The reconstruction of atomic co-ordinates from a protein stereo ribbon diagram when additional information for sufficient sidechain positions is available

We describe the application of a method for the reconstruction of three-dimensional atomic co-ordinates from a stereo ribbon diagram of a protein when additional information for some of the sidechain positions is available. The method has applications in cases where the 3D co-ordinates have not been made available by any means other than the original publication and are of interest as models for molecular replacement, homology modelling etc. The approach is, on the one hand, more general than other methods which are based on stereo figures which present specific atomic positions, but on the other hand relies on input from a specialist. Its exact implementation will depend on the figure of interest. We have applied the method to the case of the -d-galactose-binding lectin jacalin with a resultant RMS deviation, compared to the crystal structure, of 1.5 Å for the 133 C positions of the -chain and 2.6 Å for the less regular -chain. The success of the method depends on the secondary structure of the protein under consideration and the orientation of the stereo diagram itself but can be expected to reproduce the mainchain co-ordinates more accurately than the sidechains. Some ways in which the method may be generalised to other cases are discussed.     

Cu(II)-Self-assembling bipyridyl-glycoclusters and dendrimers bearing the Tn-antigen cancer marker: syntheses and lectin binding properties

Using the carbohydrate cancer marker, T_N-antigen (α-GalNAc-OR), covalently linked to a bipyridine core, square planar complexes were formed by self-assembly upon simple addition of Cu(II) sulfate. The required α-d-GalNAc-OR building block was constructed from 2-azidoethyl 2-acetamido-2-deoxy-α-d-glucopyranoside (GlcNAc) by epimerization at C-4 of a suitably protected derivative followed by conventional modifications to provide 2-aminoethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-d-galactopyranoside. The 2-aminoethyl aglycone was further elongated into a key monomer having an aminocaproic acid spacer together with their corresponding dimers using a double N-alkylation strategy of their N-bromoacetyl derivatives using mono-Boc-1,4-diaminobutane, respectively. The building blocks containing the bipyridyl dimers, having either a short or a long spacer arm, together with the tetramer built from the short spacer derivative were prepared in a convergent manner using 2,2′-bipyridine-4,4′-dicarboxylic acid chloride and the aminated sugar derivatives, respectively. Copper(II)-nucleated GalNAc derivatives containing four and eight residues were obtained from an aqueous solution of the bipyridyl derivatives. The relative inhibitory potencies of these glycodendrimers were evaluated against monomeric allyl α-d-GalNAc using a solid-phase competition assay with asialoglycophorin and horseradish peroxidase-labeled lectin Vicia villosa. The di- and tetra-valent bipyridyl clusters showed up to 87-fold increased inhibitory properties (IC₅₀ 7.14, 1.82, 4.09 μM, respectively) when compared to the monomer (IC₅₀ 158.3 μM) while the Cu(II)-complexes showed up to a 259-fold increase potencies (IC₅₀ 0.61 μM) with the octamer showing the highest affinity. However, when expressed on a per-saccharide basis, the tetramer Cu(II) nucleated derivative, possessing the longest inter-sugar distances showed the highest affinity (IC₅₀ 0.63 μM).     

Precise synthesis of heterogeneous glycopolymers with well-defined saccharide motifs in the side chain via post-polymerization modification and recognition with lectin

Heterogeneous glycopolymers with different sugar units in the side chain have been receiving considerable attention due to their potential properties in enhancing molecular recognition abilities toward a specific receptor, yet there are limited synthetic approaches to introduce different sugar motifs into the glycopolymer backbone. Herein, a series of heterogeneous glycopolymers consisting of different sugar units in the side chains were synthesized by post-polymerization modification of activated PFPA ester precursor polymers. The functionalized amines bearing two different sugar motifs have been synthesized by gradient CuAAC reaction, which could serve as a platform for achieving heterogeneous sugar units with functional control in concise steps. Isothermal titration calorimetry (ITC) measurements of the obtained glycopolymers with Concanavalin A indicated that the heterogeneous glycopolymers, poly(Man-βGlu-OH) and poly(Man-βGa-OH) bearing α-D-mannose and other non-binding β-Glucose or β-Galatose units, show higher affinities toward Concanavalin A in comparison to monoglycopolymer poly(Man-Alkyne-OH) in which the non-binding sugar motifs was substituted with non-sugar unit due to synergistic effects of non-binding sugar units. Moreover, this work allows for precise fabrication of a broad variety of glycopolymers in which it significantly broadens the library of accessible polymer structures, either homogeneous or heterogeneous glycopolymers.     

Biologically Active Heteroglycoclusters Constructed on a Pillar[5]arene‐Containing [2]Rotaxane Scaffold

A synthetic approach combining recent concepts for the preparation of multifunctional nanomolecules (click chemistry on multifunctional scaffolds) with supramolecular chemistry (self‐assembly to prepare rotaxanes) gave easy access to a large variety of sophisticated [2]rotaxane heteroglycoclusters. Specifically, compounds combining galactose and fucose have been prepared to target the two bacterial lectins (LecA and LecB) from the opportunistic pathogen Pseudomonas aeruginosa.     

Electrochemical biosensors for monitoring the recognition of glycoprotein-lectin interactions

Despite the wide applicability and specificity of lectins to carbohydrate moieties, there are few lectin specific biosensors. This is attributed to the difficulty in defining the relevant experimental parameters to measure for sensing. We hereby describe the development of direct and indirect electrochemical sensors to determine the exact trace amounts of probarley lectin (ProBL) and its conversion product wheat germ agglutinin (WGA). In addition to WGA, the antigens (ProBL) employed in this study were over expressed in bacteria, isolated from protein bodies, and purified using immobilized N-acetylglusamine in order to obtain correctly folded active lectins. The amperometric immunosensor uses cell lines producing monoclonal antibody (mAB) to the pro-region of ProBL over expressed from Escherichia coli. The efficacy and sensing characteristics of the lectin were optimized using monoclonal antibody to WGA and the resulting sensor was found to detect only ProBL in the linear range 10⁻³-10² μg mL⁻¹ and a detection limit of 10⁻³ μg mL⁻¹.     

菜豆凝集素抑制剂的设计与凝血活性

针对菜豆凝集素二聚体复合物的结构特征,利用计算机辅助药物设计的方法设计抑制剂以破坏复合物结构;进一步采用标准的Fomc保护氨基酸NT氨基的固相法合成纯化了小肽抑制剂.体外兔血红细胞凝集实验结果表明,该小肽对菜豆凝集素的凝血能力有一定的抑制效果.该方法为植物凝集素凝血研究及菜豆相关的食品安全问题提供了一个新思路.     

Degradation of Target Oligosaccharides by Anthraquinone–Lectin Hybrids with Light Switching

Anthraquinone–lectin hybrids were effectively synthesized using water‐soluble anthraquinone derivative 11 with concanavalin A (ConA) and hygrophorus russula lectin (HRL) to give anthraquinone–ConA ( 16 ) and anthraquinone–HRL ( 17 ) hybrids, respectively. These anthraquinone–lectin hybrids effectively and selectively degraded oligosaccharides containing a mannose residue as a non‐reducing terminal sugar, which has affinity for ConA and HRL, under photo‐irradiation with long‐wavelength UV light without additives and under neutral conditions. In addition, anthraquinone–HRL ( 17 ) selectively photo‐degraded only Man(α1,6)Man, which has a high affinity for HRL, among several mannosides by recognition of both the type and glycosidic linkage profile of the sugar in an oligosaccharide.     

水溶性含糖共轭聚合物的制备及应用

水溶性共轭聚合物具有优异的光学稳定性、高亮度、易于修饰和水溶性等特点,广泛应用于离子检测、蛋白检测和生物成像等领域。水溶性共轭聚合物主要通过在共轭聚合物的侧基或端基修饰水溶性的离子基团或水溶性聚合物实现其水溶性,水溶性共轭聚合物还可以通过引入功能性基团或聚合物使其具备不同的功能特性。糖化合物是天然存在的一类生物分子且大部分具有水溶性的特点,因此最近十几年来科研工作者将糖化合物引入共轭聚合物中以赋予共轭聚合物糖化合物的生物功能特性。本文总结了水溶性含糖共轭聚合物的制备方法、化学结构及其在凝集素、细菌检测和细胞荧光成像中的应用。最后总结了此类聚合物的特性、发展方向及目前所需解决的问题。     

Isolation, characterization and molecular evolution of a novel pearl shell lectin from a marine bivalve, Pteria penguin

Summary A novel lectin, PPL, was isolated from the mantle of penguin wing oyster (Pteria penguin) by affinity chromatography on mucin-Sepharose 4B and cation exchange chromatography on HiTrap SP. This lectin was estimated to be a 21-kDa monomer by gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted time of flight (MALDI-TOF) mass spectrometry. However, dynamic light scattering experiments revealed that a non-covalently linked dimer formed under high salt conditions (500 mM NaCl). Interestingly, PPL showed an increasing hemagglutinating activity with increasing salt concentration. The amino acid sequence of PPL was determined by direct protein sequence analysis and cDNA cloning. The 167-amino acid sequence included 24 lysine residues and had two tandemly repeated homologous domains (residues 20–78 and 107–165) with 44% internal homology. PPL showed sequence homology to L-rhamnose-binding lectins from fish eggs and a D-galactose-binding lectin from sea urchin eggs, with sequence identities in the range 37–48%. PPL agglutinated various animal erythrocytes independently of calcium ions. The minimum concentration of PPL needed to agglutinate rabbit erythrocytes was 0.5 μg/ml, and the most effective saccharides to inhibit the hemagglutination were D-galactose, methyl-D-galactopyranoside and N-acetyl-D-lactosamine. Lactose also inhibited hemagglutination, but L-rhamnose did so only weakly despite the sequence homology with trout egg L-rhamnose-binding lectins. The carbohydrate-binding specificity of PPL was further examined by frontal affinity chromatography using 37 different pyridylaminated oligosaccharides. PPL was found to have strong binding affinity for various oligosaccharides that have Galβ1-4Glu/GlcNAc, Galβ1-3GalNAc/GlcNAc and Galα 1-4Gal moieties in their structure. PPL had a high thermal stability and retained 50% of its hemagglutinating activity after incubation at 70°C for 100 min. It agglutinated some Gram-negative bacteria by recognizing lipopolysaccharides. Together, these results suggest that PPL is a new member of the trout egg lectin family which participates in the self-defense mechanism against bacteria and pathogens with a distinct carbohydrate-binding specificity. We conclude that the trout egg lectin family proteins, in particular their carbohydrate recognition domains, have acquired diverse carbohydrate-binding specificities during molecular evolution.