Microgels with Immobilized Glycosyltransferases for Enzymatic Glycan Synthesis.

Glycans, composed of linked monosaccharides, play crucial roles in biology and find diverse applications. Enhancing their enzymatic synthesis can be achieved by immobilizing enzymes on materials such as microgels. Here, we present microgels with immobilized glycosyltransferases, synthesized through droplet microfluidics, immobilizing enzymes either via encapsulation or postattachment. SpyTag-SpyCatcher interaction was used for enzyme binding, among others. Fluorescamine and permeability assays confirmed enzyme immobilization and microgel porosity, while enzymatic activities were determined using HPLC. The potential application of microgels in cascade reactions involving multiple enzymes was demonstrated by combining ß4GalT and α3GalT in an enzymatic reaction with high yields. Moreover, a cascade of ß4GalT and ß3GlcNAcT was successfully implemented. These results pave the way toward a modular membrane bioreactor for automated glycan synthesis containing the presented biocatalytic microgels.

Process Development for the Enzymatic Gram-Scale Production of the Unnatural Nucleotide Sugar UDP-6-Azido-GalNAc.

Azido sugars hold great promise as substrates in numerous click-chemistry applications. However, the synthesis of activated azido sugars is limited by cost and complexity. Conventional chemical activation methods are intricate and time-consuming. In response, we have developed a process for the large-scale production of UDP-6-azido-GalNAc through enzymatic nucleotide sugar synthesis on a gram scale. Our optimization strategies encompassed refining the process parameters of an enzyme cascade featuring NahK from Bifidobacterium longum and AGX1 from Homo sapiens. Using the repetitive-batch-mode technology, we synthesized up to 2.1 g of UDP-6-azido-GalNAc, achieving yields up to 97 % in five consecutive batch cycles using a single enzyme batch. The synthesis process demonstrated to have total turnover numbers (TTNs) between 4.4-4.8 g of product per gram of enzyme (gP/gE) and STYs ranging from 1.7-2.4 g per liter per hour (g*L-1*h-1). By purification of a product solution pool containing 2.6 g (4.1 mmol) UDP-6-azido-GalNAc, 2.1 g (2,122.1 mg) UDP-6-azido-GalNAc (sodium salt) with a purity of 99.96 % (HPLC) were obtained. The overall recovery after purification was 81 % (3.32 mmol). Our work establishes a robust production platform for the gram-scale synthesis of unnatural nucleotide sugars, opening new avenues for applications in glycan engineering.

Strategies for Automated Enzymatic Glycan Synthesis (AEGS).

Glycans are the most abundant biopolymers on earth and are constituents of glycoproteins, glycolipids, and proteoglycans with multiple biological functions. The availability of different complex glycan structures is of major interest in biotechnology and basic research of biological systems. High complexity, establishment of general and ubiquitous synthesis techniques, as well as sophisticated analytics, are major challenges in the development of glycan synthesis strategies. Enzymatic glycan synthesis with Leloir-glycosyltransferases is an attractive alternative to chemical synthesis as it can achieve quantitative regio- and stereoselective glycosylation in a single step. Various strategies for synthesis of a wide variety of different glycan structures has already be established and will exemplarily be discussed in the scope of this review. However, the application of enzymatic glycan synthesis in an automated system has high demands on the equipment, techniques, and methods. Different automation approaches have already been shown. However, while these techniques have been applied for several glycans, only a few strategies are able to conserve the full potential of enzymatic glycan synthesis during the process - economical and enzyme technological recycling of enzymes is still rare. In this review, we show the major challenges towards Automated Enzymatic Glycan Synthesis (AEGS). First, we discuss examples for immobilization of glycans or glycosyltransferases as an important prerequisite for the embedment and implementation in an enzyme reactor. Next, improvement of bioreactors towards automation will be described. Finally, analysis and monitoring of the synthesis process are discussed. Furthermore, automation processes and cycle design are highlighted. Accordingly, the transition of recent approaches towards a universal automated glycan synthesis platform will be projected. To this end, this review aims to describe essential key features for AEGS, evaluate the current state-of-the-art and give thought- encouraging impulses towards future full automated enzymatic glycan synthesis.

Fucose Binding Motifs on Mucin Core Glycopeptides Impact Bacterial Lectin Recognition.

Mucin glycoproteins are essential components of the mucosal barrier, which protects the host from pathogens. Throughout evolution, bacteria have developed strategies to modulate and penetrate this barrier, and cause virulence by interacting with mucin O-glycans at the epithelial cell-surface. O-fucosylated glycan epitopes on mucins are key ligands of many bacterial lectins. Here, a chemoenzymatic synthesis strategy is described to prepare a library of fucosylated mucin core glycopeptides to enable studies of mucin-interacting and fucose-binding bacterial lectins. Glycan cores with biologically important Lewis and H-antigens were prepared decorating the peptide backbone at different sites and densities. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of glycan core and peptide backbone presentation of these antigens in binding interactions with the P. aeruginosa lectin LecB and the C. difficile toxin A.

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays.

Galectins are ß-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6-3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

Enzyme cascades for the synthesis of nucleotide sugars: Updates to recent production strategies.

Nucleotide sugars play an elementary role in nature as building blocks of glycans, polysaccharides, and glycoconjugates used in the pharmaceutical, cosmetics, and food industries. As substrates of Leloir-glycosyltransferases, nucleotide sugars are essential for chemoenzymatic in vitro syntheses. However, high costs and the limited availability of nucleotide sugars prevent applications of biocatalytic cascades on a large industrial scale. Therefore, the focus is increasingly on nucleotide sugar synthesis strategies to make significant application processes feasible. The chemical synthesis of nucleotide sugars and their derivatives is well established, but the yields of these processes are usually low. Enzyme catalysis offers a suitable alternative here, and in the last 30 years, many synthesis routes for nucleotide sugars have been discovered and used for production. However, many of the published procedures shy away from assessing the practicability of their processes. With this review, we give an insight into the development of the (chemo)enzymatic nucleotide sugar synthesis pathways of the last years and present an assessment of critical process parameters such as total turnover number (TTN), space-time yield (STY), and enzyme loading.

Methods of in vitro study of galectin-glycomaterial interaction.

Galectins are a family of carbohydrate-binding lectins modulating cell events such as cell proliferation, apoptosis, adhesion or migration by cross-linking the glycan structures of cell membranes and/or extracellular matrix components. In a diseased organism, galectins are upregulated and trigger the progression of diseases such as inflammation, cancerogenesis, fibrosis, cardiovascular and metabolic disorders. Targeting galectins with glycomaterials for the aims of diagnostics or therapy is, therefore, a focus of biotechnological and biomedicinal research, and already led to candidates for clinical trials. Testing and evaluation of galectin-glycomaterial interactions require informative and versatile analytical methods at several levels of knowledge, from basic inter-molecular interaction to complex cell-based assays. This review aims to classify and characterize a selection of the most promising methods to identify the prospective glycomaterials for translating galectin targeting from the molecular level to the level of tailored in vivo assays.

Repetitive Synthesis of High-Molecular-Weight Hyaluronic Acid with Immobilized Enzyme Cascades.

Industrial hyaluronic acid (HA) production comprises either fermentation with Streptococcus strains or extraction from rooster combs. The hard-to-control product quality is an obstacle to these processes. Enzymatic syntheses of HA were developed to produce high-molecular-weight HA with low dispersity. To facilitate enzyme recovery and biocatalyst re-use, here the immobilization of cascade enzymes onto magnetic beads was used for the synthesis of uridine-5'-diphosphate-α-d-N-acetyl-glucosamine (UDP-GlcNAc), UDP-glucuronic acid (UDP-GlcA), and HA. The combination of six enzymes in the UDP-sugar cascades with integrated adenosine-5'-triphosphate-regeneration reached yields between 60 and 100 % for 5 repetitive batches, proving the productivity. Immobilized HA synthase from Pasteurella multocida produced HA in repetitive batches for three days. Combining all seven immobilized enzymes in a one-pot synthesis, HA production was demonstrated for three days with a HA concentration of up to 0.37 g L-1 , an average MW of 2.7-3.6 MDa, and a dispersity of 1.02-1.03.

Immunoprotective neo-glycoproteins: Chemoenzymatic synthesis of multivalent glycomimetics for inhibition of cancer-related galectin-3.

Galectin-3 plays a crucial role in cancerogenesis; its targeting is a prospective pathway in cancer diagnostics and therapy. Multivalent presentation of glycans was shown to strongly increase the affinity of glycoconjugates to galectin-3. Further strengthening of interaction with galectin-3 may be accomplished using artificial glycomimetics with apt aryl substitutions. We established a new, as yet undescribed chemoenzymatic method to produce selective C-3-substituted N,N'-diacetyllactosamine glycomimetics and coupled them to human serum albumin. From a library of enzymes, only ß-N-acetylhexosaminidase from Talaromyces flavus was able to efficiently synthesize the C-3-propargylated disaccharide. Various aryl residues were attached to the functionalized N,N'-diacetyllactosamine via click chemistry to assess the impact of the aromatic substitution. In ELISA-type assays with galectin-3, free glycomimetics exhibited up to 43-fold stronger inhibitory potency to Gal-3 than the lactose standard. Coupling to human serum albumin afforded multivalent neo-glycoproteins with up to 4209-fold increased inhibitory potency per glycan compared to the monovalent lactose standard. Surface plasmon resonance brought further information on the kinetics of galectin-3 inhibition. The potential of prepared neo-glycoproteins to target galectin-3 was demonstrated on colorectal adenocarcinoma DLD-1 cells. We investigated the uptake of neo-glycoproteins into cells and observed limited non-specific transport into the cytoplasm. Therefore, neo-glycoproteins primarily act as efficient scavengers of exogenous galectin-3 of cancer cells, inhibiting its interaction with the cell surface, and protecting T-lymphocytes against galectin-3-induced apoptosis. The present neo-glycoproteins combine the advantage of a straightforward synthesis, selectivity, non-toxicity, and high efficiency for targeting exogenous galectin-3, with possible application in the immunomodulatory treatment of galectin-3-overexpressing cancers.

Electrochemical Impedance Spectroscopy Biosensor Enabling Kinetic Monitoring of Fucosyltransferase Activity.

Monitoring glycosyltransferases on biosensors is of great interest for pathogen and cancer diagnostics. As a proof of concept, we here demonstrate the layer-by-layer immobilization of a multivalent neoglycoprotein (NGP) as a substrate for a bacterial fucosyltransferase (FucT) and the subsequent binding of the fucose-specific Aleuria aurantia lectin (AAL) on an electrochemical impedance spectroscopy (EIS) sensor. We report for the first time the binding kinetics of a glycosyltransferase in real-time. Highly stable EIS measurements are obtained by the modification of counter and reference electrodes with polypyrrole: polystyrene sulfonate (PPy:PSS). In detail, the N-acetyllactosamine (LacNAc)-carrying NGP was covalently immobilized on the gold working electrode and served as a substrate for the FucT-catalyzed reaction. The LacNAc epitopes were converted to Lewisx (Lex) and detected by AAL. AAL binding to the Lex epitope was further confirmed in a lectin displacement and a competitive lectin binding inhibition experiment. We monitored the individual kinetic processes via EIS. The time constant for covalent immobilization of the NGP was 653 s. The FucT kinetics was the slowest process with a time constant of 1121 s. In contrast, a short time constant of 11.8 s was determined for the interaction of AAL with the modified NGPs. When this process was competed by 400 mM fucose, the binding was significantly slowed down, as indicated by a time constant of 978 s. The kinetics for the displacement of bound AAL by free fucose was observed with a time constant of 424 s. We conclude that this novel EIS biosensor and the applied workflow has the potential to detect FucT and other GT activities in general and further monitor protein-glycan interactions, which may be useful for the detection of pathogenic bacteria and cancer cells in future biomedical applications.

Enzymatic Synthesis of Glycans and Glycoconjugates.

Glycoconjugates have great potential to improve human health in a multitude of different ways and fields. Prominent examples are human milk oligosaccharides and glycosaminoglycans. The typical choice for the production of homogeneous glycoconjugates is enzymatic synthesis. Through the availability of expression and purification protocols, recombinant Leloir glycosyltransferases are widely applied as catalysts for the synthesis of a wide range of glycoconjugates. Extensive utilization of these enzymes also depends on the availability of activated sugars as building blocks. Multi-enzyme cascades have proven a versatile technique to synthesize and in situ regenerate nucleotide sugar.In this chapter, the functions and mechanisms of Leloir glycosyltransferases are revisited, and the advantage of prokaryotic sources and production systems is discussed. Moreover, in vivo and in vitro pathways for the synthesis of nucleotide sugar are reviewed. In the second part, recent and prominent examples of the application of Leloir glycosyltransferase are given, i.e., the synthesis of glycosaminoglycans, glycoconjugate vaccines, and human milk oligosaccharides as well as the re-glycosylation of biopharmaceuticals, and the status of automated glycan assembly is revisited.

Current state on the enzymatic synthesis of glycosaminoglycans.

Glycosaminoglycans (GAGs) are linear anionic polysaccharides, and most of them show a specific sulfation pattern. GAGs have been studied for decades, and still, new biological functions are discovered. Hyaluronic acid and heparin are sold for medical or cosmetic applications. With increased market and applications, the production of GAGs stays in the focus of research groups and the industry. Common industrial GAG production relies on the extraction of animal tissue. Contamination, high dispersity, and uncontrolled sulfation pattern are still obstacles to this process. Tailored production strategies for the chemoenzymatic synthesis have been developed to address these obstacles. In recent years, enzyme cascades, including uridine-5'-diphosphate sugar syntheses, were established to obtain defined polymer size and dispersity, as well as defined sulfation patterns. Nevertheless, the complex synthesis of GAGs is still a challenging research field.

A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity.

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called 'Pig intestinal bacterial collection' (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota.

Polyethylene glycol-mediated blocking and monolayer morphology of an electrochemical aptasensor for malaria biomarker detection in human serum.

Better approaches are critically needed for in situ point-of-care diagnostic biosensors that enable primary care physicians, or even individual patients, to directly analyze biological fluids without complicated sample pretreatments. Additional purification steps consume time, consume reagents, often require other equipment, and can introduce false-negative results. Biosensors have been modified with blocking molecules to reduce biofouling; however, the effectiveness relies on their chemical composition and morphology. Here, we used a polyethylene glycol film to suppress unspecific binding from human serum on an electrochemical malaria aptasensor. A detailed study of the variation of the chemical and morphological composition of the aptamer/polyethylene glycol mixed monolayer as a function of incubation time was conducted. Higher resistance to matrix biofouling was found for polyethylene glycol than for hydrophobic alkanethiol films. The best sensor performance was observed for intermediate polyethylene glycol immobilization times. With prolonged incubation, phase separation of aptamer, and polyethylene glycol molecules locally increased the aptamer density and thereby diminished the analyte binding capability. Remarkably, polyethylene glycols do not affect the aptasensor sensitivity but enhance the complex matrix tolerance, the dynamic range, and the limit of detection. Careful tuning of the blocking molecule immobilization is crucial to achieving high aptasensor performance and biofouling resistance.

Sequence-Defined Heteromultivalent Precision Glycomacromolecules Bearing Sulfonated/Sulfated Nonglycosidic Moieties Preferentially Bind Galectin-3 and Delay Wound Healing of a Galectin-3 Positive Tumor Cell Line in an In Vitro Wound Scratch Assay.

Within this work, a new class of sequence-defined heteromultivalent glycomacromolecules bearing lactose residues and nonglycosidic motifs for probing glycoconjugate recognition in carbohydrate recognition domain (CRD) of galectin-3 is presented. Galectins, a family of ß-galactoside-binding proteins, are known to play crucial roles in different signaling pathways involved in tumor biology. Thus, research has focused on the design and synthesis of galectin-targeting ligands for use as diagnostic markers or potential therapeutics. Heteromultivalent precision glycomacromolecules have the potential to serve as ligands for galectins. In this work, multivalency and the introduction of nonglycosidic motifs bearing either neutral, amine, or sulfonated/sulfated groups are used to better understand binding in the galectin-3 CRD. Enzyme-linked immunosorbent assays and surface plasmon resonance studies are performed, revealing a positive impact of the sulfonated/sulfated nonglycosidic motifs on galectin-3 binding but not on galectin-1 binding. Selected compounds are then tested with galectin-3 positive MCF 7 breast cancer cells using an in vitro would scratch assay. Preliminary results demonstrate a differential biological effect on MCF 7 cells with high galectin-3 expression in comparison to an HEK 293 control with low galectin-3 expression, indicating the potential for sulfonated/sulfated heteromultivalent glycomacromolecules to serve as preferential ligands for galectin-3 targeting.

Glycopolymers for Efficient Inhibition of Galectin-3: In Vitro Proof of Efficacy Using Suppression of T Lymphocyte Apoptosis and Tumor Cell Migration.

The development of efficient galectin-3 (Gal-3) inhibitors draws attention in the field of anti-cancer therapy, especially due to the prominent role of extra- and intracellular Gal-3 in vital processes of cancerogenesis, such as immunosuppression, stimulation of tumor cells proliferation, survival, invasion, apoptotic resistance, and metastasis formation and progression. Here, by combining poly-LacNAc (Galß4GlcNAc)-derived oligosaccharides with N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, we synthesized multivalent glycopolymer inhibitors with a high potential to target extracellular and intracellular Gal-3. The inhibitory capabilities of the best conjugate in the studied series were in the nanomolar range proving the excellent Gal-3 inhibitory potential. Moreover, thorough investigation of the inhibitory effect in the biological conditions showed that the glycopolymers strongly inhibited Gal-3-induced apoptosis of T lymphocytes and suppressed migration and spreading of colorectal, breast, melanoma, and prostate cancer cells. In sum, the strong inhibitory activity toward Gal-3, combined with favorable pharmacokinetics of HPMA copolymers ensuring enhanced tumor accumulation via the enhanced permeability and retention effect, nominate the glycopolymers containing LacdiNAc-LacNAc (GalNAcß4GlcNAcß3Galß4GlcNAc) tetrasaccharide as promising tools for preclinical in anti-cancer therapy evaluation.

Synthesis of the Thomsen-Friedenreich-antigen (TF-antigen) and binding of Galectin-3 to TF-antigen presenting neo-glycoproteins.

The Thomsen-Friedenreich-antigen, Gal(ß1-3)GalNAc(α1-O-Ser/Thr (TF-antigen), is presented on the surface of most human cancer cell types. Its interaction with galectin 1 and galectin 3 leads to tumor cell aggregation and promotes cancer metastasis and T-cell apoptosis in epithelial tissue. To further explore multivalent binding between the TF-antigen and galectin-3, the TF-antigen was enzymatically synthesized in high yields with GalNAc(α1-EG3-azide as the acceptor substrate by use of the glycosynthase BgaC/Glu233Gly. Subsequently, it was coupled to alkynyl-functionalized bovine serum albumin via a copper(I)-catalyzed alkyne-azide cycloaddition. This procedure yielded neo-glycoproteins with tunable glycan multivalency for binding studies. Glycan densities between 2 and 53 glycan residues per protein molecule were obtained by regulated alkynyl-modification of the lysine residues of BSA. The number of coupled glycans was quantified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a trinitrobenzene sulfonic acid assay. The binding efficiency of the neo-glycoproteins with human galectin-3 and the effect of multivalency was investigated and assessed using an enzyme-linked lectin assay. Immobilized neo-glycoproteins of all modification densities showed binding of Gal-3 with increasing glycan density. However, multivalent glycan presentation did not result in a higher binding affinity. In contrast, inhibition of Gal-3 binding to asialofetuin was effective. The relative inhibitory potency was increased by a factor of 142 for neo-glycoproteins displaying 10 glycans/protein in contrast to highly decorated inhibitors with only 2-fold increase. In summary, the functionality of BSA-based neo-glycoproteins presenting the TF-antigen as multivalent inhibitors for Gal-3 was demonstrated.

Fluorinated Galactoses Inhibit Galactose-1-Phosphate Uridyltransferase and Metabolically Induce Galactosemia-like Phenotypes in HEK-293 Cells.

Genetic defects of human galactose-1-phosphate uridyltransferase (hGALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by hGALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant hGALT with a KI of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and rhMUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.