A network-based approach to identify substrate classes of bacterial glycosyltransferases

Background

Bacterial interactions with the environment- and/or host largely depend on the bacterial glycome. The specificities of a bacterial glycome are largely determined by glycosyltransferases (GTs), the enzymes involved in transferring sugar moieties from an activated donor to a specific substrate. Of these GTs their coding regions, but mainly also their substrate specificity are still largely unannotated as most sequence-based annotation flows suffer from the lack of characterized sequence motifs that can aid in the prediction of the substrate specificity.

Results

In this work, we developed an analysis flow that uses sequence-based strategies to predict novel GTs, but also exploits a network-based approach to infer the putative substrate classes of these predicted GTs. Our analysis flow was benchmarked with the well-documented GT-repertoire of Campylobacter jejuni NCTC 11168 and applied to the probiotic model Lactobacillus rhamnosus GG to expand our insights in the glycosylation potential of this bacterium. In L. rhamnosus GG we could predict 48 GTs of which eight were not previously reported. For at least 20 of these GTs a substrate relation was inferred.

Conclusions

We confirmed through experimental validation our prediction of WelI acting upstream of WelE in the biosynthesis of exopolysaccharides. We further hypothesize to have identified in L. rhamnosus GG the yet undiscovered genes involved in the biosynthesis of glucose-rich glycans and novel GTs involved in the glycosylation of proteins. Interestingly, we also predict GTs with well-known functions in peptidoglycan synthesis to also play a role in protein glycosylation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-349) contains supplementary material, which is available to authorized users.     

Statistical analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB): Characteristics and diversity of bacterial carbohydrates in comparison with mammalian glycans

Background  

There are considerable differences between bacterial and mammalian glycans. In contrast to most eukaryotic carbohydrates, bacterial glycans are often composed of repeating units with diverse functions ranging from structural reinforcement to adhesion, colonization and camouflage. Since bacterial glycans are typically displayed at the cell surface, they can interact with the environment and, therefore, have significant biomedical importance.     

Donor substrate promiscuity of bacterial β1–3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1–4-galactosyltransferases

β1–3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1–4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1–3Galβ1–4GlcβProN₃ and Galβ1–3Galβ1–4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.     

A guide into glycosciences: How chemistry,biochemistry and biology cooperate to crack the sugar code

BackgroundThe most demanding challenge in research on molecular aspects within the flow of biological information is posed by the complex carbohydrates (glycan part of cellular glycoconjugates). How the ‘message’ encoded in carbohydrate ‘letters’ is ‘read’ and ‘translated’ can only be unraveled by interdisciplinary efforts.Scope of reviewThis review provides a didactic step-by-step survey of the concept of the sugar code and the way strategic combination of experimental approaches characterizes structure–function relationships, with resources for teaching.Major conclusionsThe unsurpassed coding capacity of glycans is an ideal platform for generating a broad range of molecular ‘messages’. Structural and functional analyses of complex carbohydrates have been made possible by advances in chemical synthesis, rendering production of oligosaccharides, glycoclusters and neoglycoconjugates possible. This availability facilitates to test the glycans as ligands for natural sugar receptors (lectins). Their interaction is a means to turn sugar-encoded information into cellular effects. Glycan/lectin structures and their spatial modes of presentation underlie the exquisite specificity of the endogenous lectins in counterreceptor selection, that is, to home in on certain cellular glycoproteins or glycolipids.General significanceUnderstanding how sugar-encoded ‘messages’ are ‘read’ and ‘translated’ by lectins provides insights into fundamental mechanisms of life, with potential for medical applications.     

糖苷内切酶法合成带有均一糖链的糖蛋白和糖肽

糖基化作用是真核生物蛋白翻译后修饰的重要环节,糖链对于蛋白质的结构和功能有重要影响。目前,合成带有均一糖链的糖蛋白和糖肽的策略主要有:(1)利用糖基化的氨基酸进行固相或液相合成。(2)将氨基化的寡糖链直接与预先合成的带有糖基化位点的多肽相结合。(3)利用糖基转移酶和糖苷酶的化学酶法合成策略。以上三种方法,都有各自的优点和不足。相对而言,利用微生物来源的β-N-乙酰氨基葡萄糖苷内切酶(ENGase)合成策略是目前发展较快且更具实践意义的方法。糖苷内切酶法合成策略的研究进展包括:(1)ENGase催化机制的研究。(2)糖基供体的研究。(3)ENGase突变体的研究。(4)糖苷内切酶法的应用。     

Recent advances in synthetic glycoengineering for biological applications

Carbohydrates are involved in many important biological events such as protein maturation and trafficking, pathogen invasion, immune response, cell–cell communications, and so on. Synthetic and chemoenzymatic approaches for glycoengineering have emerged and been applied in perturbing and modulating the biological processes at the protein or cellular level. In this review, we summarize the recent advances in glycoengineering, including new strategies in chemoenzymatic synthesis of glycans, glycopeptides, glycoproteins, and other glycoconjugates. And, the progresses of cell-surface glyco-editing methods for gain of functions are also discussed.

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Chemoenzymatic synthesis and antibody detection of DNA glycoconjugates

A chemoenzymatic approach for the efficient synthesis of DNA-carbohydrate conjugates was developed and applied to an antibody-based strategy for the detection of DNA glycoconjugates. A phosphoramidite derivative of N-acetylglucosamine (GlcNAc) was synthesized and utilized to attach GlcNAc sugars to the 5'-terminus of DNA oligonucleotides by solid-phase DNA synthesis. The resulting GlcNAc-DNA conjugates were used as substrates for glycosyl transferase enzymes to synthesize DNA glycoconjugates. Treatment of GlcNAc-DNA with beta-1,4-galactosyl transferase (GalT) and UDP-Gal produced N-acetyllactosamine-modified DNA (LacNAc-DNA), which could be converted quantitatively to the trisaccharide Lewis X (LeX)-DNA conjugate by alpha-1,3-fucosyltransferase VI (FucT) and GDP-Fuc. The facile enzymatic synthesis of LeX-DNA from GlcNAc-DNA also was accomplished in a one-pot reaction by the combined action of GalT and FucT. The resulting glycoconjugates were characterized by gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and glycosidase digestion experiments. Covalent modification of the 5'-terminus of DNA with carbohydrates did not interfere with the ability of DNA glycoconjugates to hybridize with complementary DNA, as indicated by UV thermal denaturation analysis. The trisaccharide DNA glycoconjugate, LeX-DNA, was detected by a dual DNA hybridization/monoclonal antibody (mAb) detection protocol ("Southwestern"): membrane-immobilized LeX-DNA was visualized by Southern detection with a radiolabeled complementary DNA probe and by Western chemiluminescence detection with a mAb specific for the LeX antigen. The efficient chemoenzymatic synthesis of DNA glycoconjugates and the Southwestern detection protocol may facilitate the application of glycosylated DNA to cellular targeting and DNA glycoconjugate detection strategies.     

植物糖生物学与糖链植物疫苗

植物糖生物学是研究植物与糖类互作机制、植物体内糖链与糖缀合物结构及生物学功能的科学,具体涉及糖信号、糖蛋白及其糖链功能、糖基转移酶及植物凝集素等研究方向。依据相关文献及实际研究经验,简要综述植物糖生物学的最新研究进展,其中重点介绍糖链植物疫苗并阐述其应用情况及作用机制。     

Sialic acid metabolism and sialyltransferases: natural functions and applications

Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases.     

A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating Lewis(x)

Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewis(x) antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.     

Antigenic glycans in parasitic infections: implications for vaccines and diagnostics

Infections by parasitic protozoans and helminths are a major world-wide health concern, but no vaccines exist to the major human parasitic diseases, such as malaria, African trypanosomiasis, amebiasis, leishmaniasis, schistosomiasis, and lymphatic filariasis. Recent studies on a number of parasites indicate that immune responses to parasites in infected animals and humans are directed to glycan determinants within cell surface and secreted glycoconjugates and that glycoconjugates are important in host-parasite interactions. Because of the tremendous success achieved recently in generating carbohydrate-protein conjugate vaccines toward microbial infections, such as Haemophilus influenzae type b, there is renewed interest in defining parasite-derived glycans in the prospect of developing conjugate vaccines and new diagnostics for parasitic infections. Parasite-derived glycans are compelling vaccine targets because they have structural features that distinguish them from mammalian glycans. There have been exciting new developments in techniques for glycan analysis and the methods for synthesizing oligosaccharides by chemical or combined chemo-enzymatic approaches that now make it feasible to generate parasite glycans to test as vaccine candidates. Here, we highlight recent progress made in elucidating the immunogenicity of glycans from some of the major human and animal parasites, the potential for developing conjugate vaccines for parasitic infections, and the possible utilization of these novel glycans in diagnostics.     

Features and applications of bacterial sialidases

Sialidases, or neuraminidases (EC 3.2.1.18), belong to a class of glycosyl hydrolases that release terminal N-acylneuraminate residues from the glycans of glycoproteins, glycolipids, and polysaccharides. In bacteria, sialidases can be used to scavenge sialic acids as a nutrient from various sialylated substrates or to recognize sialic acids exposed on the surface of the host cell. Despite the fact that bacterial sialidases share many structural features, their biochemical properties, especially their linkage and substrate specificities, vary widely. Bacterial sialidases can catalyze the hydrolysis of terminal sialic acids linked by the α(2,3)-, α(2,6)-, or α(2,8)-linkage to a diverse range of substrates. In addition, some of these enzymes can catalyze the transfer of sialic acids from sialoglycans to asialoglycoconjugates via a transglycosylation reaction mechanism. Thus, some bacterial sialidases have been applied to synthesize complex sialyloligosaccharides through chemoenzymatic approaches and to analyze the glycan structure. In this review article, the biochemical features of bacterial sialidases and their potential applications in regioselective hydrolysis reactions as well as sialylation by transglycosylation for the synthesis of sialylated complex glycans are discussed.     

A “biphasic glycosyltransferase high-throughput screen” identifies novel anthraquinone glycosides in the diversification of phenolic natural products

The sugar moieties of many glycosylated small molecule natural products are essential for their biological activity. Glycosyltransferases (GTs) are enzymes responsible for installing these sugar moieties on a variety of biomolecules. Many GTs active on natural products are inherently substrate promiscuous and thus serve as useful tools in manipulating natural product glycosylation to generate new combinations of sugar units (glycones) and scaffold molecules (aglycones) in a process called glycodiversification. It is important to have an effective screening tool to detect the activity of promiscuous enzymes and their resulting glycoside products. Toward this aim, we developed a strategy for screening natural product GTs in a high-throughput fashion enabled by rapid isolation and detection of chromophoric or fluorescent glycosylated natural products. This involves a solvent extraction step to isolate the resulting polar glycoside product from the unreacted aglycone acceptor substrate and the detection of the formed glycoside by the innate absorbance or fluorescence of the aglycone moiety. Using our approach, we screened a collection of natural product GTs against a panel of precursors to therapeutically important molecules. Three GTs showed previously unreported promiscuity toward anthraquinones resulting in novel ε-rhodomycinone glycosides. Considering the pharmaceutical value of clinically used anthraquinone glycosides that are biosynthesized from an ε-rhodomycinone precursor, and the significance that the sugar moiety has on the biological activity of these drugs, our results are of particular importance toward the glycodiversification of therapeutics in this class. The GTs identified and the novel compounds they produce show promise toward new biocatalytic tools and therapeutics.     

Bacterial CMP-sialic acid synthetases: production,properties, and applications

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell–cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5′-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.     

Reconstitution in vitro of the GDP-fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster

The deoxyhexose sugar fucose has an important fine-tuning role in regulating the functions of glycoconjugates in disease and development in mammals. The two genetic model organisms Caenorhabditis elegans and Drosophila melanogaster also express a range of fucosylated glycans, and the nematode particularly has a number of novel forms. For the synthesis of such glycans, the formation of GDP-fucose, which is generated from GDP-mannose in three steps catalysed by two enzymes, is required. By homology we have identified and cloned cDNAs encoding these two proteins, GDP-mannose dehydratase (GMD; EC 4.2.1.47) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GER or FX protein; EC 1.1.1.271), from both Caenorhabditis and Drosophila. Whereas the nematode has two genes encoding forms of GMD (gmd-1 and gmd-2) and one GER-encoding gene (ger-1), the insect has, like mammalian species, only one homologue of each (gmd and gmer). This compares to the presence of two forms of both enzymes in Arabidopsis thaliana. All corresponding cDNAs from Caenorhabditis and Drosophila, as well as the previously uncharacterized Arabidopsis GER2, were separately expressed, and the encoded proteins found to have the predicted activity. The biochemical characterization of these enzymes is complementary to strategies aimed at manipulating the expression of fucosylated glycans in these organisms.     

Structures and mechanisms of glycosyltransferases

Glycosyltransferases (GTs) catalyze the transfer of a sugar moiety from an activated donor sugar onto saccharide and nonsaccharide acceptors. A sequence-based classification spreads GTs in many families thus reflecting the variety of molecules that can be used as acceptors. In contrast, this enzyme family is characterized by a more conserved three-dimensional architecture. Until recently, only two different folds (GT-A and GT-B) have been identified for solved crystal structures. The recent report of a structure for a bacterial sialyltransferase allows the definition of a new fold family. Progress in the elucidation of the structures and mechanisms of GTs are discussed in this review. To accommodate the growing number of crystal structures, we created the 3D-Glycosyltransferase database to gather structural information concerning this class of enzymes.     

Golgi Glycosylation

Glycosylation is a very common modification of protein and lipid, and most glycosylation reactions occur in the Golgi. Although the transfer of initial sugar(s) to glycoproteins or glycolipids occurs in the ER or on the ER membrane, the subsequent addition of the many different sugars that make up a mature glycan is accomplished in the Golgi. Golgi membranes are studded with glycosyltransferases, glycosidases, and nucleotide sugar transporters arrayed in a generally ordered manner from the cis-Golgi to the trans-Golgi network (TGN), such that each activity is able to act on specific substrate(s) generated earlier in the pathway. The spectrum of glycosyltransferases and other activities that effect glycosylation may vary with cell type, and thus the final complement of glycans on glycoconjugates is variable. In addition, glycan synthesis is affected by Golgi pH, the integrity of Golgi peripheral membrane proteins, growth factor signaling, Golgi membrane dynamics, and cellular stress. Knowledge of Golgi glycosylation has fostered the development of assays to identify mechanisms of intracellular vesicular trafficking and facilitated glycosylation engineering of recombinant glycoproteins.The Golgi is home to a multitude of glycosyltransferases (GTs), glycosidases, and nucleotide sugar transporters that function together to complete the synthesis of glycans from founding sugars covalently attached to protein or lipid in the endoplasmic reticulum (ER) (Fig. 1, sugars shaded in green). Thus, glycoproteins, glycosphingolipids (GSLs), proteoglycans, and glycophosphatidylinositol (GPI) anchors acquire their final sugar complement during passage through the Golgi. Most glycoproteins and proteoglycans are either secreted from the cell, or span the plasma membrane with their glycans becoming the molecular frontier of the cell (Fig. 1). GSLs and GPI-anchored proteins also reside in the plasma membrane, the latter being confined to the outer leaflet of the lipid bilayer. The forest of glycans at the cell surface is often called the glycocalyx and can be visualized by electron microscopy after staining for sugars.Open in a separate windowFigure 1.Glycans that mature in the Golgi. The diagram depicts simple N- and O-glycans attached to glycoproteins, proteoglycans, glycosphingolipids, and a GPI anchor in the plasma membrane. Rather rare O-glycans are found attached to EGF-like repeats (EGF; pink) or thrombospondin repeats (TSR; gray) with a particular consensus sequence. The WxxW motif in a TSR is C-mannosylated. Core regions boxed in teal are sugars added in the ER. The remaining sugars in each class of glycan are added during passage through the cis-, medial-, and trans-Golgi network (TGN) compartments of the Golgi. Abbreviations are: Man, mannose; Gal, galactose; Glc, glucose; GlcNAc, N-acetylglucosamine; GlcNH₂, Glucosamine; GlcA, glucuronic acid; IdoA, iduronic acid; GalNAc, N-acetylgalactosamine; Xyl, xylose; Fuc, Fucose; Sia, sialic acid; 3S, 3-O-sulfated; 6S, 6-O-sulfated, PO₄⁻, phosphate. (Modified from Figure 1.6 in Essentials of glycobiology, with permission from Varki and Sharon 2009.)Glycosylation is the most common posttranslational modification of proteins. Mature glycans at any one glycosylation site may be as simple as a single sugar, or as complex as a polymer of more than 200 sugars, potentially modified with phosphate, sulfate, acetate, or phosphorylcholine. Most importantly, glycans are often branched. For example, a complex N-glycan (Fig. 1) may have up to six branches or antennae, and each antenna may contain many repeating disaccharide units. This article will describe the nature of resident Golgi GTs and other activities involved in Golgi glycosylation from entry into the cis-Golgi through passage to the trans-Golgi network (TGN). The focus is on mammalian Golgi glycosylation but comparisons with yeast, Caenorhabditis elegans, and Drosophila are made where appropriate.     

Glycoconjugates for the recognition of Bacillus spores

Carbohydrates act as ligands in many biological processes, including the folding and secretion of proteins, cell-cell recognition, adhesion, and sporulation in the Bacillus genus. Fluorescent-labeled disaccharide glycoconjugates have been applied to evaluate binding to bacterial spores assuming that the spore surface is covered with carbohydrates. This study has shown that specific recognition of bacterial spores is based on interactions between disaccharide glycoconjugates acting as ligands and monosaccharide units expressed on the exterior of bacterial spores. Using fluorophore-assisted carbohydrate electrophoresis (FACE), carbohydrates that are expressed on the exterior of the spores were enumerated. The findings have an impact on how to improve ligand selection, essential for sensor development. In addition, the findings provide new information for inhibition of bacterial spores, and in general, demonstrate how carbohydrates function as recognition signals in nature.     

Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody

The HIV envelope has evolved a dense array of immunologically "self" carbohydrates that efficiently protect the virus from antibody recognition. Nonetheless, one broadly neutralising antibody, IgG1 2G12, has been shown to recognise a cluster of oligomannose glycans on the HIV-1 surface antigen gp120. Thus the self carbohydrates of HIV are now regarded as potential targets for viral neutralisation and vaccine design. Here, we show that chemical inhibition of mammalian glycoprotein synthesis, with the plant alkaloid kifunensine, creates multiple HIV (2G12) epitopes on the surface of previously non-antigenic self proteins and cells, including HIV gp120. This formally demonstrates the structural basis for self/non-self discrimination between viral and host glycans, by a neutralising antibody. Moreover, this study provides an alternative protein engineering approach to the design of a carbohydrate vaccine for HIV-1 by chemical synthesis.     

Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates

Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP?<?47 (median DP 25) and DP?<?100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MS^E analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP?<?47 and DP?<?100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates.