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.

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.

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.

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.

Microgels Sopping Up Toxins-GM1a-Functionalized Microgels as Scavengers for Cholera Toxin.

Vibrio cholerae is a Gram-negative bacterium that causes secretory diarrhea and constitutes a major health threat in the industrialized world and even more in developing countries. Its main virulence factor is the cholera toxin, which is internalized by intestinal epithelial cells after binding to the glycosphingolipid receptor GM1a on their apical surface. A potential future solution to dampen complications of cholera infection is by scavenging the cholera toxin by presenting competitive binding motifs to diminish the in vivo toxicity of V. cholerae. Here, we generate GM1a-functionalized and biocompatible microgels with diameters of 20 µm using drop-based microfluidics. The microgels are designed to exhibit a mesoporous and widely meshed network structure, allowing diffusion of the toxin protein deep into the microgel scavengers. Flow cytometry demonstrates strong and multivalent binding at high capacity of these microgels to the binding domain of the cholera toxin. Cell culture-based assays reveal the ability of these microgels to scavenge and retain the cholera toxin in direct binding competition to colorectal cells. This ability is evidenced by suppressed cyclic adenosine monophosphate production as well as reduced vacuole formation in mucus-forming colorectal HT-29 cells. Therefore, glycan-functionalized microgels show great potential as a non-antibiotic treatment for toxin-mediated infectious disorders.

Galectin-Carbohydrate Interactions in Biomedicine and Biotechnology.

Cellular communication events are mediated by interactions between cell-surface sugars and lectins, which are carbohydrate-binding proteins. Galectins are ß-galactosyl-binding lectins that bridge molecules by their sugar moieties, forming a signaling and adhesion network. Severe changes in glycosylation and galectin expression accompany major processes in oncogenesis, cardiovascular disorders, and other pathologies, making galectins attractive therapeutic targets. Here we discuss advanced strategies of chemo-enzymatic carbohydrate synthesis for creating lead glycomimetics and (neo-)glycoconjugates for galectin-1 and -3 targeting in biomedicine and biotechnology. We will describe the challenges and bottlenecks on the route into biomedical and biotechnological practice and present the first clinical candidates. The coming era will see an exciting translation of selective well-defined high-affinity galectin ligands from bench to bedside.

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.

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.

Biotinylated N-Acetyllactosamine- and N,N-Diacetyllactosamine-Based Oligosaccharides as Novel Ligands for Human Galectin-3.

Galectin inhibitor design is an emerging research field due to the involvement of galectins in cancer. Galectin-3, in particular, plays an important role in tumor progression. To generate inhibitors, modifications of the glycan structure can be introduced. Conjugation of hydrophobic compounds to saccharides has proven to be promising as increased binding of galectin-3 can be observed. In the present study, we report on neo-glycans carrying hydrophobic biotin as novel ligands for human galectin-3. We modified N-acetyllactosamine- and N,N-diacetyllactosamine-based tetrasaccharides at the C6-position of the terminal saccharide unit using selective enzymatic oxidation and subsequent chemical conjugation of biotinamidohexanoic acid hydrazide. These neo-glycans were much better bound by galectin-3 than the unmodified counterparts. High selectivity for galectin-3 over galectin-1 was also proven. We generated multivalent neo-glycoproteins by conjugation of neo-glycans to bovine serum albumin showing high affinity for galectin-3. Compared to non-biotinylated neo-glycoproteins, we achieved high binding levels of galectin-3 with a lesser amount of conjugated neo-glycans. Multivalent ligand presentation of neo-glycoproteins significantly increased the inhibitory potency towards galectin-3 binding to asialofetuin when compared to free monovalent glycans. Our findings show the positive impact of 6-biotinylation of tetrasaccharides on galectin-3 binding, which broadens the recent design approaches for producing high-affinity ligands.

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.

Characterization of a new UDP-sugar pyrophosphorylase from Hordeum vulgare (barley).

The broad substrate spectrum of UDP-sugar pyrophosphorylases from plant salvage pathways is of high interest for the synthesis of expensive nucleotide sugars by straightforward enzyme cascade reactions in combination with monosaccharide kinases. We here present a new UDP-sugar pyrophosphorylase from Hordeum vulgare with favorable biochemical properties like broad pH and temperature tolerances as well as a broad substrate spectrum and high synthesis stability. Enzyme properties were determined and reaction conditions were optimized by high-through-put multiplexed capillary electrophoresis analysis. In combination with a galactokinase UDP-α-d-galactose (UDP-Gal) was efficiently synthesized with a space-time-yield of 17g/L*h for full conversion of 10mM substrate within 20min by 1.2U of each enzyme.

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.

Galectin Binding to Neo-Glycoproteins: LacDiNAc Conjugated BSA as Ligand for Human Galectin-3.

Carbohydrate-lectin interactions are relatively weak. As they play an important role in biological recognition processes, multivalent glycan ligands are designed to enhance binding affinity and inhibitory potency. We here report on novel neo-glycoproteins based on bovine serum albumin as scaffold for multivalent presentation of ligands for galectins. We prepared two kinds of tetrasaccharides (N-acetyllactosamine and N,N-diacetyllactosamine terminated) by multi-step chemo-enzymatic synthesis utilizing recombinant glycosyltransferases. Subsequent conjugation of these glycans to lysine groups of bovine serum albumin via squaric acid diethyl ester yielded a set of 22 different neo-glycoproteins with tuned ligand density. The neo-glycoproteins were analyzed by biochemical and chromatographic methods proving various modification degrees. The neo-glycoproteins were used for binding and inhibition studies with human galectin-3 showing high affinity. Binding strength and inhibition potency are closely related to modification density and show binding enhancement by multivalent ligand presentation. At galectin-3 concentrations comparable to serum levels of cancer patients, we detect the highest avidities. Selectivity of N,N-diacetyllactosamine terminated structures towards galectin-3 in comparison to galectin-1 is demonstrated. Moreover, we also see strong inhibitory potency of our scaffolds towards galectin-3 binding. These novel neo-glycoproteins may therefore serve as selective and strong galectin-3 ligands in cancer related biomedical research.

Glycopolymer brushes for specific lectin binding by controlled multivalent presentation of N-acetyllactosamine glycan oligomers.

A new multivalent glycopolymer platform for lectin recognition is introduced in this work by combining the controlled growth of glycopolymer brushes with highly specific glycosylation reactions. Glycopolymer brushes, synthetic polymers with pendant saccharides, are prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-O-(N-acetyl-ß-d-glucosamine)ethyl methacrylate (GlcNAcEMA). Here, the fabrication of multivalent glycopolymers consisting of poly(GlcNAcEMA) is reported with additional biocatalytic elongation of the glycans directly on the silicon substrate by specific glycosylation using recombinant glycosyltransferases. The bioactivity of the surface-grafted glycans is investigated by fluorescence-linked lectin assay. Due to the multivalency of glycan ligands, the glycopolymer brushes show very selective, specific, and strong interactions with lectins. The multiarrays of the glycopolymer brushes have a large potential as a screening device to define optimal-binding environments of specific lectins or as new simplified diagnostic tools for the detection of cancer-related lectins in blood serum.