Sugar Recognition Research Articles

Sialic acid detection and theranostic activity of phenylboronic acid-based fluorescent probe in human colorectal adenocarcinoma HT-29 cells

A new probe, 4-(((3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)imino)methyl)phenyl)boronic acid (R4B) was prepared by facile condensation of 4-formylphenylboronic acid and rhodamine B hydrazide. R4B was characterized by spectroscopic methods and single crystal X-ray diffraction. The sensor R4B solution turned pink and emitted orange fluorescence only in the presence of sialic acid but remained colorless and non-fluorescent otherwise. The sugar recognition performance was investigated via UV–vis and fluorescence spectroscopic studies. Our results revealed that R4B has good affinity and selectivity for sialic acid over common monosaccharides, with a detection limit as low as 10−7 M. Furthermore, R4B selectively inhibited growth of human colorectal adenocarcinoma HT-29 (IC50 <20 µM) without significant cytotoxicity to normal human colon fibroblasts CCD-18Co. Treatment with R4B suppressed HT-29 colony formation via mitochondrial apoptosis in a time-dependent manner. Cellular imaging studies also revealed the ability of R4B as a fluorescence dye to detect intracellular sialic acid and showed mitochondria-tracking ability in HT-29 cells. In summary, R4B is a potential theranostic for the detection of intracellular sialic acid during the early incubation period, followed by induction of cancer apoptotic cell death at a later treatment point.

Structural basis of sugar recognition by SCFFBS2 ubiquitin ligase involved in NGLY1 deficiency.

The cytosolic peptide:N-glycanase (PNGase) is involved in the quality control of N-glycoproteins via the endoplasmic reticulum-associated degradation (ERAD) pathway. Mutations in the gene encoding cytosolic PNGase (NGLY1 in humans) cause NGLY1 deficiency. Recent findings indicate that the F-box protein FBS2 of the SCFFBS2 ubiquitin ligase complex can be a promising drug target for NGLY1 deficiency. Here, we determined the crystal structure of bovine FBS2 complexed with the adaptor protein SKP1 and a sugar ligand, Man3GlcNAc2, which corresponds to the core pentasaccharide of N-glycan. Our crystallographic data together with NMR data revealed the structural basis of disparate sugar-binding specificities in homologous FBS proteins and identified a potential druggable pocket for in silico docking studies. Our results provide a potential basis for the development of selective inhibitors against FBS2 in NGLY1 deficiency.

Tas1R3 Dependent and Independent Recognition of Sugars in the Urethra and the Role of Tuft Cells in this Process (Adv. Biology 6/2024)

Tas1R3 Dependent and Independent Recognition of Sugars in the Urethra and the Role of Tuft Cells in this Process (Adv. Biology 6/2024)

Structural basis for sugar perception by Drosophila gustatory receptors.

Insects rely on a family of seven transmembrane proteins called gustatory receptors (GRs) to encode different taste modalities, such as sweet and bitter. We report structures of Drosophila sweet taste receptors GR43a and GR64a in the apo and sugar-bound states. Both GRs form tetrameric sugar-gated cation channels composed of one central pore domain (PD) and four peripheral ligand-binding domains (LBDs). Whereas GR43a is specifically activated by the monosaccharide fructose that binds to a narrow pocket in LBDs, disaccharides sucrose and maltose selectively activate GR64a by binding to a larger and flatter pocket in LBDs. Sugar binding to LBDs induces local conformational changes, which are subsequently transferred to the PD to cause channel opening. Our studies reveal a structural basis for sugar recognition and activation of GRs.

From small changes to big gains: pyridinium-based tetralactam macrocycle for enhanced sugar recognition in water.

The complex distribution of functional groups in carbohydrates, coupled with their strong solvation in water, makes them challenging targets for synthetic receptors. Despite extensive research into various molecular frameworks, most synthetic carbohydrate receptors have exhibited low affinities, and their interactions with sugars in aqueous environments remain poorly understood. In this work, we present a simple pyridinium-based hydrogen-bonding receptor derived from a subtle structural modification of a well-known tetralactam macrocycle. This small structural change resulted in a dramatic enhancement of glucose binding affinity, increasing from 56 M-1 to 3001 M-1. Remarkably, the performance of our synthetic lectin surpasses that of the natural lectin, concanavalin A, by over fivefold. X-ray crystallography of the macrocycle-glucose complex reveals a distinctive hydrogen bonding pattern, which allows for a larger surface overlap between the receptor and glucose, contributing to the enhanced affinity. Furthermore, this receptor possesses allosteric binding sites, which involve chloride binding and trigger receptor aggregation. This unique allosteric process reveals the critical role of structural flexibility in this hydrogen-bonding receptor for the effective recognition of sugars. We also demonstrate the potential of this synthetic lectin as a highly sensitive glucose sensor in aqueous solutions.

Electron Transfer Channel in the Sugar Recognition System Assembled on Nano Gold Particle

Electron Transfer Channel in the Sugar Recognition System Assembled on Nano Gold Particle

L-ribose specific recognition surface constructed by pillar[5]arene-based host–guest interaction

In living organisms, chiral molecules have specific chiral conformations that produce different physiological effects. Ribose is one of the components of RNA, which mainly plays a role in regulating biological activity. Inspired by the biological recognition of sugars, functional chiral surfaces for recognizing L-ribose through non-covalent interactions were constructed. In the strategy of this study, a functional chiral gold surface based on host-guest interactions was constructed through the assembly of the host molecule single-function alynyl pillar[5]arene(SAP5) and the guest molecule (S) -mandelate-violet (SMV). The association constant of SMV and SAP5 was calculated to be 2.95×104 M-1, with a binding ratio of 1:1. By impedance and contact angle detection, the constructed functional interface has good detection effect on L-ribose in the range of 1×10-7 M to1× 10-2 M. In addition, CV was disassembled from the aromatic cavity of pillar[5]arene after adding zinc powder and it can repeat five times with good recyclability, thus achieving the organic combination of interface recognition and intelligence.

Screening and functional analysis of three Spiroplasma eriocheiris glycosylated protein interactions with Macrobrachium nipponense C-type lectins.

N-glycosylation, one of the main protein posttranslational modifications (PTMs), plays an important role in the pathogenic process of pathogens through binding and invasion of host cells or regulating the internal environment of host cells to benefit their survival. However, N-glycosylation has remained mostly unexplored in Spiroplasma eriocheiris, a novel type of pathogen which has serious adverse effects on aquaculture. In most cases, N-glycoproteins can be detected and analyzed by lectins dependent on sugar recognition domains. In this study, three Macrobrachium nipponense C-type lectins, namely, MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3, were used to screen S. eriocheiris glycosylated proteins. First, qRT-PCR results showed that the expression levels of the three kinds of lectins were all significantly up-regulated in prawn hearts when the host was against S. eriocheiris infection. A bacterial binding assay showed that purified recombinant MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3 could directly bind to S. eriocheiris in vitro. Second, three S. eriocheiris glycosylated proteins, ATP synthase subunit beta (ATP beta), molecular chaperone Dnak (Dnak) and fructose bisphosphate aldolase (FBPA), were screened and identified using the three kinds of full-length C-type lectins. Far-Western blot and coimmunoprecipitation (CO-IP) further demonstrated that there were interactions between the three lectins with ATP beta, Dnak and FBPA. Furthermore, antibody neutralization assay results showed that pretreatment of S. eriocheiris with ATP beta, Dnak and FBPA antibodies could significantly block this pathogen infection. All the above studies showed that the glycosylated protein played a vital role in the process of S. eriocheiris infection.

Characterization of chitinases from the GH18 gene family in the myxomycete Physarum polycephalum

BackgroundPhysarum polycephalum is an unusual macroscopic myxomycete expressing a large range of glycosyl hydrolases. Among them, enzymes from the GH18 family can hydrolyze chitin, an important structural component of the cell walls in fungi and in the exoskeleton of insects and crustaceans. MethodsLow stringency sequence signature search in transcriptomes was used to identify GH18 sequences related to chitinases. Identified sequences were expressed in E. coli and corresponding structures modelled. Synthetic substrates and in some cases colloidal chitin were used to characterize activities. ResultsCatalytically functional hits were sorted and their predicted structures compared. All share the TIM barrel structure of the GH18 chitinase catalytic domain, optionally fused to binding motifs, such as CBM50, CBM18, and CBM14, involved in sugar recognition. Assessment of the enzymatic activities following deletion of the C-terminal CBM14 domain of the most active clone evidenced a significant contribution of this extension to the chitinase activity. A classification based on module organization, functional and structural criteria of characterized enzymes was proposed. ConclusionsPhysarum polycephalum sequences encompassing a chitinase like GH18 signature share a modular structure involving a structurally conserved catalytic TIM barrels decorated or not by a chitin insertion domain and optionally surrounded by additional sugar binding domains. One of them plays a clear role in enhancing activities toward natural chitin. General significanceMyxomycete enzymes are currently poorly characterized and constitute a potential source for new catalysts. Among them glycosyl hydrolases have a strong potential for valorization of industrial waste as well as in therapeutic field.

A bacterial sulfoglycosidase highlights mucin O-glycan breakdown in the gut ecosystem.

Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum.

Identification and structural analysis of a carbohydrate-binding module specific to alginate, a representative of a new family, CBM96

Carbohydrate-binding modules (CBMs) are the noncatalytic modules that assist functions of the catalytic modules in carbohydrate-active enzymes, and they are usually discrete structural domains in larger multimodular enzymes. CBMs often occur in tandem in different alginate lyases belonging to the CBM families 13, 16, and 32. However, none of the currently known CBMs in alginate lyases specifically bind to an internal alginate chain. In our investigation of the multidomain alginate lyase Dp0100 carrying several ancillary domains, we identified an alginate-binding domain denoted TM6-N4 using protein truncation analysis. The structure of this CBM domain was determined at 1.35Å resolution. TM6-N4 exhibited an overall β-sandwich fold architecture with two antiparallel β-sheets. We identified an extended binding groove in the CBM using site-directed mutagenesis, docking, and surface electrostatic potential analysis. Affinity analysis revealed that residues of Lys10, Lys22, Lys25, Lys27, Lys31, Arg36, and Tyr159 located on the bottom or the wall of the shallow groove are responsible for alginate binding, and isothermal titration calorimetry analyses indicated that the binding cleft consists of six subsites for sugar recognition. This substrate binding pattern is typical for type B CBM, and it represents the first CBM domain that specifically binds internal alginate chain. Phylogenetic analysis supports that TM6-N4 constitutes the founding member of a new CBM family denoted as CBM96. Our reported structure not only facilitates the investigation of the CBM-alginate ligand recognition mechanism but also inspires the utilization of the CBM domain in biotechnical applications.

Mechanistic and Structural Insights into the Specificity and Biological Functions of Bacterial Sulfoglycosidases

Glycan sulfation is an important modification supporting the functionalities of many proteins in biology. Exo-acting 6S-GlcNAcases from human microbiota are glycosidases that participate in the removal of 6-sulfo-GlcNAc from host glycans and thereby play an important role in human health and disease. Nonetheless, mechanisms underlying their ability to recognize the sulfate group remain poorly understood. Using structural and kinetic analyses, we here reveal the catalytically important amino acids directly involved in the recognition and cleavage of 6S-GlcNAc, but not of 6-phospho-GlcNAc, in BbhII from Bifidobacterium bifidum, Bt4394 from Bacteroides thetaiotaomicron, and SGL from Prevotella spp. The defining features of their sulfate recognition motifs underpin a genomic enzymological exploration of 6S-GlcNAcases to identify a wider range of human health-associated bacterial species having 6S-GlcNAcase activity. Our data provide significant insights into distinct molecular mechanisms of sulfated sugar recognition employed by 6S-GlcNAcases from both Gram-positive and Gram-negative bacteria along with valuable information for the exploration of extensive interactions between microbiota and their host glycans.

Probing the Importance of Host Symmetry on Carbohydrate Recognition.

The design of molecular cages with low symmetry could allow for more specific tuning of their properties and better mimic the unsymmetrical and complex environment of protein pockets. However, the added value of lowering symmetry of molecular receptors has been rarely demonstrated. Herein, C3 - and C1 -symmetrical cages, presenting the same recognition sites, have been synthesized and investigated as hosts for carbohydrate recognition. Structurally related derivatives of glucose, galactose and mannose were found to have greater affinity to the receptor with the lowest symmetry than to their C3 -symmetrical analogue. According to the host cavity modelling, the C1 symmetry receptor exhibits a wider opening than its C3 -symmetrical counterpart, providing easier access and thus promoting guest proximity to binding sites. Moreover, our results show the high stereo- and substrate selectivity of the C1 symmetry cage with respect to its C3 counterpart in the recognition of sugars.

Discovery of a lectin domain that regulates enzyme activity in mouse N-acetylglucosaminyltransferase-IVa (MGAT4A)

N-Glycosylation is a common post-translational modification, and the number of GlcNAc branches in N-glycans impacts glycoprotein functions. N-Acetylglucosaminyltransferase-IVa (GnT-IVa, also designated as MGAT4A) forms a β1-4 GlcNAc branch on the α1-3 mannose arm in N-glycans. Downregulation or loss of GnT-IVa causes diabetic phenotypes by dysregulating glucose transporter-2 in pancreatic β-cells. Despite the physiological importance of GnT-IVa, its structure and catalytic mechanism are poorly understood. Here, we identify the lectin domain in mouse GnT-IVa’s C-terminal region. The crystal structure of the lectin domain shows structural similarity to a bacterial GlcNAc-binding lectin. Comprehensive glycan binding assay using 157 glycans and solution NMR reveal that the GnT-IVa lectin domain selectively interacts with the product N-glycans having a β1-4 GlcNAc branch. Point mutation of the residue critical to sugar recognition impairs the enzymatic activity, suggesting that the lectin domain is a regulatory subunit for efficient catalytic reaction. Our findings provide insights into how branching structures of N-glycans are biosynthesized.

Access to Unexplored 3D Chemical Space: cis-Selective Arene Hydrogenation for the Synthesis of Saturated Cyclic Boronic Acids.

A new class of saturated boron‐incorporated cyclic molecules has been synthesized employing an arene‐hydrogenation methodology. cis‐Selective hydrogenation of easily accessible, and biologically important molecules comprising benzoxaborole, benzoxaborinin, and benzoxaboripin derivatives is reported. Among the various catalysts tested, rhodium cyclic(alkyl)(amino)carbene [Rh‐CAAC] (1) pre‐catalyst revealed the best hydrogenation activity confirming turnover number up to 1400 with good to high diastereoselectivity. A broad range of functional groups was tolerated including sensitive substituents such as −F, −CF3, and −silyl groups. The utility of the synthesized products was demonstrated by the recognition of diols and sugars under physiological conditions. These motifs can have a substantial importance in medicinal chemistry as they possess a three‐dimensional structure, are highly stable, soluble in water, form hydrogen bonds, and interact with diols and sugars.

Crystal Structure of Human Lysozyme Complexed with N-Acetyl-α-d-glucosamine

Human lysozyme is a natural non-specific immune protein that participates in the immune response of infants against bacterial and viral infections. Lysozyme is a well-known hydrolase that cleaves peptidoglycan in bacterial cell walls. Several crystal structures of human lysozyme have been reported, but little is known regarding how it recognizes sugar molecules. In this study, the crystal structures of human lysozyme in its native and two N-acetyl-α-d-glucosamine (α-D-NAG)-bound forms were determined at 1.3 Å and 1.55/1.60 Å resolution, respectively. Human lysozyme formed a typical c-type lysozyme fold and the α-D-NAG molecule was bound to the middle of subsites C and D. The N-acetyl and glucosamine groups of α-D-NAG were stabilized by hydrophobic interactions (Val117, Ala126, and Trp127), hydrogen bonds (Asn64, Asn78, Ala126, and Val128), and water bridges. Conformational changes of Arg80, Tyr81, Val128, and Arg131 of human lysozyme were observed due to the interactions of α-D-NAG with the active-site cleft. The binding configuration of α-D-NAG in human lysozyme was distinct compared with that of other sugar-bound lysozymes. Findings from this structural analysis provide a better understanding of the sugar recognition of human lysozyme during the immune response to microbial pathogens.

MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.

The accepted notion of dNTP transport following cytoplasmic biosynthesis is ‘facilitated diffusion’; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a ‘reservoir’. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.

Structural and mechanistic insights into the substrate specificity and hydrolysis of GH31 α-N-acetylgalactosaminidase

Glycoside hydrolase family 31 (GH31) is a diversified family of anomer-retaining α-glycoside hydrolases, such as α-glucosidase and α-xylosidase, among others. Recently, GH31 α-N-acetylgalactosaminidases (Nag31s) have been identified to hydrolyze the core of mucin-type O-glycans and the crystal structure of a gut bacterium Enterococcus faecalis Nag31 has been reported. However, the mechanisms of substrate specificity and hydrolysis of Nag31s are not well investigated. Herein, we show that E. faecalis Nag31 has the ability to release N-acetylgalactosamine (GalNAc) from O-glycoproteins, such as fetuin and mucin, but has low activity against Tn antigen. Mutational analysis and crystal structures of the Michaelis complexes reveal that residues of the active site work in concert with their conformational changes to act on only α-N-acetylgalactosaminides. Docking simulations using GalNAc-attached peptides suggest that the enzyme mainly recognizes GalNAc and side chains of Ser/Thr, but not strictly other peptide residues. Moreover, quantum mechanics calculations indicate that the enzyme preferred p-nitrophenyl α-N-acetylgalactosaminide to Tn antigen and that the hydrolysis progresses through a conformational itinerary, 4C1 → 1S3 → 4C1, in GalNAc of substrates. Our results provide novel insights into the diversification of the sugar recognition and hydrolytic mechanisms of GH31 enzymes.

X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

Fluorescent-Oxaboroles: Synthesis and Optical Property by Sugar Recognition.

The optical property of fluorescent unit-conjugated aliphatic oxaboroles has been investigated. The oxaboroles provide good fluorescence quantum yields and selective recognition toward D-ribose and D-ribose containing molecules. The molecular recognition induced significant fluorescence quenching. The property of the boroles showed the possibility of the boron-based nicotinamide adenine dinucleotide (NAD) sensor probe.