCLUSION: We confirm an autosomal recessive, generalized sialylation defect due to mutations in SLC35A1. The primary neurologic presentation consisting of ataxia, intellectual disability, and seizures, in combination with bleeding diathesis and proteinuria, is discriminative from a previous case described with deficient sialic acid transporter. Our study underlines the importance of sialylation for normal CNS development and regular organ function.

Fertilization in animals is a complex sequence of several biochemical events beginning with the insemination into the female reproductive tract and, finally, leading to embryogenesis. Studies by Kitajima and co-workers (Miyata, S., Sato, C., and Kitajima, K. (2007) Trends Glycosci. Glyc, 19, 85–98) demonstrated the presence of polysialic acid (polySia) on sea urchin sperm. Based on these results, we became interested in the potential involvement of sialic acid polymers in mammalian fertilization. Therefore, we isolated human sperm and performed analyses, including Western blotting and mild 1,2-diamino-4,5-methylenedioxybenzene-HPLC, that revealed the presence α2,8-linked polySia chains. Further analysis by a glyco-proteomics approach led to the identification of two polySia carriers. Interestingly, besides the neural cell adhesion molecule, the polysialyltransferase ST8SiaII has also been found to be a target for polysialylation. Further analysis of testis and epididymis tissue sections demonstrated that only epithelial cells of the caput were polySia-positive. During the epididymal transit, polySia carriers were partially integrated into the sperm membrane of the postacrosomal region. Because polySia is known to counteract histone as well as neutrophil extracellular trap-mediated cytotoxicity against host cells, which plays a role after insemination, we propose that polySia in semen represents a cytoprotective element to increase the number of vital sperm. Background: Polysialylated glycoproteins play an import role during numerous biological processes. Results: Polysialylated ST8SiaII and NCAM are components of mammalian semen and are partially associated with spermatozoa. Conclusion: Polysialic acid represents a further glyco-motif in mammalian ejaculates, which is known to influence the immune system. Significance: Administration of polysialic acid during insemination might be useful to increase the number of spermatozoa escaping the female immune system.

Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. Crucial virulence determinants of pathogenic Nm strains are the polysaccharide capsules that support invasion by hindering complement attack. In NmW-135 and NmY the capsules are built from the repeating units (→ 6)-α-D-Gal-(1 → 4)-α-Neu5Ac-(2 →)n and (→ 6)-α-D-Glc-(1 → 4)-α-Neu5Ac-(2 →)n, respectively. These unusual heteropolymers represent unique examples of a conjugation between sialic acid and hexosyl-sugars in a polymer chain. Moreover, despite the various catalytic strategies needed for sialic acid and hexose transfer, single enzymes (SiaDW-135/Y) have been identified to form these heteropolymers. Here we used SiaDW-135 as a model system to delineate structure-function relationships. In size exclusion chromatography active SiaDW-135 migrated as a monomer. Fold recognition programs suggested two separate glycosyltransferase domains, both containing a GT-B-fold. Based on conserved motifs predicted folds could be classified as a hexosyl- and sialyltransferase. To analyze enzyme properties and interplay of the two identified glycosyltransferase domains, saturation transfer difference NMR and mutational studies were carried out. Simultaneous and independent binding of UDP-Gal and CMP-Sia was seen in the absence of an acceptor as well as when the catalytic cycle was allowed to proceed. Enzyme variants with only one functionality were generated by site-directed mutagenesis and shown to complement each other in trans when combined in an in vitro test system. Together the data strongly suggests that SiaDW-135 has evolved by fusion of two independent ancestral genes encoding sialyl- and galactosyltransferase activity.

The glycan polysialic acid is well-known as a unique posttranslational modification of the neural cell adhesion molecule NCAM. Despite remarkable acceptor specificity, however, a few other proteins can be targets of polysialylation. Here, we recapitulate the biosynthesis of polysialic acid by the two polysialyltransferases ST8SIA2 and ST8SIA4 and highlight the increasing evidence that variation in the human ST8SIA2 gene is linked to schizophrenia and possibly other neuropsychiatric disorders. Moreover, we summarize the knowledge on the role of NCAM polysialylation in brain development gained by the analysis of NCAM- and polysialyltransferase-deficient mouse models. The last part of this review is focused on recent advances in identifying SynCAM 1 and neuropilin-2 as novel acceptors of polysialic acid in NG2 cells of the perinatal brain and in dendritic cells of the immune system, respectively.

The enteropathogenic Escherichia coli K92 synthesizes a unique capsule consisting of polysialic acid (polySia) with alternating α2,8- and α2,9-linkages. The fact that a single enzyme is responsible for the synthesis of these alternating regioisomeric linkages raises questions as to how this controlled bifunctionality is achieved mechanistically. Aiming to identify the sequence elements responsible for dual regiospecificity, we have utilized a high-throughput polysialyltransferase (polyST) activity screen to explore the relevant sequence space between this enzyme and its close monofunctional homolog from E. coli K1. The linkage specificity of selected mutants was subsequently confirmed using a polySia permethylation linkage analysis technique. We have identified a single amino acid exchange at residue 52 that toggles these enzymes between mono and dual regiospecificity. The results have implications for the mechanism by which the E. coli K92 polyST achieves bifunctional elongation.

Signal transducer and activator of transcription 3 (STAT3) is an important mediator of cardiac survival pathways. Reduced levels of STAT3 in patients with end-stage heart failure suggest a clinical relevance of STAT3 deficiency for cardiac disease. The recent identification of STAT3 as a mitochondrial protein which is important for full activity of mitochondrial complex I has opened a new field for the investigation of how STAT3 functions in cardioprotection. The goal of this study was to establish a cell culture model with a reduced STAT3 expression, and to use this model for the investigation of mitochondrial and mitochondrial-associated functions under STAT3 deficiency. In the murine cardiomyogenic cell line HL-1, the expression of STAT3 was silenced by lentiviral transduction with anti-STAT3 shRNA (STAT3 KD cells). STAT3 mRNA and protein levels were significantly reduced in HL-1 STAT3 KD cells compared to HL-1 cells transduced with a control shRNA. Spectrophotometric and polarographic assays with mitochondrial enriched fractions and intact cells showed reduced activities of respiratory chain complexes I, II, III and IV in HL-1 STAT3 KD cells. At ultrastructural level, a severe damage of mitochondrial integrity was observed, combined with a significant increase in autophagolysosomes in STAT3-deficient HL-1 cells. Our results demonstrate that the HL-1 STAT3 KD cell line is a good model to study cellular consequences of STAT3 deficiency. Moreover, this is the first study to show that STAT3 deficiency leads to a disruption of mitochondrial ultrastructure and increased autophagy.

Uridine diphosphate-glucose pyrophosphorylase (UGP) occupies a central position in carbohydrate metabolism in all kingdoms of life, since its product uridine diphosphate-glucose (UDP-glucose) is essential in a number of anabolic and catabolic pathways and is a precursor for other sugar nucleotides. Its significance as a virulence factor in protists and bacteria has given momentum to the search for species-specific inhibitors. These attempts are, however, hampered by high structural conservation of the active site architecture. A feature that discriminates UGPs of different species is the quaternary organization. While UGPs in protists are monomers, di- and tetrameric forms exist in bacteria, and crystal structures obtained for the enzyme from yeast and human identified octameric UGPs. These octamers are formed by contacts between highly conserved amino acids in the C-terminal β-helix. Still under debate is the question whether octamerization is required for the functionality of the human enzyme. Here, we used single amino acid replacements in the C-terminal β-helix to interrogate the impact of highly conserved residues on octamer formation and functional activity of human UGP (hUGP). Replacements were guided by the sequence of Arabidopsis thaliana UGP, known to be active as a monomer. Correlating the data obtained in blue native PAGE, size exclusion chromatography and enzymatic activity testing, we prove that the octamer is the active enzyme form. This new insight into structure-function relationships in hUGP does not only improve the understanding of the catalysis of this important enzyme, but in addition broadens the basis for studies aimed at designing drugs that selectively inhibit UGPs from pathogens.

Summary West syndrome consists of infantile spasms, hypsarrhythmia, and developmental arrest. Most patients remain mentally retarded and many develop Lennox-Gastaut syndrome. Using homozygosity mapping followed by exome sequencing we identified an ST3GAL3 mutation in three infants with West syndrome. ST3GAL3 encodes a sialyltransferase involved in the biosynthesis of sialyl-Lewis epitopes on cell surface–expressed glycoproteins. The mutation affected an essential sialyl-motif and abolished enzymatic activity. Abnormalities in proteins involved in forebrain γ-aminobutyric acid (GABA)ergic synaptic growth and function were recently proposed to account for infantile spasms. Dysfunctional ST3GAL3 may thus result in perturbation of the posttranslational sialylation of proteins in these pathways.