Concanavalin A is synthesized as a glycoprotein precursor

Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (M_r) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with M_r 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-M_r polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 M_r. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [³H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, -mannosidase or -N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (M_r=30000). The 4000 decrease in M_r is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.Abbreviations Con A concanavalin A - Glc glucose - GlcNAc N-acetylglucosamine - IgG immunoglobulin G - Man mannose - M_r relative molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Localization of wheat-germ agglutinin in developing wheat embryos and those cultured in abscisic acid

The time course of appearance of wheat-germ agglutinin (WGA) in the various embryonic tissues during embryogenesis in Triticum aestivum L. was studied by sensitive immunofluorescence and peroxidase-antiperoxidase detection systems. The radicle, root cap and coleorhiza first accumulated WGA in early Stage II (8-10 d post-anthesis) prior to the main period of embryo growth, while WGA was found in the epiblast and coleoptile in early and late State III, respectively. Stage III is characterized by maximum embryo growth, followed by desiccation which occurs in Stage IV. When Stage-II embryos were precociously germinated in the absence of abscisic acid (ABA) no WGA was detected in the coleoptile and epiblast of the young seedlings. In the presence of ABA, Stage-II embryos did not germinate but WGA precociously accumulated in the coleoptile and epiblast. The levels and distribution of WGA in the resulting embryo resembled those in a fully mature, dry embryo (Stage V). Barley possesses a seed lectin similar to WGA, but it is never detected in coleoptiles. Some but not all of the barley cultivars tested were found to accumulate lectin in this organ of mature embryos when treated with ABA. Thus, ABA appears to be involved in the highly regulated temporal and spatial expression of WGA during embryogenesis in cereals.Abbreviations ABA abscisic acid - DIC differential interference contrast - PAP peroxidase-antiperoxidase - WGA wheat-germ agglutinin     

Characterization of a wheat germ agglutinin-like lectin from adult wheat plants

Radioimmuno-and enzyme-linked immunosorbent assays show that a substantial amount of wheat germ agglutinin(WGA)-like protein is present at the base of the shoot and in the roots of adult wheat (Triticum aestivum L.) plants. The protein can be purified by hapten-and antibody-mediated affinity procedures. It forms an arc of identity with the embryo lectin upon Ouchterlony double-diffusion and is an active lectin that agglutinates trypsinized erythrocytes in an N-acetylglucosamine-and chitin-inhibitable manner. Reduced and carboxyamidated protein comigrates with the 18-kdalton subunits of embryo lectin on sodium dodecyl sulfate-polyacrylamide gels. Invivo labeling of 9-d-old, hydroponically grown plants with ³⁵S-labeled sulfate demonstrates that at least some of the WGA-like protein is synthesized de novo. Immunocytochemistry with rabbit anti-WGA and colloidal-gold-conjugated second antibody shows that cross-reactive protein is present at the tips of new adventitious roots. In reactive cells, the lectin is localized near the inner surface of the vacuole membrane. Wheat plants contain up to 100 ng of WGA-like protein after the first week of growth, but the level fluctuates thereafter. Since most of the lectin is present at the base of the shoot and much less is found in older roots, these fluctuations may be the consequence of changes in the initiation of new advantitious roots.Abbreviations ELISA enzyme-linked immunosorbent assay - GlcNAc N-acetylglucosamine - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat germ agglutinin     

Transport and processing of the glycosylated precursor of Concanavalin A in jack-bean

Concanavalin A (ConA) is a tetrameric lectin which is synthesized in the developing cotyledons of jack bean (Canavalia ensiformis L.) as a glycosylated precursor, pro-concanavalin A (pro-ConA). The processing of pro-ConA involves the excision of a small glycopeptide from the center of the pro-ConA molecule, and the ligation of the two polypeptides. In this paper, we show that pro-ConA is associated with the endoplasmic reticulum/Golgi fraction of the cells, and that the processing of pro-ConA occurs in the protein bodies. Processing is a complex process and different intermediate-sized polypeptides appear at different times during cotyledon development. The ConA-related polypeptides which accumulate during seed development may be the products of alternate processing events or breakdown products of ConA, rather than precursors of ConA. When glycosylation is prevented by tunicamycin, there is very little transport of pro-ConA out of the endoplasmic reticulum/Golgi system to the protein bodies; the unglycosylated pro-ConA which is transported is slowly processed. Tunicamycin does not prevent the transport of canavalin (a protein which is not glycosylated) or the transport and processing of the small amounts of glycosylated pro-ConA synthesized in the presence of the drug. This is, to our knowledge, the first demonstration that the transport of a glycoprotein in plant cells is dependent on the presence of the glycan.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - GlcN glucosamine - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis supported by a grant from NATO     

Patatin,a major soluble protein of the potato (Solanum tuberosum L.) tuber is synthesized as a larger precursor

Antibodies were raised against the highly purified glycoprotein patatin. They were used to characterize the product synthesized in a wheatgerm cell-free translation system, programmed with polyadenylated RNA from potato tubers. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that the immunoprecipitated protein had a molecular mass of 43 kDa compared to 40 for the authentic patatin. It is assumed that patatin is synthesized in vivo as a larger precursor which is processed to the mature protein by cleavage of a signal peptide. Our results are in agreement with sequence-analysis data of patatin complementary DNA which indicate a signal peptide of about 23 amino acids (Mignery et al., 1984; Nucleic Acids Res. 12, 7987–8000).Abbreviation Poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Preliminary results were published in Mitteilungsband, Botaniker Tagung in Wien, p. 180, Wien, September 1984     

Appearance of wheat-germ agglutinin during maturation of field-grown wheat

Field-grown wheat (Triticum aestivum L.) has been used as a developmental system to study the appearance of wheat-germ agglutinin during grain maturation. The lectin appears at the mid-grain growth period (30–34 days post-anthesis) and continues to be synthesised throughout the late stages of maturation and desiccation. An acidic endopeptidase activity, inhibited by pepstatin-phenanthroline is present in extracts of embryo and endosperm throughout maturation. After in-vivo labelling of immature embryos with [³⁵S]methionine for 3 h and extraction in the presence of proteinase inhibitors, immunoprecipitates with anti-wheat-germ agglutinin were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, and found to contain three ³⁵S-labelled polypeptides of M_r 46000, 18000 and 13000. Comparison of two-dimensional tryptic maps of ¹²⁵I-labelled peptides indicate the three polypeptides are closely related.Abbreviations dpa days post-anthesis - PBS phosphate-buffered saline - RIA radioimmunoassay - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

Wheat germ agglutinin uptake by cytotoxic lymphocytes,a quantitative model of receptor-mediated endocytosis

Summary The binding and uptake of fluorescence labeled wheat germ agglutinin into cytotoxic T-cells was measured by single cell cytophotometric analysis. The intensity of fluorescence in these cells increased continuously over 24 hrs, indicating a permanent turnover of the ligands for WGA. Although the labeling of the cells was intense, no change in the proliferation rate of this interleukin-2 dependent cell line was observed. Therefore no interaction between the interleukin-2 receptor and other receptors regulating the cellular proliferation with the lectin is likely.Abbreviations au arbitary units - CTLL-1 murine cytotoxic interleukin-2 dependent cell line - FCS fetal calf serum - FITC fluoresceinisothiocyanate - HEPES hydroxyethylpiperazine-ethanesulfonic acid - MHC major histocompatibility complex - WGA wheat germ agglutinin     

Sambucus nigra agglutinin is located in protein bodies in the phloem parenchyma of the bark

The bark of some young woody stems contains storage proteins which are subject to an annual rhythm: they accumulate in the autumn and are mobilized in the spring. We show here that the bark phoem-parenchyma cells of Sambucus nigra L. contain numerous protein bodies, and that the bark lectin (S. nigra agglutinin) which undergoes an annual rhythm is localized in these protein bodies. The protein bodies in the cotyledons of legume seeds also contain lectin, indicating that lectins may be storage compounds themselves or may have a function in storage and-or mobilization processes.Abbreviations PBS phosphate-buffered saline - IgG immunoglobulin - SNA Sambucus nigra agglutinin     

Dynamics of the changes of electrophysical properties of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 cells at their binding with wheat germ agglutinin

The electrooptical properties of Azospirillum brasilense Sp7 cell suspensions, have been studied at a specific interaction with wheat germ agglutinin (WGA), using the dependences between the changes of optical densities of cell suspensions at the electric orientation of cells and the orienting field frequencies of 740, 1000, 1450, 2000, and 2800 kHz. It was shown that the electrooptical (EO) properties of cell suspensions changed at the interaction of A. brasilense Sp7 cells with WGA and that the EO signal value changed irrespective of the cultivation conditions. At the same time, the dynamics of the changes of the EO properties of microbial suspensions was different for microbial cells grown under different conditions. It may be evidence of the differences in the cell surface properties of microbial cells, and of the dependence, between bacterial response to lectin and growth conditions. The possibility of using the EO analysis of bacterial suspensions for the study of the high-specific binding of polypeptide molecular signals with the bacterial target cells and for assessment of the dynamics of this process has been demonstrated.     

Wheat germ agglutinin (WGA) gene expression and ABA accumulation in the developing embryos of wheat (Triticum aestivum) in response to drought

Expression of wheat germ agglutinin (WGA) gene inthe developing embryos of wheat (Triticumaestivum L. cv. C-306) was studied in relation toabscisic acid (ABA) accumulation under water stressconditions. Imposition of water stress resulted inelevated ABA levels in the embryos at threedevelopmental stages (18, 24 and 30 DPA). On thecontrary, the effect of drought stress on WGAaccumulation was stage dependent with significantincrease in WGA content being observed at only 24 DPA. Our results suggest that apart from ABA, otherfactors which are temporally expressed, are alsoinvolved in regulation of WGA gene expression.     

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.     

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.     

Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos

Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.Abbreviations db-cAMP dibutyryl-cAMP - FTA flagellar tip activation - Mab monoclonal antibody - mt ^–/mt⁺ mating-type minus/plus - WGA wheat-germ agglutinin We gratefully acknowledge the fruitful discussions with Dr. Rainer Gilles of the Department of Biochemistry at the University of Cologne (FRG), and the advice generously given by Dr. Roel van Driel of the Department of Biochemistry at the University of Amsterdam (The Netherlands).     

Subunit unbinding mechanics of dimeric wheat germ agglutinin (WGA) studied by atomic force microscopy

Wheat germ agglutinin (WGA) is an oligomeric lectin widely used as a model of sugar moieties in biochemistry. Subunit association is important for the crosslinking function of WGA, so we used atomic force microscopy to measure the subunit unbinding force of dimeric WGA. We found that the average unbinding force of dimeric WGA is ∼55 pN at ∼1 nN/s loading rate, whereas this unbinding force is increased at least up to 100 pN when WGA is bound to glycophorin A. Moreover, the dissociation rate constant of WGA was calculated to be 1–2 × 10⁻² s⁻¹, suggesting that dimer dissociation is relatively fast.     

Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process

Summary. Paramecium bursaria cells harbor several hundred symbiotic algae in their cytoplasm. Algae-free cells can be reinfected with algae isolated from algae-bearing cells or cultivated Chlorella species through the digestive vacuoles. To determine the relationship between the infectivity of various Chlorella species and the nature of their cell wall components, algae-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella species and observed for the establishment of endosymbiosis at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana C-212 and C. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, C. kessleri C-208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp. strain C-201 derived from Spongilla fluviatilis could not be maintained. These infection-incapable strains could escape from the host digestive vacuole but failed to localize beneath the host cell membrane and were eventually digested. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin, GS-II, or concanavalin A, with or without pretreatment with 0.4 N NaOH, showed no relationship between their infectivity and the stainability with these lectins. Our results indicate that the infectivity of Chlorella species for P. bursaria is not based on the sugar residues on their cell wall and on the alkali-insoluble part of the cell wall components, but on their ability to localize just beneath the host cell membrane after escaping from the host digestive vacuole. Correspondence and reprints: Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Wheat lectin as a factor in plant-microbial communication and a stress response protein

Wheat lectin (wheat germ agglutinin, WGA), a representative of a broad group of cereal lectins, is excreted by plant roots into the surrounding medium and interacts with both pathogenic microflora and growth-stimulating rhizobacteria. WGA was found to serve as a molecular signal for the rhizobacterium Azospirillum brasilense, which forms endophytic and associative symbioses with wheat plants. The bacterial response to the lectin was pleiotropic: WGA at concentrations from 10^?10 to 10^?6 M exerted a dose-dependent effect on a range of processes in the bacterium that are important for the establishment and functioning of symbiosis. Plants with different WGA content differed in their responses to severe nitrogen starvation and to seed treatment with Azospirillum.     

Isolation and characterization of glycoprotein lectins from the bark of three species of elder,Sambucus ebulus,S. nigra and S. racemosa

Lectins have been isolated from the bark of three members of the family Caprifoliaceae, Sambucus nigra (elder), S. racemosa (red-berried elder) and S. ebulus (dwarf elder), by affinity chromatography on fetuin-agarose, ion-exchange and gel-filtration chromatography. They are all glycoproteins of M _r 140 000 made up of at least four subunits. The lectin have similar but not identical amino-acid compositions and the carbohydrate content varies between 12% and 19% (w/w), the main sugars being (N-acetyl)glucosamine, mannose, fucose and xylose. Inhibition studies of hemagglutination with various mono- and oligosaccharides have shown that N-acetylgalactosamine and galactose together with galactose-containing oligosaccharides are the most effective inhibitors. There are some differences in specificity, in particular S. ebulus agglutinin is inhibited to the same degree by galactosamine, N-acetylgalactosamine and by galactose.Abbreviations PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SEA S. ebulus agglutinin - SNA S. nigra agglutinin - SRA S. racemosa agglutinin     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.