Macrophage- and CD4+ T cell-derived SIV differ in glycosylation, infectivity and neutralization sensitivity.

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4+ T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N-glycans in SIV from CD4+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.

A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule.

Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.

Sialic acids on B cells are crucial for their survival and provide protection against apoptosis.

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.

Rescue of synaptic and cognitive functions in polysialic acid-deficient mice and dementia models by short polysialic acid fragments.

Dysregulated cortical expression of the neural cell adhesion molecule (NCAM) and deficits of its associated polysialic acid (polySia) have been found in Alzheimer's disease and schizophrenia. However, the functional role of polySia in cortical synaptic plasticity remains poorly understood. Here, we show that acute enzymatic removal of polySia in medial prefrontal cortex (mPFC) slices leads to increased transmission mediated by the GluN1/GluN2B subtype of N-methyl-d-aspartate receptors (NMDARs), increased NMDAR-mediated extrasynaptic tonic currents, and impaired long-term potentiation (LTP). The latter could be fully rescued by pharmacological suppression of GluN1/GluN2B receptors, or by application of short soluble polySia fragments that inhibited opening of GluN1/GluN2B channels. These treatments and augmentation of synaptic NMDARs with the glycine transporter type 1 (GlyT1) inhibitor sarcosine also restored LTP in mice deficient in polysialyltransferase ST8SIA4. Furthermore, the impaired performance of polySia-deficient mice and two models of Alzheimer's disease in the mPFC-dependent cognitive tasks could be rescued by intranasal administration of polySia fragments. Our data demonstrate the essential role of polySia-NCAM in the balancing of signaling through synaptic/extrasynaptic NMDARs in mPFC and highlight the therapeutic potential of short polySia fragments to restrain GluN1/GluN2B-mediated signaling.

An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1.

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-ß(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.

Sialic acids in the brain: gangliosides and polysialic acid in nervous system development, stability, disease, and regeneration.

Every cell in nature carries a rich surface coat of glycans, its glycocalyx, which constitutes the cell's interface with its environment. In eukaryotes, the glycocalyx is composed of glycolipids, glycoproteins, and proteoglycans, the compositions of which vary among different tissues and cell types. Many of the linear and branched glycans on cell surface glycoproteins and glycolipids of vertebrates are terminated with sialic acids, nine-carbon sugars with a carboxylic acid, a glycerol side-chain, and an N-acyl group that, along with their display at the outmost end of cell surface glycans, provide for varied molecular interactions. Among their functions, sialic acids regulate cell-cell interactions, modulate the activities of their glycoprotein and glycolipid scaffolds as well as other cell surface molecules, and are receptors for pathogens and toxins. In the brain, two families of sialoglycans are of particular interest: gangliosides and polysialic acid. Gangliosides, sialylated glycosphingolipids, are the most abundant sialoglycans of nerve cells. Mouse genetic studies and human disorders of ganglioside metabolism implicate gangliosides in axon-myelin interactions, axon stability, axon regeneration, and the modulation of nerve cell excitability. Polysialic acid is a unique homopolymer that reaches >90 sialic acid residues attached to select glycoproteins, especially the neural cell adhesion molecule in the brain. Molecular, cellular, and genetic studies implicate polysialic acid in the control of cell-cell and cell-matrix interactions, intermolecular interactions at cell surfaces, and interactions with other molecules in the cellular environment. Polysialic acid is essential for appropriate brain development, and polymorphisms in the human genes responsible for polysialic acid biosynthesis are associated with psychiatric disorders including schizophrenia, autism, and bipolar disorder. Polysialic acid also appears to play a role in adult brain plasticity, including regeneration. Together, vertebrate brain sialoglycans are key regulatory components that contribute to proper development, maintenance, and health of the nervous system.

Efficient solid-phase synthesis of meningococcal capsular oligosaccharides enables simple and fast chemoenzymatic vaccine production.

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.

Approach for Profiling of Glycosphingolipid Glycosylation by Multiplexed Capillary Gel Electrophoresis Coupled to Laser-Induced Fluorescence Detection To Identify Cell-Surface Markers of Human Pluripotent Stem Cells and Derived Cardiomyocytes.

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.

Engineering of complex protein sialylation in plants.

Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.

Polysialylation at Early Stages of Oligodendrocyte Differentiation Promotes Myelin Repair.

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.

A sweet development in Notch regulation.

The transmembrane signaling protein Notch, which is crucial for embryonic cell fate decisions, has 36 extracellular EGF domains that are glycosylated in variable and complex ways. A new study shows that O-fucose and O-glucose stabilize the repeats but that extension of glucose by xylose weakens stability, explained by the binding of the glycan to a protein groove. This work shows how different types of glycosylation can distinctly influence protein stability and structure.

Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells.

BACKGROUND: Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo. RESULTS: IBs directed against the polySTs prevented the translocation of the enzymes from the ER to the Golgi-apparatus. Co-immunoprecipitation of ST8SiaII and ST8SiaIV with the corresponding IBs confirmed the intracellular interaction with their cognate antigens. In CHO cells overexpressing ST8SiaII and ST8SiaIV, respectively, the transfection with αST8SiaII-IB or αST8SiaIV-IB inhibited significantly the cell surface expression of polysialylated NCAM. Furthermore stable expression of ST8SiaII-IB, ST8SiaIV-IB and luciferase in the rhabdomyosarcoma cell line TE671 reduced cell surface expression of polySia and delayed tumor growth if cells were xenografted into C57BL/6 J RAG-2 mice. CONCLUSION: Data obtained strongly indicate that αST8SiaII-IB and αST8SiaIV-IB are promising experimental tools to analyze the individual role of the two enzymes during brain development and during migration and proliferation of tumor cells.

A crucial role for polysialic acid in developmental interneuron migration and the establishment of interneuron densities in the mouse prefrontal cortex.

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.

Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen.

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.

The capsule polymerase CslB of Neisseria meningitidis serogroup L catalyzes the synthesis of a complex trimeric repeating unit comprising glycosidic and phosphodiester linkages.

Neisseria meningitidis is a human pathogen causing bacterial meningitis and sepsis. The capsular polysaccharide surrounding N. meningitidis is a major virulence factor. The capsular polysaccharide consists of polyhexosamine phosphates in N. meningitidis serogroups A and X. The capsule polymerases (CPs) of these serogroups are members of the Stealth protein family comprising d-hexose-1-phosphate transferases from bacterial and protozoan pathogens. CslA, one of two putative CPs of the pathophysiologically less relevant N. meningitidis serogroup L, is one of the smallest known Stealth proteins and caught our attention for structure-function analyses. Because the N. meningitidis serogroup L capsule polymer consists of a trimeric repeating unit ([→3)-ß-d-GlcNAc-(1→3)-ß-d-GlcNAc-(1→3)-α-d-GlcNAc-(1→OPO3→]n), we speculated that the two predicted CPs (CslA and CslB) work together in polymer production. Consequently, both enzymes were cloned, overexpressed, and purified as recombinant proteins. Contrary to our expectation, enzymatic testing identified CslB to be sufficient to catalyze the synthesis of the complex trimeric N. meningitidis serogroup L capsule polymer repeating unit. No polymerase activity was detected for CslA, although the enzyme facilitated the hydrolysis of UDP-GlcNAc. Bioinformatics analyses identified two glycosyltransferase (GT) domains in CslB. The N-terminal domain modeled with 100% confidence onto a number of GT-A folded proteins, whereas the C-terminal domain modeled with 100% confidence onto TagF, a GT-B folded teichoic acid polymerase from Staphylococcus epidermidis. Amino acid positions known to have critical catalytic functions in the template proteins were conserved in CslB, and their point mutation abolished enzyme activity. CslB represents an enzyme of so far unique complexity regarding both the catalyzed reaction and enzyme architecture.

Exclusive Decoration of Simian Immunodeficiency Virus Env with High-Mannose Type N-Glycans Is Not Compatible with Mucosal Transmission in Rhesus Macaques.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope (Env) proteins are extensively decorated with N-glycans, predominantly of the high-mannose type. However, it is unclear how high-mannose N-glycans on Env impact viral spread. We show that exclusive modification of SIV Env with these N-glycans reduces viral infectivity and abrogates mucosal transmission, despite increasing viral capture by immune cell lectins. Thus, high-mannose N-glycans have opposed effects on SIV infectivity and lectin reactivity, and a balance might be required for efficient mucosal transmission.

Engineering the product profile of a polysialyltransferase.

Oligo- and polysaccharides have myriad applications as therapeutic reagents from glycoconjugate vaccines to matrices for tissue engineering. Polysaccharide length may vary over several orders of magnitude and is a critical determinant of both their physical properties and biological activities. Therefore, the tailored synthesis of oligo- and polysaccharides of defined size is a major goal for glycoengineering. By mutagenesis and screening of a bacterial polysialyltransferase (polyST), we identified a single-residue switch that controls the size distribution of polymeric products. Specific substitutions at this site yielded distributive enzymes that synthesize polysaccharides with narrow size distribution ideal for glycoengineering applications. Mechanistic investigation revealed that the wild-type enzyme has an extended binding site that accommodates at least 20 residues of the growing polymer; changes in affinity along this binding site allow fine-tuning of the enzyme's product distribution.

Dissection of hexosyl- and sialyltransferase domains in the bifunctional capsule polymerases from Neisseria meningitidis W and Y defines a new sialyltransferase family.

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).

Molecular cloning and functional characterization of components of the capsule biosynthesis complex of Neisseria meningitidis serogroup A: toward in vitro vaccine production.

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 (1)H NMR, (31)P 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.