Selective Lectin Binding Research Articles

Synthetic Glycomacromolecules of Defined Valency, Absolute Configuration, and Topology Distinguish between Human Lectins.

Carbohydrate-binding proteins (lectins) play vital roles in cell recognition and signaling, including pathogen binding and innate immunity. Thus, targeting lectins, especially those on the surface of immune cells, could advance immunology and drug discovery. Lectins are typically oligomeric; therefore, many of the most potent ligands are multivalent. An effective strategy for lectin targeting is to display multiple copies of a single glycan epitope on a polymer backbone; however, a drawback to such multivalent ligands is they cannot distinguish between lectins that share monosaccharide binding selectivity (e.g., mannose-binding lectins) as they often lack molecular precision. Here, we describe the development of an iterative exponential growth (IEG) synthetic strategy that enables facile access to synthetic glycomacromolecules with precisely defined and tunable sizes up to 22.5 kDa, compositions, topologies, and absolute configurations. Twelve discrete mannosylated “glyco-IEGmers” are synthesized and screened for binding to a panel of mannoside-binding immune lectins (DC-SIGN, DC-SIGNR, MBL, SP-D, langerin, dectin-2, mincle, and DEC-205). In many cases, the glyco-IEGmers had distinct length, stereochemistry, and topology-dependent lectin-binding preferences. To understand these differences, we used molecular dynamics and density functional theory simulations of octameric glyco-IEGmers, which revealed dramatic effects of glyco-IEGmer stereochemistry and topology on solution structure and reveal an interplay between conformational diversity and chiral recognition in selective lectin binding. Ligand function also could be controlled by chemical substitution: by tuning the side chains of glyco-IEGmers that bind DC-SIGN, we could alter their cellular trafficking through alteration of their aggregation state. These results highlight the power of precision synthetic oligomer/polymer synthesis for selective biological targeting, motivating the development of next-generation glycomacromolecules tailored for specific immunological or other therapeutic applications.

Design of Thermoresponsive Elastin-Like Glycopolypeptides for Selective Lectin Binding and Sorting.

Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.

Block-Sequence-Specific Glycopolypeptides with Selective Lectin Binding Properties.

Glycopolypeptides with defined block sequences were prepared by sequential addition of two different N-carboxyanhydrides (NCAs), followed by selective deprotection and functionalization of predefined positions within the polypeptide backbone. The sequential arrangement of the galactose units and the block-sequence length have been systematically varied. All the glycopolypeptides have been obtained with a similar overall composition and comparable molecular weights. Circular dichroism measurements revealed some dependence of the secondary structure on the primary composition of the glycopolypeptides at physiological pH. While statistical, diblock, and tetrablock glycopolypeptides adopted a random coil conformation, the octablock glycopolypeptide was mostly α-helical. The ability to selectively bind to lectins was investigated by turbidity measurements as well as surface plasmon resonance (SPR) studies. It was found that the extent of binding was dependent on the position of the galactose units and thus the primary glycopolypeptide structure. The octablock glycopolypeptide favored interaction with lectin RCA120 while the tetrablock glycopolypeptide demonstrated the strongest binding activity to Galectin-3. The results suggest that different lectins are very sensitive to glyco coding and that precise control of carbohydrate units in synthetic polymeric glycopeptides will remain important.

Bio-recognizable and photo-cleavable block copolymers based on sugar and poly(4-substituted-ε-caprolactone) bearing a photo-cleavable junction

In this study, we synthesized bio-recognizable and photo-cleavable amphiphilic block copolymers containing photodegradable linkers, 5-hydroxy-2-nitrobenzyl alcohol, as junction points between bio-recognizable hydrophilic glucose (or maltose) and hydrophobic poly(4-substituted-e-caprolactone) chains, by using a combination of ring-opening polymerization and nucleophilic substitution reactions. When the polymer solutions were exposed to ultraviolet (UV) irradiation, substantial structural and morphological changes were observed in the particles. The copolymers were biodegradable and biocompatible, and they could self-assemble into spherical photo-responsive micelles. Fluorescence emission measurements indicated the release of Nile red, a hydrophobic dye, encapsulated by the Glyco-ONB-PXCL micelles, in response to irradiation caused by the disruption of the micelles. Selective lectin binding experiments confirmed that the glycosylated Glyco-ONB-PXCL could be used to bio-recognition applications. The nanoparticles were associated with negligible levels of toxicity at concentrations of less than 30 μg mL−1. The confocal microscopy and flow cytometry results showed that the uptake of doxorubicin (DOX)-loaded micelles by HeLa cells was faster than that of free DOX.

Thermoresponsive glycopolypeptides with temperature controlled selective lectin binding properties

Linear polypeptide poly(γ-propargyl-l-glutamate) (PPLG) was synthesized by the ring opening polymerization (ROP) of the corresponding N-carboxy anhydride (NCA). Azide-functional galactose and 1-azido-2-(2-methoxyethoxy)ethane (mEO2) were co-clicked on the polypeptide at different ratios to introduce selective binding and thermoresponsive properties. Selective binding was demonstrated by turbidity assays with Ricinus Communis Agglutinin (RCA120) lectin. Cloud point measurements confirmed the cloud point temperature (Tcp) of the polymers increasing with the ratio of mEO2 to galactose. Moreover, lectin binding experiments at temperatures above and below Tcp suggest that binding is suppressed above Tcp. This opens opportunities to design functional materials with temperature controlled biological response.

Bioactive saccharide-conjugated polypeptide micelles for acid-triggered doxorubicin delivery.

The synthesis and self-assembly of lactobionolactone-conjugated poly(l-glutamic acid)-b-poly(l-phenylalanine) amphiphilic block copolypeptides (Lac-PGA-b-PPhe) and their evaluation for anticancer drug doxorubicin (DOX) delivery have been investigated. Lactobionolactone was functionalized with the azide group and successfully conjugated with the terminal alkyne groups on the polypeptides through click reaction and these amphiphilic glycopolypeptides self-assembled to form micelles with bioactive galactose units on the particle surface as confirmed by selective lectin binding experiments. Drug release experiments showed that DOX released faster from saccharide-conjugated micelles under acidic conditions than under neutral conditions. The DOX-loaded, saccharide-conjugated micelles exhibited higher cytotoxicity toward HepG2 tumor cells than free DOX and saccharide-free micelles loaded with DOX at low concentrations, suggesting that the saccharide-conjugated micelles can effectively bind to the cells through specific recognition and subsequently the higher uptake of saccharide-conjugated micelles led to higher drug release and cytotoxicity under pH-sensitive conditions.

Selective binding of lectins to normal and neoplastic urothelium in rat and mouse bladder carcinogenesis models

Bladder cancer adjuvant intravesical therapy could be optimized by more selective targeting of neoplastic tissue via specific binding of lectins to plasma membrane carbohydrates. Our aim was to establish rat and mouse models of bladder carcinogenesis to investigate in vivo and ex vivo binding of selected lectins to the luminal surface of normal and neoplastic urothelium. Male rats and mice were treated with 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water and used for ex vivo and in vivo lectin binding experiments. Urinary bladder samples were also used for paraffin embedding, scanning electron microscopy and immunofluorescence labelling of uroplakins. During carcinogenesis, the structure of the urinary bladder luminal surface changed from microridges to microvilli and ropy ridges and the expression of urothelial-specific glycoproteins uroplakins was decreased. Ex vivo and in vivo lectin binding experiments gave comparable results. Jacalin (lectin from Artocarpus integrifolia) exhibited the highest selectivity for neoplastic compared to normal urothelium of rats and mice. The binding of lectin from Amaranthus caudatus decreased in rat model and increased in mouse carcinogenesis model, indicating interspecies variations of plasma membrane glycosylation. Lectin from Datura stramonium showed higher affinity for neoplastic urothelium compared to the normal in rat and mouse model. The BBN-induced animal models of bladder carcinogenesis offer a promising approach for lectin binding experiments and further lectin-mediated targeted drug delivery research. Moreover, in vivo lectin binding experiments are comparable to ex vivo experiments, which should be considered when planning and optimizing future research.

Precision glycopolymers and their interactions with lectins

Glycopolymers are synthetic polymers with carbohydrate groups attached and have a great potential to be utilized in drug delivery applications and nanomedicines as a result of their recognition properties. Sugar code is running in living entities with extreme precision and every small difference in the oligosaccharide structure significantly affects the recognition by lectins. Therefore, controlling the sequence of sugars along the polymer backbone is vital to achieve selective lectin binding and cell signalling. The preparation of sequence controlled glycopolymers has recently been in the focus of several research groups and the cons and pros of three recently published approaches are discussed in this minireview.

Synthesis of glycopoly(pseudo amino acid)s and their interaction with lectins

In this study, we developed a novel bioeliminable amphiphilic glycopoly(pseudo amino acid), which was synthesized by the condensation polymerization of N-benzyloxycarbonyl-4-hydroxyl-l-proline (NZHpr) followed by the coupling of an alkynyl-functional sugar derivative to the azido-end group, P(NZHpr)n. The glycosylated P(NZHpr)n polymers formed micelles in the aqueous phase. Selective lectin binding experiments confirmed that the glycosylated P(NZHpr)n can be used in biorecognition applications. The DOX-loaded micelles facilitated improved uptake of DOX by HeLa cells within 2h and were primarily retained in the cytoplasm, whereas free DOX tended to accumulate in the nuclei. The DOX-loaded glucosylated P(NZHpr)n micelles exhibited a substantially lower cytotoxicity compared to free DOX.

Biologically Active Polymersomes from Amphiphilic Glycopeptides

Polypeptide block copolymers with different block length ratios were obtained by sequential ring-opening polymerization of benzyl-L-glutamate and propargylglycine (PG) N-carboxyanhydrides. Glycosylation of the poly(PG) block was obtained by Huisgens cycloaddition "click" reaction using azide-functionalized galactose. All copolymers were self-assembled using the nanoprecipitation method to obtain spherical and wormlike micelles as well as polymersomes depending on the block length ratio and the nanoprecipitation conditions. These structures display bioactive galactose units in the polymersome shell, as proven by selective lectin binding experiments.

Photochemical Microcontact Printing by Thiol−Ene and Thiol−Yne Click Chemistry

This article describes the microstructured immobilization of functional thiols on alkene- and alkyne-terminated self-assembled monolayers on silicon oxide substrates by photochemical microcontact printing. A photochemical thiol-ene or thiol-yne “click” reaction was locally induced in the area of contact between stamp and substrate by irradiation with UV light (365 nm). The immobilization reaction by photochemical microcontact printing was verified by contact angle measurements, X-ray photoelectron spectroscopy, atomic force microscopy, and time-of-flight secondary ion mass spectrometry. The reaction rate of photochemical microcontact printing by thiol-ene chemistry was studied using time dependent contact angle measurements. The selective binding of lectins to galactoside microarrays prepared by photochemical microcontact printing was also demonstrated. It was found that photochemical microcontact printing results in a high surface coverage of functional thiols within 30 s of printing even for dilute (mM) ink solutions.

Hydrolytically Stable Bioactive Synthetic Glycopeptide Homo- and Copolymers by Combination of NCA Polymerization and Click Reaction

The synthesis of poly(dl-propargylglycine) and poly(γ-benzyl-l-glutamate-co-dl-propargylglycine) was performed by NCA polymerization at 0 °C to yield well-defined polypeptides with polydispersity indices below 1.3. FTIR results confirm β-sheet and α-helical conformation of the homopolymer and copolymer, respectively. The subsequent glycosylation was achieved by Huisgen [3 + 2] cylcoaddition (“click” reaction) with azide-functional galactose. FTIR, NMR, SEC, and MALDI−ToF analyses verify the successful glycosylation and suggest a high efficiency of the click reaction. The homoglycopeptide was found to be water-soluble and to form aggregates in water above a critical concentration of 0.079 mg/mL. Selective lectin binding experiments confirmed that the glycopeptides can be used in biorecognotion applications. Moreover, the selective hydrolysis of the benzyl ester groups in the copolymer was achieved without loss of the galactose.

Carbohydrate Microarray on Glass: A Tool for Carbohydrate-Lectin Interactions

A simple method to immobilize carbohydrates on a glass surface to obtain a carbohydrate microarray is described. The array was used to study carbohydrate-lectin interactions. The glass surface was modified with aldehyde terminated linker groups of various chain lengths. Coupling of carbohydrates with an amino terminated alkyl spacer to the aldehyde terminated glass followed by reductive amination resulted in carbohydrate microarrays. Fluorescently labeled (FI-TC) lectins (concanavalin A and Arachis hypogaea) were used to study specific carbohydrate-lectin interactions. contact angle, atomic force microscopy (AFM) and confocal laser fluorescence microscopy (CLFM) techniques were used in this study to monitor the modification of the glass and the successful selective binding of lectins to the carbohydrate microarray.

Selective binding of lectins to embryonic chicken vasculature.

Chicken embryos are an excellent model system for studies related to vascular morphogenesis. Development in ovo allows manipulations otherwise difficult in mammals, and the use of chicken-quail chimeras offers an additional advantage to this experimental system. Furthermore, the chicken chorioallantoic membrane has been extensively used for in vivo assays of angiogenesis. Surprisingly, few markers are available for a comprehensive visualization of the vasculature. Here we report the use of lectins for identification of embryonic chicken blood vessels. Nine lectins were evaluated using intravascular perfusion and directly on sections. Our results indicate that Lens culinaris agglutinin, concanavalin A, and wheat germ agglutinin can be used effectively for visualization of vessels of early chicken embryos (E2.5-E4). At later developmental stages, Lens culinaris agglutinin is a better choice because it displays equal affinity for the endothelia of arteries, veins, and capillaries. The findings presented here expand our understanding of lectin specificity in the endothelium of avian species and provide information as to the use of these reagents to obtain comprehensive labeling of the embryonic and chorioallantoic membrane vasculature.

In vitro proliferation of ICR mouse sertoli cells supplemented with recombinant h-FSH (rh-FSH) at different concentrations

Objective: Sertoli cells are important nurse cells in the seminiferous tubules for male germ cell growth and differentiation into spermatozoa. In contrast to the germ cells, Sertoli cells have receptors for follicle stimulating hormone, which are the main hormonal regulators of spermatogenesis. Hormonal control of spermatogenesis involves control of the maturation and supporting activities of Sertoli cells. Impairment of Sertoli cell function has severe consequences for spermatogenesis. In adult testes, the final number of Sertoli cells correlates with the increase of germ cell production and total sperm output. In this study, we have cultured Sertoli cells for studying the growth kinetics of these cells in culture condition in vitro. Design: With the aim of setting up a proliferation assay to investigate the control of Sertoli cell division, we used an easy and rapid technique, based on the use of selective lectin binding which allows to obtain functional Sertoli cells. Materials/Methods: Datura stramonium agglutin (DSA)-coated dish were used to rapidly single Sertoli cells from immature mouse testis. Isolated Sertoli cells were cultured for 17 days in Eagle’s minimal essential medium (EMEM) with supplements according treatment groups. The proliferative activity of Sertoli cells to FSH responsiveness was examined at each different concentration( 0, 37.5, 75, 150, 300 and 600 mIU/ml) of recombinant h-FSH (rh-FSH). The cell purity of Sertoli cell cultured was examined by immunocytochemical procedure using mouse monoclonal anticytokeratin Pan. Results: The rh-FSH dose of 37.5 mIU/ml and 75 mIU/ml showed the dose dependent increase of Sertoli cells. The supplemented rh-FSH was found to stimulate Sertoli cell proliferation in culture condition. But, the proliferation of cells in rh-FSH dose of 300 mIU/ml and 600 mIU/ml were not showed in this study. Hyper-dose of rh-FSH was showed detrimental effect of the Sertoli cell proliferation. Conclusions: The Sertoli cell culture using immunocytochemistry and DSA-coated dish is useful tool in studies of growth kinetics and cell proliferation in vitro. Also the optimal dose of rh-FSH was important factor for Sertoli cell proliferation.

Expression of N-acetylgalactosamine Residues on Ectoderm Cell Surfaces during Neurulation in the Bantam Chick Embryo.

The expression of cell surface N-acetylgalactosamine (GalNAc), which residues on the ectoderm in the neural plate stage and neural tube stage of bantam embryos, was examined using fluorescein isothiocyanatelabeled lectins BPA, MPA, WFA, HPA, DBA, SJA and LFA before and after neuraminidase treatment. The results showed selective lectin binding on ectoderms of both stages and indicated that the neuroectoderm ac quired different carbohydrate compositions from those in the surface-ectoderm during neurulation. The results also may indicate that GalNAc residues are small in quantity at the terminal position on the bantam neurula ectoderm and many of them hold the penultimate position which is masked by sialic acid. The role of masking on the GalNAc residues during neural tube closure is discussed.

Cell surface glycoconjugates in the olfactory system of lungfish Protopterus annectens Owen

AbstractFranceschini, V. Lazzari, M. and Ciani, F. 2000. Cell surface glycoconjugates in the olfactory system of lungfish Protopterus annectens Owen. —Acta Zoologica (Stockholm) 81: 131–137Lectin binding was performed on the olfactory system of lungfish Protopterus annectens to identify specific glycoconjugates on the cell surface of olfactory receptor cells. The lectin histochemical patterns and the Western blot analysis indicate that the receptor cells of the olfactory mucosa are characterized by high density of α‐N‐acetyl‐ d‐galactosamine residues on the saccharidic chains of the surface glycoproteins. Other lectins display a regional pattern between the regions of the olfactory bulbs. This different histochemical lectin pattern might be due to a different regional segregation of the olfactory projections. On the other hand it could allow the identification of an area corresponding to the accessory olfactory bulb of terrestrial vertebrates in the ventrolateral region of Protopterus olfactory bulb. The presence in the dipnoan olfactory system of a vomeronasal organ homologous to the organ in amphibians is discussed. Moreover, the selective lectin binding on the surface of primary olfactory neurones suggests that specific cell surface glycoproteins may have a role in the axonal growth due to the continuous cycle of proliferation and the death of olfactory receptor cells.

Glycoconjugate and adhesion molecule changes during the cell cycle in a human embryonic epithelial cell line

The changes in the expression of glycoconjugates and adhesion molecules were studied by selective lectin binding and immunocytochemical reactions in a human embryonic epithelial cell line (EUE cells), synchronized in the cell cycle phases. The results can be summarized as follows: most of the tested lectins display a more diffuse binding for the cytoplasm in G 1 than S and G 2 phases; in the S and particularly in G 2 phases the cytoplasm glycoconjugates are rearranged around the nucleus; cells in mitosis always show a strong binding towards all tested lectins. Cellular fibronectin and its receptor β 1 integrin are well expressed in G 1, but the strongest reaction is observed in the S phase. The immunoreactions for laminin and uvomorulin (L-CAM) are poorly positive in all cell cycle phases. The immunocytochemical reaction for heparan sulfate is positive, with a stronger reaction in S and G 2 than in G 1; on the contrary a diffuse staining with the anti-dermatan sulfate proteoglycan antibody appears unchanged during the cell cycle.

The masking effect of sialic acid on Con A, PNA and SBA ectoderm binding sites during neurulation in the bantam chick embryo.

The masking effect of sialic acid on cell surface carbohydrates localized on the ectoderm in stage 6-11 bantam embryos was examined using fluorescein isothiocyanate-labeled Con A, PNA, SBA, LFA, and LPA before and after neuraminidase treatment. The results showed selective lectin binding on both the neuroectoderm and the surface ectoderm. In general, these lectin-binding sites increased or were at least expressed on neuroectoderm during neurulation. On the apical surfaces of the developing neuroectoderm, masked Con A-binding sites were evident from the earliest stage and rapidly increased. These sites coexisted with unmasked binding sites which gradually increased. Masked PNA sites were rarely observed but became abundant in later stages, even though coexistent unmasked sites also rapidly increased. Masked SBA sites were poorly observable in the early stage and gradually increased thereafter, whereas unmasked sites were expressed at later stages. On the basal surfaces masked Con A sites were evident in the early stages but gradually decreased in later stages, whereas unmasked sites were relatively abundant and increased thereafter. Masked PNA sites were evident and increased very rapidly, whereas unmasked sites became observable up to the latest stage. Masked SBA sites were minimal in all three stages, and unmasked sites expressed themselves slightly at later stages. The change in composition of carbohydrates on the developing neuroectoderm was obviously different from that on the developing surface ectoderm. On the contact surface of the neural ridge, the number of masked sites of penultimate sugars was large at Con A sites, slight at PNA and SBA sites, which coexisted with unmasked sugar chain terminals in the areas where Con A sites were moderate and where PNA and SBA sites were poor. Finally, the role of masking on binding sites for Con A, PNA and SBA during neural tube closure is discussed, and the observation that the apparent masking effect on three lectin binding sites did not correspond to the content of sialic acid detected by LFA and LPA is a subject for further study.

Bandeiraea simplicifolia lectin I and Vicia villosa agglutinin bind specifically to the vomeronasal axons in the accessory olfactory bulb of the rat

The binding of 21 lectins to the accessory olfactory bulb (AOB) of the rat was examined by histochemistry. Two lectins [ Bandeiraea simplicifolia lectin I (BSL-I) and Vicia villosa agglutinin (VVA)] bound specifically to the vomeronasal (VN) axons in the AOB. Seven lectins ( Datura stramonium lectin, Erythrina cristagalli lectin, Lycoperisicon esculentum lectin, Ricinus communis agglutinin I, soybean agglutinin, Solanum tuberosum lectin, and Ulex europaeus agglutinin) bound to both VN axons in AOB and olfactory axons in the main olfactory bulb. BSL-I and VVA are useful as the marker of VN axons. This selective binding of lectins indicates the presence of specific glycoconjugates on the surface of VN axons.