The characterisation of a galactokinase from Streptomyces coelicolor

Promiscuous galactokinases (GalKs), which catalyse the ATP dependent phosphorylation of galactose in nature, have been widely exploited in biotechnology for the rapid synthesis of diverse sugar-1-phosphates. This work focuses on the characterisation of a bacterial GalK from Streptomyces coelicolor (ScGalK), which was overproduced in Escherichia coli and shown to phosphorylate galactose. ScGalK displayed a broad substrate tolerance, with activity towards Gal, GalN, Gal3D, GalNAc, Man and L-Ara. Most interestingly, ScGalK demonstrated a high activity over a broad pH and temperature range, suggesting that the enzyme could be highly amenable to multi-enzyme systems

Using Automated Glycan Assembly (AGA) for the Practical Synthesis of Heparan Sulfate Oligosaccharide Precursors

Herein we report synthesis of complex heparan sulfate oligosaccharide precursors by automated glycan assembly using disaccharide donor building blocks. Rapid access to a hexasaccharide was achieved through iterative solid phase glycosylations on a photolabile resin using Glyconeer™, an automated oligosaccharide synthesiser, followed by photochemical cleavage and glycan purification using simple flash column chromatography

Biocatalytic Transfer of Pseudaminic Acid (Pse5Ac7Ac) Using Promiscuous Sialyltransferases in a Chemoenzymatic Approach to Pse5Ac7Ac-Containing Glycosides

Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host–pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein, we describe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors, enabling the enzymatic synthesis of Pse5Ac7Ac-containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late-stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot two-enzyme process using biosynthetic enzymes. Finally, screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac-terminated glycosides.</p

Probing Thermodynamics, Kinetics and Structural Details of Multivalent Lectin-Glycan Interactions by Quantum Dot-FRET

Multivalent lectin-glycan interactions (MLGIs) are widely employed for bio- recognition and discrimination, but they are also exploited by pathogens to infect host cells. Their biophysical details (e.g. thermodynamics, kinetics, binding modes and binding site orientation) are thus highly valuable, not only for elucidating the underlying mechanisms, but also for guiding the design of multivalent therapeutics against specific MLGIs. However, these details are not readily available due to the limitations of conventional biophysical techniques in probing such complex, flexible interactions. We have recently established densely glycosylated quantum dots (glycan-QDs) as novel structural probes for MLGIs. Using a pair of important, almost identical tetrameric lectins, DC-SIGN and DC-SIGNR, as the model lectins, we have shown that glycan-QDs can not only provide quantitative binding affinities but also dissect their distinct binding modes: DC-SIGN binds simultaneously with one glycan-QD whereas DC-SIGNR inter-cross-links. Herein, we further extend the capacity of the glycan-QD probes to investigate how binding mode affects the binding thermodynamics and kinetics, and probe a structural basis of their binding nature. We show that, while both lectin-glycan-QD interactions are enthalpy driven, with similar binding enthalpy changes (~4 times that of monovalent binding measured by ITC), DC-SIGN binding pays a lesser entropy penalty than DC-SIGNR, giving rise to a stronger affinity. We also reveal that a short C-terminal segment at the flexible junction between the tetramerization domain and glycan binding domain in DC-SIGN, absent in DC-SIGNR, plays a critical role in maintaining DC-SIGN’s glycan-QD binding properties: its removal leads to an entirely different binding enthalpy and entropy profile, despite maintaining the same binding mode. Furthermore, we show that the simultaneous lectin-glycan-QD binding partners give single 2nd-order kon rates which rapidly reach saturation, whereas cross-linking partners give two distinct on-rates: a rapid initial association step, followed by a much slower secondary interaction. Together, our work have established glycan-QDs as a powerful new biophysical platform for solution-based MLGI studies which can provide a wide range of important biophysical parameters

Polyvalent Glycan-Quantum Rods as Multifunctional Mechanistic Probes for Multivalent Glycan-Lectin Recognitions

Multivalent lectin-glycan interactions (MLGIs) are widespread and vital for biology, and also hold the key to many therapeutic applications. However, the underlying structural and biophysical mechanisms for many MLGIs remain poorly understood, limiting our ability to design glycoconjugates that can potently target specific MLGIs for therapeutic intervention. Glycosylated nanoparticles have recently emerged as a powerful biophysical probe for MLGIs, although how nanoparticle shape affects MLGI mechanisms remain largely unexplored. Herein, we have prepared fluorescent quantum rods (QRs), densely coated with -1,2-manno-biose ligands (denoted as QR-DiMan), as a new multifunctional probe to investigate how scaffold geometry affects the MLGI of a pair of closely-related, important tetrameric viral receptors, DC-SIGN and DC-SIGNR. We have previously shown that a DiMan-capped spherical quantum dot (QD-DiMan) gives weak crosslinking interactions with DC-SIGNR but strong simultaneous binding with DC-SIGN. Against the elongated QR scaffold, DC-SIGN retains a similarly strong simultaneous binding of all four binding sites with a single QR-DiMan (apparent K_d ~0.5 nM, ~1.8 million fold stronger than the corresponding monovalent binding), while DC-SIGNR is able to achieve both weak crosslinking and strong individual binding interactions, resulting in a larger binding affinity enhancement than that with QD-DiMan. S/TEM analysis of QR-DiMan-lectin assemblies reveals that DC-SIGNR’s different binding modes arise from the different surface curvatures of the QR scaffold. The glycan display at the spherical ends present too high a steric barrier for DC-SIGNR to bind with all four binding sites, thus it crosslinks between two QR-DiMan to maximize binding multivalency, whereas the more planar character of the cylindrical center allows the glycans to bridge all binding sites in DC-SIGNR. This work thus establishes glycosylated QRs as a powerful new biophysical probe for MLGIs, not only to provide quantitative binding affinities and binding modes, but also to demonstrate the specificity of multivalent lectins in discriminating different glycan displays dictated by the scaffold curvature. This work thus highlights the importance of nano-scaffold shape on MLGIs

Probing Scaffold Size Effects on Multivalent Lectin-Glycan Binding Affinity, Thermodynamics and Antiviral Potency Using Polyvalent Glycan-Gold Nanoparticles

Multivalent lectin-glycan interactions (MLGIs) are pivotal for viral infections and immune regulation. Their structural and biophysical data are thus highly valuable, not only for the understanding of basic mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information for some important MGLIs remain poorly understood, which has greatly limited the research progress in this area. We have recently developed densely glycosylated nanoparticles (e.g., ~4 nm quantum dot (QD) or ~5 nm gold nanoparticle (GNP)) as new mechanistic probes for MLGIs. Using two important tetrameric viral receptors, DC-SIGN and DC-SIGNR as model lectins, we have shown these probes not only can offer sensitive fluorescence readouts for MLGI affinity quantification, but also reveal key structural information (e.g., binding site orientation and binding mode) that are very useful for MLGI targeting. However, the relatively small sizes of scaffolds may not be optimal for maximizing MLGI affinity and targeting specificity. Herein, using -manno-1,2-biose (DiMan) functionalized GNPs (GNP-DiMan) probes, we have systematically studied how GNP scaffold size (e.g., 5, 13, and 27 nm) and glycan density (e.g., 100, 75, 50 and 25%) determine their MLGI affinities, thermodynamics, and antiviral properties. We have developed a new GNP fluorescence quenching assay format for quantifying MLGI affinity to minimize the potential interference from GNP’s strong inner filter effect, revealing that increasing GNP size is highly beneficial to enhance MLGI affinity. We have further determined the MLGI thermodynamics by combining temperature-dependent affinity measurement and Van’t Hoff analysis, revealing that GNP-DiMan-DC-SIGN/R binding is enthalpy driven. Finally, we find that increasing GNP size significantly enhances the antiviral potency. Notably, the DiMan functionalised 27 nm GNP (G27-DiMan) potently and robustly blocks both DC-SIGN and DC-SIGNR mediated pseudo-Ebola virus cellular entry with an EC50 of ~23 and ~49 pM, respectively, placing it the most potent glycoconjugate entry inhibitor against DC-SIGN/R mediated Ebola cellular infections

A Polyvalent Nano-Lectin Potently Neutralizes SARS-CoV-2 by Targeting Glycans on the Viral Spike Protein

Mutations in spike (S) protein epitopes allow SARS-CoV-2 variants to evade antibody responses induced by infection and/or vaccination. In contrast, glycosylation sites in the S protein are conserved across SARS-CoV-2 variants, making glycans a potential robust target for developing antivirals. However, this target has not been adequately exploited for SARS-CoV-2, mostly due to intrinsically weak monovalent protein-glycan inter-actions. We hypothesize that polyvalent nano-lectins with flexibly linked carbohydrate-recognition-domains (CRDs) can adjust their relative positions and bind multivalently to S protein glycans, potentially exerting potent antiviral activity. Herein, we displayed the CRDs of DC-SIGN, a dendritic cell lectin known to bind to diverse viruses, polyvalently onto 13 nm gold nanoparticles (named as G13-CRD). G13-CRD bound strongly and specifically to target glycan-coated quantum dots with sub-nM Kd. Moreover, G13-CRD neutralized particles pseudo-typed with the S proteins of Wuhan Hu-1, B1, Delta variant and Omicron subvariant BA.1 with low nM EC50. In contrast, natural tetrameric DC-SIGN and its G13 conjugate were ineffective. Further, G13-CRD potently and completely inhibited authentic SARS-CoV-2 Wuhan Hu-1 and BA.1, with <10 pM and <10 nM EC50, respectively. These results identify G13-CRD as a polyvalent nano-lectin with broad activity against SARS-CoV-2 variants that merits further exploration as a novel approach to antiviral therapy

Probing Multivalent Lectin-Carbohydrate Binding via Multifunctional Glycan-Gold Nanoparticles: Implications for Blocking Virus Infection

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g. lectin’s binding site orientation, binding mode, and inter-binding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely-related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (inter-crosslinking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF) has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection

Site-Selective C-C Modification of Proteins at Neutral pH Using Organocatalyst-Mediated Cross Aldol Ligations

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing dynamic biological mechanisms. The general use of chemical methods for the functionalisation of proteins remains limited however by the frequent requirement for complicated reaction partners to be present in large excess, and harsh reaction conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH under biocompatible conditions. OPAL enables rapid chemical modification of proteins within an hour using simple aldehyde probes in minimal excess, and is utilised here in the selective affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the b-hydroxy aldehyde product of the OPAL can be functionalised a second time at neutral pH in a subsequent organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the ‘chemical mimicry’ of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis. <br /