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.

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.

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.

How bacterial pathogens of the gastrointestinal tract use the mucosal glyco-code to harness mucus and microbiota: New ways to study an ancient bag of tricks.

During the last decades, the flourishing scientific field of molecular pathogenesis brought groundbreaking knowledge of the mechanisms of pathogenicity and the underlying bacterial virulence factors to cause infectious diseases. However, a major paradigm shift is currently occurring after it became increasingly evident that bacterial-host and host-host cell interactions including immune responses orchestrated by defined virulence factors are not the sole drivers of infectious disease development. Strong evidence has been collected that information and nutrient flow within complex microbial communities, as well as to and from host cells and matrices are equally important for successful infection. This particularly holds true for gastrointestinal (GI) pathogens and the GI microbiota interacting and communicating with each other as well as with the host GI mucus and mucosa. Gut-adapted pathogens appear to have developed powerful and specific strategies to interact with human GI mucus including the microbiota for nutrient acquisition, mucosal adhesion, inter-species communication and traversing the mucus barrier. This review covers the existing evidence on these topics and explores the mutual dynamics of host GI mucus, the mucosal habitat and incoming acute and chronic pathogens during GI infections. A particular focus is placed on the role of carbohydrates in diverse mucosal interaction, communication and competition processes. Novel techniques to analyze and synthesize mucus-derived carbohydrates and to generate mucus mimetics are introduced. Finally, open questions and future objectives for pathogen - host GI mucus research will be discussed.

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.

Enzymatic Synthesis of 2-(ß-Galactosyl)-ethyl Methacrylate by ß-Galactosidase from Pyrococcus woesei and Application for Glycopolymer Synthesis and Lectin Studies.

Glycosidases have long been used for the synthesis of glycosides by transglycosylation reactions. Especially glycosidases from hyperthermophilic bacteria are useful for reactions under extreme reaction conditions, e.g., in the presence of organic solvents. We herein report the facile enzymatic synthesis and purification of 2-(ß-galactosyl)-ethyl methacrylate (Gal-EMA) with the recombinant hyperthermostable glycosidase from Pyrococcus woesei in high yields. Optimized reaction conditions resulted in gram-scale synthesis of the galactosylated monomer with 88% transglycosylation yield. The product Gal-EMA was characterized by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. Gal-EMA was utilized to synthesize sugar-functionalized acrylate polymers with defined amounts of incorporated galactose (0-100%). Analysis of the binding affinity of the lectin RCA120 from Ricinus communis to the glycopolymers using an enzyme-linked lectin assay (ELLA) revealed KD values between 0.24 and 6.2 nM, depending on the amount of incorporated Gal-EMA. The potential of Gal-EMA for the synthesis of acrylate-functionalized glycan oligomers was demonstrated by sequential elongation of the terminal galactose by two glycosyltransferases, resulting in the terminal glycan N-acetyllactosamine (LacNAc) epitope. In conclusion, the enzymatic synthesis of Gal-EMA opens new routes to a series of novel monomeric building blocks for the synthesis of glycan-functionalized polyacrylates.

High-Affinity N-(2-Hydroxypropyl)methacrylamide Copolymers with Tailored N-Acetyllactosamine Presentation Discriminate between Galectins.

N-Acetyllactosamine (LacNAc; Galß4GlcNAc) is a typical disaccharide ligand of galectins. The most abundant members of these human lectins, galectin-1 (Gal-1) and galectin-3 (Gal-3), participate in a number of pathologies including cancerogenesis and metastatic formation. In this study, we synthesized a series of fifteen N-(2-hydroxypropyl)methacrylamide (HPMA)-based glycopolymers with varying LacNAc amounts and presentations and evaluated the impact of their architecture on the binding affinity to Gal-1 and Gal-3. The controlled radical reversible addition-fragmentation chain transfer copolymerization technique afforded linear polymer precursors with comparable molecular weight (Mn ≈ 22,000 g mol-1) and narrow dispersity (D̵ ≈ 1.1). The precursors were conjugated with the functionalized LacNAc disaccharide (4-22 mol % content in glycopolymer) prepared by enzymatic synthesis under catalysis by ß-galactosidase from Bacillus circulans. The structure-affinity relationship study based on the enzyme-linked immunosorbent assay revealed that the type of LacNAc presentation, individual or clustered on bi- or trivalent linkers, brings a clear discrimination (almost 300-fold) between Gal-1 and Gal-3, reaching avidity to Gal-1 in the nanomolar range. Whereas Gal-1 strongly preferred a dense presentation of individually distributed LacNAc epitopes, Gal-3 preferred a clustered LacNAc presentation. Such a strong galectin preference based just on the structure of a multivalent glycopolymer type is exceptional. The prepared nontoxic, nonimmunogenic, and biocompatible glycopolymers are prospective for therapeutic applications requiring selectivity for one particular galectin.

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.

Identifying Efficient Clostridium difficile Toxin A Binders with a Multivalent Neo-Glycoprotein Glycan Library.

Clostridium difficile infections cause gastrointestinal disorders and can lead to life-threatening conditions. The symptoms can vary from severe diarrhea to the formation of pseudomembranous colitis and therefore trigger a need for new therapies. The initial step of disease is the binding of the bacterial enterotoxins toxin A and B to the cell surface of epithelial intestinal cells. Scavenging of the toxins is crucial to inhibit their fatal effect in the human body and circumvent the administration of antibiotics. Cell surface glycans are common as ligands for TcdA. Although crucial for carbohydrate-protein interactions, a multivalent presentation of glycans for binding has been hardly considered. Here, we establish a neo-glycoprotein-based glycan library to identify an effective multivalent glycan ligand for TcdA. It comprises 40 different glycan epitopes based on N-acetyllactosamine precursors. Nine structures exhibit strong binding of the receptor domain. Among them, the Lewisy-Lewisx-epitope shows the best performance for binding both the receptor domain and the holotoxin. Therefore, the glycan was synthesized de novo and coupled to BSA as a scaffold for multivalent presentation. The corresponding neo-glycoprotein facilitates the proper scavenging of TcdA in vitro and effectively protects HT29 cells from TcdA-induced cell damage.

Key Factors for A One-Pot Enzyme Cascade Synthesis of High Molecular Weight Hyaluronic Acid.

In the last decades, interest in medical or cosmetic applications of hyaluronic acid (HA) has increased. Size and dispersity are key characteristics of biological function. In contrast to extraction from animal tissue or bacterial fermentation, enzymatic in vitro synthesis is the choice to produce defined HA. Here we present a one-pot enzyme cascade with six enzymes for the synthesis of HA from the cheap monosaccharides glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). The combination of two enzyme modules, providing the precursors UDP-GlcA and UDP-GlcNAc, respectively, with hyaluronan synthase from Pasteurella multocida (PmHAS), was optimized to meet the kinetic requirements of PmHAS for high HA productivity and molecular weight. The Mg2+ concentration and the pH value were found as key factors. The HA product can be tailored by different conditions: 25 mM Mg2+ and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-NaOH pH 8 result into an HA product with high Mw HA (1.55 MDa) and low dispersity (1.05). Whereas with 15 mM Mg2+ and HEPES-NaOH pH 8.5, we reached the highest HA concentration (2.7 g/L) with a yield of 86.3%. Our comprehensive data set lays the basis for larger scale enzymatic HA synthesis.

Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides.

Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple "routine" method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of [13C6]galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-[15N]GlcNAc and its application for the enzymatic synthesis of [13C6/15N]lacto-N-neo-tetraose for the first time. An exemplary application was selected-analysis of tetraose in complex biological mixtures-to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations.

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High-Molecular-Weight Hyaluronic Acid.

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

Thiodigalactoside-Bovine Serum Albumin Conjugates as High-Potency Inhibitors of Galectin-3: An Outstanding Example of Multivalent Presentation of Small Molecule Inhibitors.

Galectin inhibitors are urgently needed to understand the mode of action and druggability of different galectins, but potent and selective agents still evade researchers. Small-sized inhibitors based on thiodigalactoside (TDG) have shown their potential while modifications at their C3 position indicated a strategy to improve selectivity and potency. Considering the role of galectins as glycoprotein traffic police, involved in multivalent bridging interactions, we aimed to create multivalent versions of the potent TDG inhibitors. We herein present for the first time the multivalent attachment of a TDG derivative using bovine serum albumin (BSA) as the scaffold. An efficient synthetic method is presented to obtain a novel type of neoglycosylated proteins loaded with different numbers of TDG moieties. A polyethylene glycol (PEG)-spacer is introduced between the TDG and the protein scaffold maintaining appropriate accessibility for an adequate galectin interaction. The novel conjugates were evaluated in galectin binding and inhibition studies in vitro. The conjugate with a moderate density of 19 conjugated TDGs was identified as one of the most potent multivalent Gal-3 inhibitors so far, with a clear demonstration of the benefit of a multivalent ligand presentation. The described method may facilitate the development of specific galectin inhibitors and their application in biomedical research.

KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose.

Sulfated polysaccharides such as cellulose can mimic the functionalities of pathophysiologically important glycosaminoglycans. Enzymatic sulfation offers a green chemistry route to selective (mono)sulfation of oligosaccharides (e.g., cellobiose as a building block of cellulose) in aqueous solution, at ambient temperature, and high chemoselectivity. Here, we report the first KnowVolution campaign for the aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense to advance ASTB toward a synthetically attractive biocatalyst. The generated final recombination variant (ASTB-M5) carries two amino acid substitutions (Leu446Pro and Val579Lys) leading to an up to 7.6-fold increase in specific activity (6.15 U mg-1 ) that was obtained with one round of KnowVolution. Mass spectrometry analysis confirmed a monosulfated product of cellobiose and structure elucidation by NMR confirmed the sulfation at the positions C-3 or C-4 of GlcNAc-linker-tBoc as opposed to the preferred C-6 by chemical means. Computational analysis suggested an important role of Leu446Pro in substrate-binding and recognized Val579Lys as a distal substitution.

High level in vivo mucin-type glycosylation in Escherichia coli.

BACKGROUND: Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. RESULTS: The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. CONCLUSION: In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.

Poly-N-Acetyllactosamine Neo-Glycoproteins as Nanomolar Ligands of Human Galectin-3: Binding Kinetics and Modeling.

Galectin-3 (Gal-3) is recognized as a prognostic marker in several cancer types. Its involvement in tumor development and proliferation makes this lectin a promising target for early cancer diagnosis and anti-cancer therapies. Gal-3 recognizes poly-N-acetyllactosamine (LacNAc)-based carbohydrate motifs of glycoproteins and glycolipids with a high specificity for internal LacNAc epitopes. This study analyzes the mode and kinetics of binding of Gal-3 to a series of multivalent neo-glycoproteins presenting complex poly-LacNAc-based oligosaccharide ligands on a scaffold of bovine serum albumin. These neo-glycoproteins rank among the strongest Gal-3 ligands reported, with Kd reaching sub-nanomolar values as determined by surface plasmon resonance. Significant differences in the binding kinetics were observed within the ligand series, showing the tetrasaccharide capped with N,N'-diacetyllactosamine (LacdiNAc) as the strongest ligand of Gal-3 in this study. A molecular model of the Gal-3 carbohydrate recognition domain with docked oligosaccharide ligands is presented that shows the relations in the binding site at the molecular level. The neo-glycoproteins presented herein may be applied for selective recognition of Gal-3 both on the cell surface and in blood serum.

Binding characteristics of galectin-3 fusion proteins.

Galectin-3 modulates cell adhesion and signaling events by specific binding and cross-linking galactoside containing carbohydrate ligands. Proteolytic cleavage by metalloproteinases yields in vivo N-terminally truncated galectin-3 still bearing the carbohydrate recognition domain. Truncated galectin-3 has been demonstrated to act in vivo as a negative inhibitor of galectin-3 due to higher affinity for carbohydrate ligands. We here present our studies on a series of 12 human galectin-3 protein constructs. Truncated galectin-3 (∆1-62 and ∆1-116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) display altered binding efficiencies (ratio of maximum binding signal and apparent affinity constant Kd) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Δ1-62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Δ1-116) shows only weak binding. We demonstrate here for the first time that SNAP-tag and YFP fusions of galectin-3 and truncated galectin-3 proteins improve binding efficiencies to ASF. SNAP-tagged galectin-3, galectin-3(Δ1-62) and galectin-3(Δ1-116) are found with significant (3- to 6-fold) higher binding efficiencies in SPR when compared with native galectin-3. Fusion of truncated galectin-3 with YFP renders binding properties similar to native galectin-3, whereas in combination with SNAP-tag improved binding characteristics are obtained. Our results emphasize the importance of the N-terminal domain of human galectin-3 for ligand binding. Most importantly, in combination with fusion proteins suitable for the design of diagnostic and therapeutic tools binding properties can be beneficially tuned. The resulting novel protein tools may be advantageous for potential galectin-3 directed applications in tumor diagnostics and therapy.

Tailored Multivalent Neo-Glycoproteins: Synthesis, Evaluation, and Application of a Library of Galectin-3-Binding Glycan Ligands.

Galectin-3 (Gal-3), a member of the ß-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galß1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcß1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.

Glycan-Functionalized Microgels for Scavenging and Specific Binding of Lectins.

Lectins are proteins with a well-defined carbohydrate recognition domain. Many microbial proteins such as bacterial toxins possess lectin or lectin-like binding domains to interact with cell membranes that are decorated with glycan recognition motifs. We report a straightforward way to prepare monodisperse and biocompatible polyethylene glycol microgels, which carry glycan motifs for specific binding to lectins. The sugar-functionalized colloids exhibit a wide mesh size and a highly accessible volume. The microgels are prepared via drop-based microfluidics combined with radical polymerization. GSII and ECL are used as model lectins that bind specifically to the corresponding carbohydrates, namely, GlcNAc and LacNAc. LacNAc microgels bind ECL with a high capacity and high affinity (Kd ≈ 0.5 to 1 µM), suggesting multivalent binding of the lectin to the LacNAc-decorated flexible microgel network. Glycan-functionalized microgels present a useful tool for lectin scavenging in biomedical applications.

Enzymatic Synthesis of N-Acetyllactosamine (LacNAc) Type 1 Oligomers and Characterization as Multivalent Galectin Ligands.

Repeats of the disaccharide unit N-acetyllactosamine (LacNAc) occur as type 1 (Galß1, 3GlcNAc) and type 2 (Galß1, 4GlcNAc) glycosylation motifs on glycoproteins and glycolipids. The LacNAc motif acts as binding ligand for lectins and is involved in many biological recognition events. To the best of our knowledge, we present, for the first time, the synthesis of LacNAc type 1 oligomers using recombinant ß1,3-galactosyltransferase from Escherichia coli and ß1,3-N-acetylglucosaminyltranferase from Helicobacter pylori. Tetrasaccharide glycans presenting LacNAc type 1 repeats or LacNAc type 1 at the reducing or non-reducing end, respectively, were conjugated to bovine serum albumin as a protein scaffold by squarate linker chemistry. The resulting multivalent LacNAc type 1 presenting neo-glycoproteins were further studied for specific binding of the tumor-associated human galectin 3 (Gal-3) and its truncated counterpart Gal-3∆ in an enzyme-linked lectin assay (ELLA). We observed a significantly increased affinity of Gal-3∆ towards the multivalent neo-glycoprotein presenting LacNAc type 1 repeating units. This is the first evidence for differences in glycan selectivity of Gal-3∆ and Gal-3 and may be further utilized for tracing Gal-3∆ during tumor progression and therapy.