Wheat Germ Agglutinin Binding Sites Research Articles

Cover Feature: Photoswitching Affinity and Mechanism of Multivalent Lectin Ligands (Chem. Eur. J. 27/2022)

Photochemical cis–trans isomerization was used to switch a divalent lectin ligand between its two forms, which show contrasting binding properties to the plant lectin wheat germ agglutinin (WGA), as visualized by the yin-yang symbol. Whereas the E isomer (obtained under irradiation with green light) is able to bridge adjacent binding sites of WGA leading to a high-affinity chelating binding mode, the Z isomer (obtained under UV light irradiation) crosslinks two lectin molecules associated with a much lower binding affinity. More information can be found in the Research Article by V. Wittmann and co-authors (DOI: 10.1002/chem.202200267).

Photoswitching Affinity and Mechanism of Multivalent Lectin Ligands

Multivalent receptor–ligand binding is a key principle in a plethora of biological recognition processes. Immense binding affinities can be achieved with the correct spatial orientation of the ligands. Accordingly, the incorporation of photoswitches, which can be used to reversibly change the spatial orientation of molecules, into multivalent ligands is a means to alter the binding affinity and possibly also the binding mode of such ligands. We report a divalent ligand for the model lectin wheat germ agglutinin (WGA) containing an arylazopyrazole photoswitch. This switch, which has recently been introduced as an alternative to the more commonly used azobenzene moiety, is characterized by almost quantitative E/Z photoswitching in both directions, high quantum yields, and high thermal stability of the Z isomer. The ligand was designed in a way that only one of the isomers is able to bridge adjacent binding sites of WGA leading to a chelating binding mode. Photoswitching induces an unprecedentedly high change in lectin binding affinity as determined by isothermal titration calorimetry (ITC). Furthermore, additional dynamic light scattering (DLS) data suggest that the binding mode of the ligand changes from chelating binding of the E isomer to crosslinking binding of the Z isomer.

血小板活性化の初期反応部位の研究 : 血小板細胞におけるカチオン化フェリチンとwheat germ agglutininの結合部位(杏林大学学位論文要旨ならびに審査要旨)

血小板活性化の初期反応部位の研究 : 血小板細胞におけるカチオン化フェリチンとwheat germ agglutininの結合部位(杏林大学学位論文要旨ならびに審査要旨)

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.

Polar Distribution Dynamics of Con A Binding Sites in Embryo Sacs is Temporally Coupled by the Fertilization Process

The binding site distribution of concanavalin agglutinin (Con A) and wheat germ agglutinin (WGA) on embryo sacs at various developmental stages of Torenia fournieri L was studied by using a cooled Charge Coupled Device (CCD) and fluorescent Con A and WGA probes. The distribution patterns of Con A and WGA binding sites on embryo sacs changed during the fertilization process. The fluorescent signal indicating Con A binding sites was distributed evenly on the surface of the embryo sac wall before anthesis, was much denser on the micropylar end of the embryo sac wall and looked like a corona on the day of anthesis. After pollination, stronger fluorescence was present on the micropylar end of the embryo sac wall and the filiform apparatus (FA), showing an obvious polar distribution. When the pollen tube entered the embryo sac and reached a synergid, the fluorescence was still concentrated on the micropylar end and FA, and started to appear on the synergid. After fertilization, the polar distribution of the fluorescence gradually disappeared and an even distribution pattern was observed again on the embryo sac wall. These results revealed that the dynamic distribution of Con A binding sites was temporally coupled with the process of fertilization. WGA binding site distribution on the embryo sac was also investigated and showed a simple pattern but also regularly changed during the process of fertilization. The variation of these lectin binding sites during the fertilization process suggests that lectin binding site interactions may play a role in the process.

In vitro and in situ localization of concanavalin A and wheat germ agglutinin binding sites on the surface of female cells in Torenia fournieri L.

The interaction between lectins and their specific binding sites is believed to play a critical role in fertilization in animals and some lower plants. However, for higher plants there is no information on lectins or their binding sites related to female gametes and fertilization. The present work was designed as a first attempt to reveal the general pattern of lectin binding site distribution on the surface of female cells, namely egg cells, central cells, and synergids of Torenia fournieri and, especially, to investigate the possible effects of cell isolation procedure on the distribution of lectin binding sites. Therefore, concanavalin A (Con A) and wheat germ agglutinin (WAG) binding sites on the surface of both in vitro and in situ living female cells were localized by using fluorescein isothiocyanate (FITC) conjugated Con A and WGA as probes. We demonstrated that enzymatic treatment and isolation procedures did not notably modify the surface character of the female cells and the distribution of Con ...

Clearance of senescent erythrocytes: wheat germ agglutinin distribution on young and old human erythrocytes.

To add an additional aspect to the process of recognition and removal of senescent human erythrocytes from the circulation, the binding of wheat germ agglutinin (WGA) to separated young, old and sialidase-treated human erythrocytes is evaluated with the immune-electron microscopical method. WGA/gold conjugate binding to old erythrocytes was lower (27%) than to young erythrocytes and even lower following treatment with sialidase (82%), exhibiting a clustered, non-continuous labeling pattern in all three erythrocyte populations, thus showing a possible redistribution of WGA binding sites. The decrease in bound WGA/gold particles correlates well with the previously reported decrease in surface sialic acid on old erythrocytes. The binding of WGA/gold are indicative of the changes occurring on erythrocyte membrane surfaces when interacting with different agglutinins.

Chick Ciliary Neurotrophic Factor Is Secreted via a Nonclassical Pathway

In contrast to mammalian ciliary neurotrophic factors (CNTFs), chick CNTF is secreted, although it lacks an N-terminal signal. We determined that a 52 aa region of chick CNTF containing an internal hydrophobic domain could direct secretion of rat CNTF. Using a stable cell line that overexpressed chick CNTF, we found that chick CNTF immunoreactivity was punctate throughout the cytosol. Cellular fractionation confirmed chick CNTF to be protected by vesicles. Chick CNTF did not colocalize with fibronectin, calreticulin, wheat germ agglutinin binding sites, or with transferrin receptor. The distribution of chick CNTF was altered neither by brefeldin A nor by chloroquine treatment. Although the punctate pattern of chick CNTF immunoreactivity was not due to reuptake, chick CNTF could be found in a cellular compartment labeled after a brief incubation with dextran microbeads. When synthesized in vitro, chick CNTF did not translocate into microsomes. We conclude that chick CNTF is secreted via a nonclassical pathway.

Reorganization of actin cytoskeleton at the growing end of the cleavage furrow of Xenopus egg during cytokinesis.

We studied reorganization of actin-myosin cytoskeleton at the growing ends of the cleavage furrow of Xenopus eggs in order to understand how the contractile ring is formed during cytokinesis. Reorganization of F-actin structures during the furrow formation was demonstrated by rhodamine-phalloidin staining of the cleavage furrow and by time-lapse scanning with laser scanning microscopy of F-actin structures in the cleavage furrow of live eggs to which rhodamine-G-actin had been injected. Actin filaments assemble to form small clusters that we call 'F-actin patches' at the growing end of the furrow. In live recordings, we observed emergence and rapid growth of F-actin patches in the furrow region. These patches then align in tandem, elongate and fuse with each other to form short F-actin bundles. The short bundles then form long F-actin bundles that compose the contractile ring. During the furrow formation, a cortical movement towards the division plane occurs at the growing ends of the furrow, as shown by monitoring wheatgerm agglutinin-conjugated fluorescent beads attached to the egg surface. As a result, wheatgerm agglutinin-binding sites accumulate and form 'bleb-like' structures on the surface of the furrow region. The F-actin patch forms and grows underneath this structure. The slope of F-actin accumulation in the interior region of the furrow exceeds that of accumulation of the cortex transported by the cortical movement. In addition, rhodamine-G-actin microinjected at the growing end is immediately incorporated into the F-actin patches. These data, together with the rapid growth of F-actin patches in the live image, suggest that actin polymerization occurs in the contractile ring formation. Distribution of myosin II in the cleavage furrow was also examined by immunofluorescence microscopy. Myosin II assembles as spots at the growing end underneath the bleb-like structure. It was suggested that myosin is transported and accumulates as spots by way of the cortical movement. F-actin accumulates at the position of the myosin spot a little later as the F-actin patches. The myosin spots and the F-actin patches are then simultaneously reorganized to form the contractile ring bundles

GP-83 and GP-39, two glycoproteins secreted by human epididymis are conjugated to spermatozoa during maturation.

Surface glycoconjugates of spermatozoa are modified during epididymal maturation, which is closely related to the development of sperm function. In addition, recognition of surface glycoconjugates is one of very critical events in sperm-oocyte interaction. The binding of carbohydrate-specific lectins to the human sperm surface during epididymal maturation has been investigated. However, the glycoproteins responsible for lectin binding in sperm maturation are not well documented. This study used wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (Con-A) to identify sperm maturation-related glycoproteins in human epididymis. Histochemical localization revealed that the binding sites of WGA, PNA and Con-A were mainly in the principal cells and luminal contents of the human epididymis, but not in the interstitial regions. Each lectin displayed a fairly distinct regional localization. On Western blots probed with WGA and Con-A, glycoproteins of 83 kDa (GP-83) and 39 kDa (GP-39) were identified in the sperm extracts, epididymal fluid and tissue extracts of the corpus and cauda epididymides, but not in the caput. PNA identified GP-83 in the same manner as WGA and Con-A, but did not recognize GP-39. These results suggest that lectin-binding glycoproteins GP-83 and GP-39 found on mature spermatozoa may be secreted by the principal cells of corpus and cauda epididymis, and conjugated to spermatozoa during their transit in human epididymis.

Differentiation of Lectin Binding Patterns in Developing Oocytes of the Giant Freshwater Prawn, Macrobrachium rosenbergii.

Five different peroxidase-labeled lectins, Helix pomatia agglutinin (HPA), Concanavalin A agglutinin (Con A), Ricinus communis agglutinin (RCA-I), Wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I), were used to investigate alterations of the lectin binding patterns during oocyte development of the giant freshwater prawn, Macrobrachium rosenbergii. HPA, Con A and RCA-I binding sites were detected in the cytoplasm of developing oocytes; whereas HPA, WGA and UEA-I binding sites were localized on the oocyte surfaces. Although, HPA binding to the cytoplasm of developing oocytes could be detected at all stages of development, significant differentiation of the binding occurred at vitellogenic stage. Con A or RCA-I binding to the cytoplasm was first detected at early vitellogenic stage but the patterns of binding were altered at later stage. Moreover, HPA, Con A or RCA-I was reactive to different regions of the cytoplasm and produced different types of the reaction products. Formation of new surface components at vitellogenic stage was also implicated. The oocyte surfaces began to be reactive to HPA at early vitellogenic stage and were intensely reactive to HPA, WGA and UEA-I at late vitellogenic stage. Furthermore, affinity of HPA or RCA-I to the nucleolus was also detected at late vitellogenic stage.

The wheat germ agglutinin binding sites and development of the mitochondria-rich cells in gills of tilapia ( Oreochromis mossambicus)

By labelling with gold-colloid-conjugated wheat germ agglutinin (WGA), the WGA-binding sites were identified in the apical region of certain mitochondria-rich (MR) cells in gills of freshwater-adapted tilapia (Oreochromis mossambicus). Bromo-deoxyuridine (BrdU) injection was used to discern the differentiation of the WGA binding sites during development of MR cells. Double labeling of gill sections of fish 2, 4, 6, and 8 days after a single BrdU injection showed that the BrdU labeled MR cells 2 and 4 days after injection were mostly WGA negative, whereas 6 and 8 days after injection, WGA and BrdU-labeled MR cell increased gradually in number. Furthermore, the ratio of the WGA-positive to total MR-cells was higher in gills of tilapia adapted to low calcium ([Ca] = 0.015 mm) freshwater than in hard freshwater ([Ca] = 1.898 mm). The results indicate that WGA binding site may be a marker expressed late during differentiation of MR cells, and physiologically functional in assisting uptake of calcium when environmental calcium is low. According to their WGA binding and also characteristics their likely function in calcium absorption, the WGA positive MR cells shown in this study are considered to be similar to the β cells described by Pisam et al. (1995).

Distribution of concanavalin A and wheat germ agglutinin binding sites in the rat peripheral nerve fibres revealed by lectin/glycoprotein-gold histochemistry.

The affinity of rat peripheral nerve fibres for concanavalin A (Con A) and wheat germ agglutinin (WGA) was tested in semi-thin sections of Epon-embedded material. A two-step post-embedding technique was used. As a first step, Con A and WGA were used in pure form. As a second step, peroxidase-gold (for Con A) and ovomucoid-gold (for WGA) complexes were applied. The lectin-binding sites, visualized by means of signal amplification with the photochemical silver reaction, were associated mainly with the myelin sheaths and the surfaces of Schwann cells.

Role of glycoconjugates in adherence ofSalmonella pullorumto the intestinal epithelium of chicks

1. The distribution of glycoconjugates on the surface of Salmonella pullorum and the ileal epithelium of chicks was demonstrated by lectin cytochemistry. The role of glycoconjugates in adherence of S. pullorum to the ileal epithelium was determined by a sugar inhibition assay using the scanning electron microscope. 2. S. pullorum exhibited binding to Concanavalin A (Con A) and wheat germ agglutinin (WGA) but not to soyabean agglutinin (SBA), Ricinus communis agglutinin (RCA) and Ulex europaeus agglutinin (UEA). 3. The ileal epithelium of chicks bound WGA, Con A and SBA. The binding sites for WGA were on the brush border and cytoplasm of columnar enterocytes as well as on goblet cells. The binding of Con A was confined to the cytoplasm of columnar enterocytes, while SBA bound, in a limited way, to the brush border of columnar enterocytes. 4. After an oral dose of S. pullorum, adherence to the ileal epithelium was inhibited by methyl alpha-D-mannopyranoside.

Enrichment and functional characterization of Sca-1+WGA+, Lin-WGA+, Lin- Sca-1+, and Lin-Sca-1+WGA+ bone marrow cells from mice with an Ly-6a haplotype

Approximately 4% to 5% of all bone marrow (BM) cells and 8% to 9% of low density BM cells from FVB/N and BALB/c mice (Ly-6a haplotype) show high to intermediate expression of Ly-6E.1 antigen, recognized by the Sca-1 antibody. Functional properties of enriched cells expressing Ly- 6E.1-allelic form of Sca-1 antigen were analyzed and correlated with the properties of cells expressing the carbohydrate binding sites for the lectin wheat-germ agglutinin (WGA). Using equilibrium density centrifugation and fluorescence-activated cell sorting, Sca-1+WGA+, Lin- WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ cells were isolated and their splenic colony-forming unit (CFU-S) cell content, radioprotection ability, and long-term reconstitution capacity determined. Enriched Sca- 1+WGA+, Lin-WGA+, Lin-Sca-1+ and Lin-Sca-1+WGA+ cells gave rise to 1 CFU-S12 cell out of 26, 20, 21, and 15 sorted cells, respectively. When transplanted into lethally irradiated recipients (100 to 500 cells/mouse) all populations rescued 70% to 100% of recipients in a 30- day radioprotection assay and mediated survival of 40% to 80% of recipients 6 months after transplantation. Using transgenic mice as cell donors we have shown that 12 to 16 weeks after transplantation of 100 Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ cells, 40% to 80% of recipients had donor cells in BM, spleen, thymus, and lymph nodes. These results indicate that the population of cells expressing Ly-6E.1 form of Sca-1 antigen in two analyzed mouse strains with Ly-6a haplotype contains CFU-S and long-term repopulating cells. Furthermore, the data suggest that, at least in FVB/N mice, day-12 CFU-S cells and cells with long-term repopulating capacity simultaneously express Ly- 6E.1 form of Sca-1 antigen and WGA-binding molecules.

Enrichment and Functional Characterization of Sca-1 + WGA +, Lin WGA +, Lin− Sca-1+, and Lin− Sea-1+ WGA+ Bone Marrow Cells From Mice With an Ly-6a Haplotype

AbstractApproximately 4% to 5% of all bone marrow (BM) cells and 8% to 9% of low density BM cells from FVB/N and BALB/c mice (Ly-6a haplotype) show high to intermediate expression of Ly-6E.1 antigen, recognized by the Sca-1 antibody. Functional properties of enriched cells expressing Ly-6E.1 -allelic form of Sca-1 antigen were analyzed and correlated with the properties of cells expressing the carbohydrate binding sites for the lectin wheat-germ agglutinin (WGA). Using equilibrium density centrifugation and fluorescence-activated cell sorting, Sca-1+WGA+, Lin−WGA+, Lin−Sca-1+, and Lin−Sca-1 +WGA+ cells were isolated and their splenic colony-forming unit (CFU-S) cell content, ra-dioprotection ability, and long-term reconstitution capacity determined. Enriched Sca-1+WGA+, Lin−WGA+, Lin−Sca-1 + and Lin “Sca-1 +WGA+ cells gave rise to 1 CFU-S12 cell out of 26, 20, 21, and 15 sorted cells, respectively. When transplanted into lethally irradiated recipients (100 to 500 cells/mouse) all populations rescued 70% to 100% of recipients in a 30-day radioprotection assay and mediated survival of 40% to 80% of recipients 6 months after transplantation. Using transgenic mice as cell donors we have shown that 12 to 16 weeks after transplantation of 100 Sca-1+WGA+, Lin−WGA+, Lin−Sca-1+, and Lin ”Sca-1 + WGA+ cells, 40% to 80% of recipients had donor cells in BM, spleen, thymus, and lymph nodes. These results indicate that the population of cells expressing Ly-6E.1 form of Sca-1 antigen in two analyzed mouse strains with Ly-6a haplotype contains CFU-S and long-term repopulating cells. Furthermore, the data suggest that, at least in FVB/N mice, day-12 CFU-S cells and cells with long-term repopulating capacity simultaneously express Ly-6E.1 form of Sca-1 antigen and WGA-binding molecules.

The role of lymphocyte surface binding sites for wheat germ agglutinin in the negative regulation of cancer patients.

The role of lymphocyte surface binding sites for wheat germ agglutinin (WGA) in the negative regulation of cancer patients was investigated. The number of WGA binding sites on the surface of each lymphocyte ranged from 10(7) to 10(8). Fluorescein isothiocyanate (FITC)-conjugated WGA, bound to the majority of peripheral blood lymphocytes (PBL) with two peaks of fluorescent intensity was expressed either dimly or brightly. The increase in lymphocytes brightly expressing WGA fluorescent intensity (WGA bright lymphocytes) significantly correlated with the number of WGA binding sites. The suppression of lymphocyte proliferation mediated by the purified soluble suppressor factor (SSF) significantly correlated with an increase in the WGA bright lymphocyte population (P < 0.05). A significantly greater number of WGA bright lymphocytes in PBL was found in patients with esophageal, gastric, breast, or colon cancer, than in those with benign diseases or in healthy controls. Furthermore, an increase in WGA bright lymphocytes was found in subsets expressing the antigens CD8 dimly or CD16. Thus, it is suggested that the number of WGA binding sites may increase mainly on the surface of effector cells such as NK cells and CD8-positive killer T cells in cancer patients, triggering the negative regulation mediated by SSF.

Isolation and enrichment of two sublobular compartment-specific endothelial cell subpopulations from liver sinusoids

Similar to the well-recognized phenotypical heterogeneity of hepatocytes, in situ sublobular variations have recently been detected in the cell structure, fenestration patterns, filtrating efficiency, surface glycosylation, scavenger function and pathological responses of the sinusoidal lining endothelium. However, unlike other liver cell populations, until now no endothelial cell subpopulations had been isolated or defined with clarity, much less with sublobular/acinar zone-related differential properties. On the basis of our previous studies showing that periportal segments of mouse liver sinusoids express a significantly higher number of wheat germ agglutinin-binding sites than do perivenous ones, we used this differential feature for in vitro labeling of the specific sublobular derivation of isolated sinusoidal lining endothelial cells to correlate their original lobular position with other features determined on flow cytometry, centrifugal elutriation, discontinuous arabinogalactan density gradients and electron microscopy. Our results revealed additional heterogeneous properties whose association with high or low wheat germ agglutinin-binding capacity made it possible to define in vitro two dominant endothelial cell subpopulations that appear similar to the differential features in the periportal and perivenous sinusoidal segments. Type 1 endothelial cells had low forward angle light scatter and high integrated side scatter, low cytoplasmic porosity index (12% +/- 5%) and high wheat germ agglutinin-binding efficiency (160 +/- 35 fluorescence intensity units/cell size); these findings are similar to what was observed in situ in the periportal sinusoidal endothelium. On the other hand, type 2 endothelial cells, with high forward angle light scatter and low integrated side scatter, had a high cytoplasmic porosity index (25% +/- 8%) and low wheat germ agglutinin-binding efficiency (60 +/- 15 fluorescence intensity units/cell size), findings similar to in situ observations of the perivenous sinusoidal lining endothelium. Moreover, these physical and morphological differences entail different cell sedimentation behaviors: type 1 endothelial cell sedimented at high centrifugal elutriation counterflow rates (23 to 37 ml/min) and high arabinogalactan density gradient levels (10% to 15%), whereas type 2 endothelial cell sedimented at low counterflow rates (18 to 23 ml/min) and low density levels (6% to 10%). The combination of these separation procedures made it possible to isolate a 90%-enriched type 1 endothelial cell population in the 12% to 15% interphase of the 23 and 37 ml/min elutriation flow rates and a 75%-enriched type 2 endothelial cell population in the 6% to 10% interphase of the 18 and 23 ml/min flow rates.

Immunocytochemical demonstration of wheat germ agglutinin (WGA)-receptors on the cell surface of guinea pig peritoneal macrophages.

WGA-receptors on the surface of resident guinea pig peritoneal macrophages were investigated. Macrophages were characterized by the endogenous peroxidase pattern. Two different immunogold techniques, the indirect WGA-anti WGA-protein A-gold technique and the direct WGA/BSA-gold technique were compared to evaluate their suitability for quantification of WGA binding sites on the cell surface. Because of the higher binding specificity the direct technique should be preferred for quantitative studies.

In vivo effects of tunicamycin on chondrocytes of rat mandibular condyles as revealed by lectin cytochemistry.

The in vivo effects of tunicamycin on the glycosylation of proteoglycans and link protein in rat mandibular condylar chondrocytes were studied by ultrastructural lectin histochemistry. The binding of wheat-germ agglutinin was shown by using anti-lectin antibody followed by protein A-gold complex. In normal rats, wheat-germ agglutinin labeling was restricted to trans cisternae and vacuoles of the Golgi complex, whereas it was observed in neither the cis region of the Golgi complex nor in the rough endoplasmic reticulum. By 3 h after the drug administration, wheat-germ agglutinin binding sites on the disorganized Golgi vacuoles were dramatically reduced in number. At 6 h after the drug administration, the lectin binding sites on the Golgi vacuoles were restored. These results demonstrate that the in vivo use of tunicamycin in combination with histochemical analysis using lectin probes is of significant value for the study of protein glycosylation in chondrocytes of the rat mandibular condyle.