Lectin Binding Sites Research Articles

Ultrastructural Localization of Lectin Binding Sites of the Acrosomal Membrane System of Boar Spermatozoa*

The distribution of the main lectin receptors of boar spermatozoa were analyzed by concanavalin A and Ricinus communis agglutinin-120 visualized for light microscopy by enzymatic amplification with 3-amino-9-ethylcarbazole. The lectin binding sites of the acrosomal membranes in addition to the plasma membrane were displayed by electron microscopy using the lectin-peroxidase and lectin-ferritin technique. Lectin binding was particularly prominent at the anterior part of the head. It could be shown that both acrosomal membranes contain receptor sites for concanavalin A and Ricinus communis-agglutinin-120. Additionally, the whole cell surface could be labeled with both lectins.

Lectin-Binding Sites in Normal Human Testis/Lektinbindungsstellen normaler humaner Hoden

Nine fluorescein isothiocyanate (FITC)-labelled lectins have been used to investigate the distribution of glycoconjugates in unfixed frozen and Bouin-fixed sections of normal human testis. Interstitial cells and lamina propria of seminiferous tubuli were stained by PNA, HPA, RCA II, SBA, ConA, and WGA indicating an abundance of the following glycoconjugates: N-GlcNAc, N-GalNAc, Gal, and Man. The germinative cells were stained cytoplasmatically by ConA (Alpha-D-Man/-Glc). Sertoli cells showed the same pattern with ConA. Early spermatids fixed PNA and RCA II in the acrosomal region. Elongated spermatids fixed WGA on their acrosomes and fainty on the flagellae too indicating abundance of N-GlcNAc residues. The findings argue for differentiation-related modifications of lectin-binding sites on germinative cells and the usefulness of Bouin-fixed samples for lectin histochemistry.

Lectin-Binding Sites in Testis of Men with Acrosomeless Round-Headed Spermatozoa/Testikuläre Lektinbindungsstellen bei Männern mit akrosomenlosen Rundkopfspermatozoen

We report herein about lectin histochemistry of seminiferous epithelia in two infertile men with exlusely acrosomeless round-headed spermatozoa. FITC-conjugated lectins (ConA, PNA, RCA II, WGA) have been employed on tissue sections of Bouin fixed testicular biopsies. RCA II gave a dot-like fluorescence of the acrosomal region and WGA gave a cap-like acrosomal fluorescence of spermatids. PNA-a marker of acrosomal differentiation-failed to stain spermatids. The binding of ConA to germ cells was not influenced by this syndrome. In conclusion, the syndrome of acrosomeless round-headed spermatozoa is associated with selective perturbations of testicular lectin-binding sites. They might contribute of the inability of sperm cells to adhere to and penetrate into ova.

Glycomimetics and Glycodendrimers as High Affinity Microbial Anti‐adhesins

Adhesion to epithelial surface is often the first step in bacterial and viral infection. In this process, the microbes use a variety of proteins for interaction with host carbohydrates presented as glycoconjugates on cell surfaces. Crystal structures of adhesin and lectin binding sites in complexes with oligosaccharide open the route for design and synthesis of glycomimetics, glycodendrimers, and glycopolymers that are able to block infection at an early stage.

Jacalin: a lectin mitogenic for human CD4 T lymphoctyes

The major protein component of seeds from jackfruit is the lectin jacalin. Jackfruit crude extracts are known to stimulate human lymphocytes, but the mitogenic properties of purified jacalin have not been studied in detail so far. Study of the proliferative response of cell populations from normal human peripheral blood to purified jacalin showed it to be mitogenic through an interaction with lymphocytes by its lectin-binding site, as shown by inhibition by IgA. Jacalin failed to stimulate B cells to proliferate and to undergo plasma cell maturation. It induced a proliferation of CD4 (and not CD8) lymphocytes, as shown by phenotypic analysis of cells recovered after culture and by studies of the response of isolated T cell subpopulations. The proliferative response to jacalin was autologous monocyte-dependent. The kinetics of jacalin-induced DNA synthesis, expression of CD25 and interleukin-2 secretion was shifted by comparison with that induced by phytohaemagglutinin. The reason for the restricted responsiveness of CD4 T cells is presently unclear; jacalin bound to all blood cells and did not significantly co-cap with CD1, CD2, CD3, CD4, CD8 and CD38, and jacalin response was neither enhanced nor inhibited by antibodies to these surface antigens.

Imaging Glycosylation

The glycosylation state of individual antibodies was imaged using an atomic force microscope with a probe modified with lectins and an image acquisition system that permits simultaneous acquisition of sample topography data along with a map of lectin binding sites.

Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

Isolation of de-exined pollen and cytological studies of the pollen intines of Pinus bungeana Zucc. Ex Endl. and Picea wilsonii Mast

To study the cytological and biochemical characteristics of intine, pollen deprived of exine, or de-exined pollen, was isolated from the gymnosperms Pinus bungeana and Picea wilsonii. The factors influencing the isolation rate were examined. Cellulose, callose, pectin, and arabinogalactan proteins (AGPs) were localized in this material using fluorescent probes, and components of the isolated intine were further analyzed by Fourier transform infrared (FTIR) microspectroscopy. The isolation protocol was repeatable and reliable. Cellulose was found to be evenly distributed on the surface of the intine, as indicated by strong calcofluor White ST (CW) fluorescence, and aniline blue staining revealed that callose was present on the intine of P. bungeana but not on that of P. wilsonii. Immunolabeling revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present on the pollen intine, as well as esterified pectin recognized by the monoclonal antibody JIM7, and AGPs recognized by the LM2 antibody. Two lectin binding sites, the concanavalin agglutinin (Con A) and soybean agglutinin (SBA) binding sites, were present on the intine surface, but no wheat germ agglutinin (WGA) binding sites were detectable. These results were confirmed by FTIR analysis.

Binding studies with Ulex europaeus agglutinin I (UEA-I) of the vascular endothelium of the synovial membrane

The lectin binding sites of the synovium of patients with rheumatoid arthritis and osteoarthritis were investigated. It was shown that Ulex europaeus agglutinin is a constant marker of the vascular endothelium and is not induced during the course of inflammatory process in rheumatoid arthritis.

Why are metastases in cirrhotic livers so rare?

Metastases in livers with cirrhosis are a rare event in comparison to metastases in normal livers. This fact is of great importance for clinical differential diagnosis of tumours of the liver. High metalloproteinase inhibitor contents and especially altered lectins or lectin binding sites in cirrhosis of the liver may help to explain the rare event "metastasis in cirrhosis".

Interaction of lectin from Viscum album L. with arabinogalactan-proteins from Echinacea purpurea (L.) Moench

Today mistletoe extracts play an important role in adjuvant cancer treatment because of their immunostimulating properties. Clinical studies have shown that mistletoe lectin I enhances the secretion of cytokines and interleukins and increases the number of natural killer cells. As recognition and binding of mistletoe lectins to sugar chains present on the cell surface of immunogenic cells is the first step in biological activity, knowledge on sugar binding properties of lectins is significant in determining their medicinal potential. In order to enhance knowledge on possible binding sites of mistletoe lectins, we examined interaction of ML I with an arabinogalactan-protein (AGP) from Echinacea purpurea.

Neurokinin 1 receptor, IB4 lectin and nitric oxide synthase localizations in whole-mount preparation of the rat meninges following noxious stimulation

Distribution and immunocytochemical characterization of the nerve fibers and their terminals in Wistar rats (n=15) meninges were studied by using neurokinin 1 receptor, IB4 lectin and nitric oxide synthase labeling after the application of noxious peripheral stimulation. For immunocytochemistry, free floating specimens were incubated with neurokinin 1 receptor, nitric oxide synthase and IB4 lectin antibodies and then observed with avidin-biotin-peroxidase method. The neuronal markers stained in all unmyelinated nerve fibres throughout the meninges. Neurokinin 1 receptor- and IB4 lectinimmunoreactive nerve fibers were localized in all leptomeningeal compartments, where they terminated close to the subarachnoid space or within the connective tissue, thus suggesting extensive distribution of nerve fibres in the meninges. Neuronal nitric oxide synthase-positive nerve fibers and a prominent mast cell population were seen in the rat leptomeninx and dura. The presence of a significant number of putative nociceptive fibres suggests a possible role for nociceptive function in the context of pathophysiological aspects. The distributions pattern of IB4 lectin binding sites, neurokinin 1 receptor and nitric oxide synthase could be due to the function of particular segments in the pathogenesis of meningeal vascular network.

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.

Enriched Environmental Conditions Ameliorate Age-Dependent Alterations in the Somatosensory System, but Do Not Affect the Motor System of the Rat Spinal Cord

At the behavioral level, old rats are known to show a number of age-related changes such as characteristic impairments in the sensorimotor system, which is most strikingly expressed in a walking impairment involving the hind limbs. We have previously shown that sensorimotor impairments involving the hind limbs are in part reversible following housing in an enriched environment. The question arises as to whether these effects are accompanied by cytological alterations in the spinal cord. During aging, we found an increase in cyclooxygenase with nitrotyrosine in the fasciculus gracilis and lateral funiculus. The somatosensory tracts and dorsal horn were more affected by myelin-degrading processes than the other parts of the spinal cord. The accumulation of lipofuscin seemed to depend on age, but independent of the environment. In contrast, the age-related calretinin immunoreactivity loss in the substantia gelatinosa from both the cervical and the lumbar enlargements was significantly reduced in stimulating environmental conditions. Morphologically, the lectin binding sites of the perineuronal nets seemed to be decreased. The results provide evidence that the somatosensory system of the spinal cord is affected by age-dependent alterations independently of the motor system.

NMR Investigation of the Bound Conformation of Natural and Synthetic Oligomannosides to Banana Lectin

AbstractThe conformational behaviour of three mannose‐containing oligosaccharides, namely, the α1→3[α1→6] trisaccharide, a heptasaccharide with α1→2, α1→3 and α1→6 linkages and a tetrasaccharide consisting of α1→3 and α1→2 linkages, when bound to banana lectin (BanLec) has been evaluated by trNOE NMR methods and docking calculations. It was found that the molecular recognition event involves a conformational selection process with only one of the conformations present in the free state of the sugar being recognised at the lectin binding site. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Lectin histochemistry of metastatic adenocarcinomas of the lung

Several clinical studies indicate that primary tumour cells with high metastatic potential often show aberrant glycosylation as detected by lectin histochemistry. However, it is unclear whether aberrant glycosylation is still present in metastatic deposits. The aim of the present investigation was thus to analyse a possible association between the presence of lectin binding sites of pulmonary adenocarcinoma cells and their lymph node and haematogenous metastatic cells. For this purpose, the expression of HPA, PHA-L and UEA-I was assessed in primary tumours, lymph node metastases and haematogenous metastases of 96 patients with metastatic adenocarcinomas of the lung that underwent surgery between 1999 and 2002. Besides, lectin-binding data and other known prognostic factors were correlated with survival. We found a significant positive correlation between the binding of the lectins HPA (p=0.002), PHA-L (p<0.00001) and UEA-I (p<0.00001) to the cells of the primary tumour and to their lymph node metastases. There was a positive correlation between the binding of HPA to the cells of the primary tumour and the haematogenous metastases as well. Patients with tumours which did not show HPA binding sites had a median overall survival of 27.9 months (95%-CI 7.7-infinity months). Patients with a HPA binding tumour had a median overall survival of 20.9 months (95%-CI 18.5-28.7 months). This is the first investigation to demonstrate a positive correlation between the binding of the lectins HPA, PHA-L and UEA-I to the cells of the primary tumour and to their lymph node metastases. Expression of HPA binding sites is also preserved in the haematogenous metastases. In summary, our results support the hypothesis that altered glycosylation of the membrane-bound glycoproteins of the tumour cells is associated with, but not sufficient for promotion of lymphogenic and haematogenous metastasis.

Modifications of surface carbohydrates on bovine spermatozoa mediated by oviductal fluid: a flow cytometric study using lectins

The objective of the present study was to characterize and quantify changes in exposed saccharide residues of bovine sperm during capacitation in oviductal fluid (ODF) using flow cytometry (FC). Bovine sperm were incubated with 0% or 50% non-luteal ODF for 30 min or 3.5 h. After incubation, sperm were labelled with 11 fluorescein isothiocyanate-labelled lectins and evaluated for lectin binding with FC. Furthermore, inhibiting sugars were used to determine specificity of lectin binding to oligosaccharides on the sperm surface. After 30 min incubation, there was a 91% decrease in fluorescence intensity of labelled sperm incubated in WGA, a 76% decline for Con A, 75% decline for BS-I and a 36% decline for DBA. These differences remained approximately the same over the 3.5-h incubation. Interestingly, although there was no reduction in UEA-I binding at 30 min, a significant reduction (23%) was observed at 3.5 h. Con A fluorescence was mostly inhibited with either alpha-d-glucose or alpha-d-mannose (86% and 90% respectively). BS-I fluorescence was reduced after prior incubation of the control samples with N-acetyl-galactosamine and galactose by 74% and 80% respectively. After prior incubation with N-acetyl-galactosamine DBA fluorescence reduced by 18% in the control samples. With UEA-I no fluorescence reduction was observed after prior incubation with l-fucose. We have demonstrated that capacitation of bovine sperm in ODF is accompanied by a quantitative reduction in individual lectin binding sites. These modifications may be crucial to the subsequent signalling events involved with sperm-zona binding, zona penetration or interaction with the oolema.

Sialidase expression in activated human T lymphocytes influences production of IFN-γ

Sialidases influence cellular activity by removing terminal sialic acid from glycoproteins and glycolipids. Four genetically distinct sialidases (Neu1-4) have been identified in mammalian cells. In this study, we demonstrate that only lysosomal Neu1 and plasma membrane-associated Neu3 are detected in freshly isolated and activated human T lymphocytes. Activation of lymphocytes by exposure to anti-CD3 and anti-CD28 IgG resulted in a ninefold increase in Neu1-specific activity after growth of cells in culture for 5 days. In contrast, the activity of Neu3 changed minimally in activated lymphocytes. The increase in Neu1 enzyme activity correlated with increased synthesis of Neu1-specific mRNA. Neu1 was present on the surface of freshly isolated and activated CD4 and CD8 T lymphocytes, as determined by staining intact cells with anti-Neu1 IgG and analysis by flow cytometry and by Western blot analysis of biotin-labeled cell surface proteins. Cell surface Neu1 was found tightly associated with a subunit of protective protein/cathepsin A (PPCA). Compared with freshly isolated lymphocytes, activated cells expressed more surface binding sites for galactose-recognizing lectins Erythrina cristagalli (ECA) and Arachis hypogaea. Growth of cells in the presence of sialidase inhibitors 2,3-dehydro-2-deoxy-N-acetylneuraminic acid or 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid resulted in a smaller increase in number of ECA-binding sites and a greater amount of cell surface sialic acid in activated cells. Inhibition of sialidase activity also resulted in reduced expression of IFN-gamma in activated cells. The down-regulation of IFN-gamma occurred at the transcriptional level. Thus, sialidase activity in activated T lymphocytes contributes to the hyposialylation of specific cell surface glycoconjugates and to the production of IFN-gamma.

POLYMORPHONUCLEAR LEUKOCYTE DYSFUNCTION SYNDROME IN PATIENTS WITH INCREASING SEPSIS SEVERITY

Production of oxygen radicals is required for both microbicidal and tissue-toxic effector functions of granulocytes. Inasmuch as an ambivalent role of polymorphonuclear leukocytes (PMNs) may become apparent during sepsis, we studied levels of hydrogen peroxide (H2O2) production by PMNs depending upon the nature of different particulate and soluble stimuli in patients with increasing sepsis severity. Patients with sepsis (n = 15), severe sepsis (n = 12), or septic shock (n = 33) were prospectively enrolled in the study. Healthy volunteers of comparable age and sex served as controls (n = 50). Unopsonized and opsonized zymosan particles were used to assess adhesion, phagocytosis, and the associated H2O2 production. Zymosan particles are rich in beta-glucans and lectin structures that are known to trigger H2O2 production via two major non-toll-like receptor pathogen recognition receptors, comprising the lectin-binding site in the alpha-chain (CD11b) of the complement receptor type 3 and the more recently identified nonclassical C-type lectin, dectin-1. To determine H2O2 production upon cell activation by soluble stimuli, PMNs were activated by the chemotactic tripeptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) alone or after priming of cells by preincubation with tumor necrosis factor alpha. To get insight into the changes of fMLP receptor classical intracellular signaling pathways, PMNs were also incubated with the calcium ionophore A23187 and the phorbol ester phorbol myristate acetate, bypassing receptor-dependent signal transduction to directly activate calcium/calmodulin kinase- and protein kinase C-dependent pathways, respectively. As compared with healthy volunteers, levels of H2O2 production by PMNs from septic patients varied depending upon the nature of the activating signal: reduced (zymosan), unchanged (phorbol myristate acetate, opsonized zymosan), and enhanced (spontaneous, fMLP, fMLP + tumor necrosis factor alpha, A23187), with the changes most pronounced in patients with septic shock. Specifically, phagocytosis of zymosan and the associated H2O2 production were significantly decreased whereas spontaneous and stimulated H2O2 production elicited by soluble stimuli strongly increased. Thus, these findings suggest the development of a PMN dysfunction syndrome in patients with increasing sepsis severity. Moreover, as binding of zymosan particles to the PMNs' surface remained unchanged despite increasingly suppressed phagocytosis and associated H2O2 production, observed effects are likely to reflect defects in signaling by the lectin-binding site of CD11b and/or the beta-glucan receptor dectin-1, respectively.

Nanoparticle-Based Sensing of Glycan−Lectin Interactions

Here we present the first report on nanoparticle-based biosensing of glycan markers of diseases. The protocol relies on the competition between a nanocrystal (CdS)-tagged sugar and the target sugar for the binding sites of surface-confined lectin and monitoring the extent of competition through highly sensitive electrochemical detection of the captured nanocrystal. This development is expected to allow decentralized detection of carbohydrate moieties and lectin-carbohydrate interactions to be performed more rapidly, sensitively, inexpensively, and reliably.