The inhibitory effect of some lectins on the differentiation of Friend erythroleukemia cells

Growth and differentiation of Friend cells can be inhibited by treatment with concanavalin A and wheat-germ agglutinin. This inhibition is specific for cells that are in the early stages of the differentiation process (24–48 h after the addition of dimethyl sulfoxide) and is reversible upon treatment with the sugars competitive for these lectins. These results suggest a regulatory role for some plasma membrane-bound glycoproteins early in the differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide.     

麦胚凝集素作用下膜血型糖蛋白A构象变化研究

为进一步验证麦胚凝集素作用下膜血型糖蛋白Ａ空间构象的变化,本文用傅里叶变换红外技术,定量测定了麦胚凝集素作用下,溶液血型糖蛋白Ａ和脂蛋白体膜血型糖蛋白Ａ的二级结构的改变,发现麦胚凝集素结合于血型糖蛋白Ａ导致血型糖蛋白Ａα—螺旋减少,β—结构增加,麦胚凝集素的抑制剂Ｎ—乙酰葡萄糖胺对麦胚凝集素诱导的血型糖蛋白Ａ二级结构的改变有抑制作用。本文同时用扫描隧道显微术直接观察了麦胚凝集素结合前后膜血型糖蛋白Ａ分子形态的变化。     

Influence of lectins on the binding of 125I-labeled EGF to human fibroblasts.

Lectins that interact with mannose (concanavalin A), galactose (ricin, abrin), or N-acetylglucosamine (wheat germ agglutinin) block ¹²⁵I-labeled EGF binding to the surface of cultured human fibroblasts at 37° or 5°. Lectins specific for fucose or N-acetylgalactosamine, soybean agglutinin or gorse lectin, respectively, do not interfere with growth factor binding. The inhibition of ¹²⁵I-labeled EGF binding by concanavalin A at 37° or 5° could be reversed rapidly by the addition of α-methyl mannoside. The results suggest that the fibroblast membrane receptor for EGF is, or is closely associated with, a glycoprotein or glycolipid that contains mannose, galactose and N-acetylglucosamine residues.     

麦胚凝集素作用下膜血型糖蛋白A构象变化研究

为进一步验证麦胚凝集素作用下膜血型糖蛋白Ａ空间构象的变化,本文用傅里叶变换红外技术,定量测定了麦胚凝集素作用下,溶液血型糖蛋白Ａ和脂蛋白体膜血型糖蛋白Ａ的二级结构的改变,发现麦胚凝集素结合于血型糖蛋白Ａ导致血型糖蛋白Ａα—螺旋减少,β—结构增加,麦胚凝集素的抑制剂Ｎ—乙酰葡萄糖胺对麦胚凝集素诱导的血型糖蛋白Ａ二级结构的改变有抑制作用。本文同时用扫描隧道显微术直接观察了麦胚凝集素结合前后膜血型糖蛋白Ａ分子形态的变化。     

Exploring the oviductal fluid proteome by a lectin‐based affinity approach

The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein, we assessed the combined use of Concanavalin A and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel‐based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography‐tandem mass spectrometry (nLC‐ESI‐MS/MS) further allowed the reliable identification of 134 nonbound as well as 130 lectin‐bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin‐bound fraction were known glycoproteins (p‐value [FDR] = 1.45E‐31). The low variance of the number of peptide spectrum matches for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3% for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin‐based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF.     

Lectin activity in pollen from plants representative of thirty orders of spermatophyta

Summary Pollen extracts from a variety of species representative of thirty orders of spermatophyta, including gymnosperms, dicotyledons and monocotyledons, were examined for the presence of lectin activity by means of a hemagglutination assay. Hemagglutinating activity (HA) was detected in the pollen extracts of all the species examined, indicating that lectins are generally present in the pollen of spermatophyta. The response of this pollen hemagglutinating activity to the sugars and glycoproteins tested as potential inhibitors was identical in all species examined. Moreover, the hemagglutinating activity of pollen extracts from eight species which had been selected as representative of the gymnosperms and both subclasses of angiosperms exhibited similar properties (e.g. distribution by differential centrifugation, stability to heat, response to bivalent ions). The bulk of the hemagglutinating activity was always recovered in the pellet after centrifugation at 1000 g for 5 min. Although sequential treatments with 1% Triton X-100 and 1 M KCl were ineffective, subsequent incubation of the pellet with saline phosphate buffer released hemagglutinating activity. The solubilized hemagglutinating activity was destroyed by protease treatment, indicating that the substance(s) responsible for the activity is (are) protein in nature and, consequently, might be considered to be a lectin. The sugar specifity of the pollen lectin activity from wheat, potato and bean was compared with that of wheat germ agglutinin (WGA), potato agglutinin and bean agglutinin — the lectins present in sporophytic tissues of these plants. For all three plants, the response of the pollen lectin activity to sugars and glycoproteins was different from that shown by the lectin from sporophytic tissues.Abbreviations HA Hemagglutinating activity - PBS 150 mM Na-phosphate buffer (pH 7.2) containing 0.9% NaCl - PHA Phaseolus vulgaris agglutinin - STA Solanum tuberosum agglutinin - WGA wheat germ agglutinin     

Gluten,a lectin with oligomannosyl specificity and the causative agent of gluten-sensitive enteropathy

The pathogenesis of gluten-sensitive enteropathy or coeliac disease is as yet unknown. We can demonstrate by laser nephelometric measurements that gluten has lectin-like properties. Gluten binds ‘high-mannose type’ glycoproteins and the complex formation is inhibitable by mannan. As known for other lectins the reaction is absolutely Ca-dependent. Glycoproteins from the immature crypt cells from the small intestine are highly more reactive than glycoproteins from the mature villous zone. The possibility of a genetically determined deficiency of the growth-dependent N-acetyl-glucosaminyltransferase-1 as the pathogenic factor of the gluten-sensitive enteropathy is discussed.     

Lectin Analysis of Surface Saccharides in Two Trichomonas vaginalis Strains Differing in Pathogenicity*

SYNOPSIS. Surface saccharides in 2 Trichomonas vaginalis strains, the moderately pathogenic, JH34A, and the mild, JH162A, were analyzed with the aid of plant lectins. Concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), castor bean agglutinin (CBA), and lectin from the garden pea (GPA) were employed in agglutination tests and in treatment of ultrathin sections for electron microscopy according to the horseradish peroxidase-3,3′-diaminobenzidine method. With Con A and WGA, small quantitative differences were noted between the 2 strains in the results of agglutination and in the reaction-product deposits observed by electron microscopy. Distribution of the binding sites for the 2 lectins was also somewhat different in the JH34A and JH162A trichomonads. In general, the reactions with the more pathogenic strain were slightly stronger. Although the reactions with SBA and CBA lectins were weaker than those with Con A or WGA, they provided the means for qualitative differentiation between the 2 trichomonad strains. SBA alone agglutinated the JH34A strain and formed demonstrable deposits on the cell surfaces. On the other hand, only CBA reacted with JH162A flagellates. The garden pea lectin failed to bind to the surface of either strain. On the basis of results obtained with the control preparations incubated in the presence of specific inhibitors, it was concluded that both strains had α-methyl-D-mannoside and/or α-methyl-D-mannoside-like as well as N-acetyl-D-glucosamine residues on their surfaces. In addition, JH34A strain had D-lactose-containing residues while JH162A trichomonads had residues with D-galactose. Neither strain appeared to possess residues containing N-acetyl-D-galactosamine.     

Lectin-Induced Inhibition of Nerve Growth Factor Binding by Receptors of Sympathetic Ganglia

Abstract: Nerve growth factor (NGF) initiates a pleiotypic response in numerous tissues derived from the neural crest by binding to specific plasma membrane receptors. In sympathetic ganglia this receptor has been characterized as a highly asymmetric, minimally hydrophobic, intrinsic membrane protein with a molecular weight of 135,000 (Costrini et al., 1979b). To further characterize this moiety we assessed the effects of lectins on ¹²⁵I-NGF specific binding to preparations of particulate and nonionic detergent-extracted micro-somal receptors of rabbit superior cervical ganglia (SCG). Concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean agglutinin or Ulex europaeus I, induced a concentration-related, carbohydrate-specific decrease in ¹²⁵T-NGF binding. Following Con A exposure, ¹²⁵I-NGF specific binding to particulate SCG receptors was maximally reduced to 23% of control values. WGA similarly reduced NGF binding to particulate microsomal receptors to 37% of control values. Scatchard analysis of growth factor binding following Con A exposure indicated that this lectin effect was principally due to a sixfold reduction in maximum receptor affinity. Lectin-associated impairment of NGF binding was also demonstrated by using a Triton X-100 solubilized receptor preparation. These results provide evidence that the high-affinity-state NGF receptor of SCG is a glycoprotein containing N -acetylglucosamine and α-D-mannopyranoside residues. These residues are probably located in close proximity to the growth factor binding region of the NGF receptor.     

Single-step Purification by Lectin Affinity and Deglycosylation Analysis of Recombinant Human and Porcine Deoxyribonucleases I Expressed in COS-7 Cells

Human and porcine recombinant deoxyribonucleases I (DNases I) were expressed in COS-7 cells, and purified by a single-step procedure. Since affinities for concanavalin A (Con A) and wheatgerm agglutinin (WGA) were strong in these recombinant DNases I, purification using Con A–WGA mixture-agarose column was performed. By this method, the enzymes in culture medium could quickly be isolated to apparent homogeneity in approx. 10 min. From 1 ml of culture medium, about 20–30 μg of purified DNase I with a specific activity ranging from 22000 to 41000 units/mg were obtained. The purified DNases I were subjected to enzymatic deglycosylation by either peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). The recombinant enzyme was cleaved by PNGase F, but not by Endo H, indicating that the recombinant enzymes are modified by N-linked complex-type carbohydrate moieties. In the human recombinant DNase I, activity was decreased by PNGase F-treatment, while that of the porcine DNase I remained unaffected. The thermal stability of the human enzyme was extremely susceptible to heat following PNGase F-treatment, as was the porcine enzyme to a lesser extent. This study suggests that N-linked complex-type carbohydrate moieties may contribute to the enzymatic activity and/or thermal stability of recombinant DNases I.     

Analysis of glycoproteins in human serum by means of glycospecific magnetic bead separation and LC-MALDI-TOF/TOF analysis with automated glycopeptide detection.

Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively.     

Lectin-induced alterations on the proliferation of three human prostatic cancer cell lines

Summary While lectins are known to influence the cell growth of several types of normal and neoplastic tissues, their roles in the case of prostatic cancer cells remain relatively unexplored. In the present work, we report thein vitro influence of five lectins, namely peanut (PNA), wheat germ (WGA), Concanavalin A (Con A),Griffonia simplicifolia (GSA-IA₄), andPhaseolus vulgaris (PHA-L) agglutinins, on the cell proliferation of one androgen-sensitive (LNCaP) and two androgen-insensitive (PC-3 and DU 145) human prostatic cancer cell lines cultured in either 10% or 1% fetal bovine serum (FBS)-supplemented media. The cell proliferation was assessed by means of the colorimetric 3-(4,5-dimethythiazol-2-yle)2,5-diphenyltetrazolium bromide. (MTT) assay. Four lectin concentrations were tested (i.e., 0.1, 1, 10, and 100 μg/ml) at five experimental states (i.e., 2, 3, 5, 7, and 9 d following the addition of each lectin to the culture media). Our results demonstrated that the five lectins under study had a globally significant dose-dependent toxic effect on prostatic cancer cell proliferation. Nevertheless, low doses of GSA-IA₄ and PHA-L significantly (P<0.05 toP<0.001) increased the cell proliferation of confluent PC-3 cells. Increasing the FBS from 1% to 10% in the culture media significantly antagonized lectin-induced toxicity in the three prostatic cell lines. In conclusion, the present data strongly suggest that some lectins might influence the proliferation of prostatic carcinoma cells. In addition, because lectins are present in our diet, and are able to pass into the systemic circulation and thus reach the prostate, the present results suggest that some lectins might exert an influence on prostate cancer growth under clinical conditions.     

Crystallization and preliminary X-ray diffraction studies of soybean agglutinin

Soybean agglutinin crystallizes in the monoclinic space group C2 with unit cell dimensions $\text{a = 118.6}\text{A}\text{?}\text{, b = 88.9}\text{A}\text{?}\text{, c = 165.9}\text{A}\text{?}\text{, β = 103.0 °}$ and one tetramer of 120,000 M_r per asymmetric unit. The crystals are suitable for high-resolution work.     

Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni

Summary Using the label-fracture technique, an ultrastructural comparison was made of the number and distribution of wheat germ agglutinin (WGA)-binding sites between human normal and sickle red blood cells. The WGA was adsorbed to colloidal gold, and quantitative analysis of the electron micrographs revealed that more binding sites were present on the sickle erythrocytes than on the normal erythrocytes. Moreover, the sites were more clustered on the sickle red cells than on the normal red cells. Use of another lectin, Bandieraea simplicifolia-II, revealed that it did not bind to normal or sickle red cells. Because of the affinity of the WGA for sialic acid residues, it is probable that the WGA is binding to a transmembrane sialoglycoprotein, glycophorin A. The conformation and/or distribution of the glycophorin A molecules may be altered by the sickle hemoglobin that binds to the red cell membrane. Hence, as detected by WGA, new surface receptors, which could play a role in the adhesion of sickle cells to endothelium may be exposed.     

Measurement of the lateral mobility of cell surface components in single living cells by fluorescence recovery after photobleaching

The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin-receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGA-receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 × 10^?11 to 2 × 10^?10 cm²/sec. Both the percentage of mobile WGA-receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-receptor (Con A-receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.     

Fasciola hepatica: tegumental alterations as a consequence of lectin binding

Adult flukes, Fasciola hepatica, incubated in Hedon - Fleig saline containing concanavalin A (Con A) for 10 and 45 min, respectively, exhibited severe alterations to tegumental morphology involving increased secretory activity, blebbing of the apical plasma membrane, increased total surface area, and swelling of the basal infolds . The effects of Con A were prevented by the addition of alpha-methyl-D-mannoside to the incubating medium. Similar, but less pronounced, effects were caused by wheat germ agglutinin (WGA) binding. Con A and WGA binding indicate the presence of mannose, glucosamine, or glucose moieties and of N-acetylglucosamine. The effects of lectin binding were similar to the early effects of antibody attachment, and it was considered that accelerated membrane turnover was occurring in both cases. Swelling of the basal infolds was thought to be a result of increased apical surface membrane and/or increased permeability due to lectin binding.     

Studies on the agglutinating activity of pancreatic extracts and its relevance to function

Summary Agglutination of washed rabbit erythrocytes caused by pancreatic acinar cell extract was inhibited by glucose, maltose and cellobiose. Process of elimination and purification divulged that the acinar cell enzyme -amylase was responsible for attributing the agglutinin activity. Assay of enzyme and agglutinin activity data of different fractions of re-purified -amylase eluted from HPLC column showed that both the activity resides in the same fraction which suggests that the enzyme binds to the glucosyl residues of the rabbit erythrocytes via the carbohydrate binding/catalytic sites. Similar properties of other glycosidases were also noted.