Con A-binding Proteins Research Articles

A single glycoprotein from Puccinia graminis f. sp. tritici cell walls elicits the hypersensitive lignification response in wheat

Con A-binding glycoproteins were detected in germ tube walls of Puccinia graminis f. sp. tritici and appeared to be potent inducers of the hypersensitive lignification response. This response, which is typical of the large resistance reaction in wheat leaves, was preceded by a increase in extractable phenylalanine ammonia-lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway. Germ tube glycoproteins were isolated from homogenized fungal cell walls by differential centrifugation and affinity chromatography on a Con A-Sepharose column. The most active glycoprotein was purified on a Mono Q anion-exchange column. The relative molecular mass of the molecule was determined in SDS-polyacrylamide gels to be 67 kD. The carbohydrate portion consists mainly of mannose (50%) and galactose (47%) and contains the active part of the glycoprotein as demonstrated by the inability of pronase and trypsin digestion to influence activity. An elicitor-active glycoprotein with identical molecular mass and Con A-binding properties was isolated from intercellular fluids of rust-infected plants of the susceptible wheat cultivar Little Club indicating that the elicitor is released from fungal cell walls during infection. We discuss the role of Con A-binding proteins as inducers of non-host resistance in cereals.

The major concanavalin A-binding surface glycoprotein of Leishmania donovani chagasi promastigotes is involved in attachment to human macrophages.

Leishmania donovani, the protozoan causing visceral leishmaniasis, is an obligate intracellular parasite of mammalian macrophages. Considerable evidence has suggested that the ingestion of L. donovani promastigotes by macrophages occurs via receptors on the surface of the phagocyte. During this study, a glycoconjugate that may be involved in the receptor-mediated ingestion of L. donovani chagasi promastigotes was isolated from the parasite membrane. Octyl glucoside-soluble extracts of promastigote membranes contained a predominant doublet migrating at 60 kDa, seen by SDS-PAGE. The 60-kDa molecule was the major externally disposed promastigote surface protein labeled by 125I, and it was the major Con A-binding protein on L. donovani chagasi, as determined by Con A binding to parasite proteins transferred to nitrocellulose. Attachment of promastigotes to human monocyte-derived macrophages was inhibited by varying concentrations of the membrane extract containing both proteins, and adsorption of extracts on Con A-Sepharose resulted in both removal of the 60,000 Mr glycoprotein and loss of the ability of extracts to inhibit promastigote attachment to human macrophages. After further purification of the 60-kDa glycoprotein by gel filtration, its inhibitory activity increased 45-fold over the unpurified membrane extract. Examination of Con A blots of stationary phase promastigotes isolated from an infected hamster revealed a marked loss in the major Con A-binding glycoprotein over 4 mo in in vitro culture after isolation from the rodent host, corresponding to a loss in infectivity of the promastigotes for hamsters. The results suggest that the major Con A-binding surface glycoprotein from L. donovani chagasi promastigotes is important in attachment to human macrophages, and may be a factor in parasite virulence for a mammalian host.

Concanavalin A receptor ‘tipping’ in Tetrahymena and its relationship to cell adhesion during conjugation

Abstract Shortly after mixing cells of complementary mating types of Tetrahymena, the cells develop the ability to pair, a process inhibited by ConA, and the region joining the cells becomes ringed with ConA receptors. This study examines the arrival of ConA receptors at the conjugation junction by looking at cells in the period between mixing and pairing. By brief incubations with F-ConA at intervals after mixing, it was ascertained that some cells had fluorescent tips as early as 15 min. A kinetic analysis revealed that ‘tipping’ occurs in a manner that appears to be related to subsequent cell pairing. Cytoskeletal frameworks (CFs) were isolated under conditions in which ConA receptors remain attached. Western blot analysis of these structures revealed four major and several minor ConA-binding proteins. However, between mixing and the establishment of over 80 % paired cells, changes occurring in the banding pattern were slight. This indicates that new populations of ConA receptors are not produced to any great extent after mixing. Head-on examination of CFs showed that it was possible to monitor simultaneously the process of tip transformation (widening of the nonciliated area of the tip) and ConA-receptor localization. ConA receptors originate posterior to the tip, begin to occupy the surface of the tip in clusters as the tip widens and eventually coat the transformed tip. Finally, as cells join pairs, the receptors relocate to a ring around the conjugation junction. These data suggest that ConA receptors accumulate at the anterior tips and then concentrate at the edge of the junction. This could provide a mechanism for controlling cell-cell adhesion.

High-performance affinity chromatography of human serum concanavalin a binding proteins

A high-performance concanavalin A (Con A) affinity column Gelpack GL-L55C (Hitachi Kasei Industries) was successfully used for the fractionation of human serum Con A-binding proteins. Serum proteins that have strong affinity to Con A (ca. 11% of the recovered proteins) could be fractionated within 80 min. By analysing the eluates from the column by micro two-dimensional electrophoresis, followed by blotting and Con A staining, the specificity of the column was effectively visualized. Although the protein-binding capacity of the column gradually decreased during repeated loading of serum or tissue extracts, the specificity of the column to Con A-binding proteins did not change. Serum lipoproteins have been eluted from the column with 6 M urea, suggesting that the capacity decrease is caused by the binding of lipids or lipoproteins to the column.

Infection-related proteins in intercellular washing fluids from stem rust-affected wheat leaves: race-associated protein differences revealed by PAGE and Con A blotting

Primary leaves of wheat were infiltrated with buffer and subjected to gentle centrifugation to yield intercellular washing fluid (IWF). Proteins in these fluids were separated by isoelectric focusing-polyacrylamide gel electrophoresis, stained with Coomassie brilliant blue, or first blotted onto nitrocellulose membranes and then probed for concanavalin A (Con A) binding. β- d-Xylosidase isozymes endogenous in IWF served as internal standards for comparison of Con A-binding proteins between samples. Intercellular washing fluid from leaves infected with each of four races of the stem rust fungus contained infection-related proteins. Many of these, including several of the most prominent Con A-binding proteins, differed between the four samples either in amount or in relative mobility. To confirm that these differences were race-associated, leaves were infected with five isolates of one race and similarly analysed. The five isolates had been collected in different years and in widely separated geographic areas to emphasize inter-isolate differences, if they exist. Leaves infected with each of the five isolates yielded indistinguishable patterns of Con A-binding IWF proteins.

A-CAM: a 135-kD receptor of intercellular adherens junctions. I. Immunoelectron microscopic localization and biochemical studies.

The recently described adherens junction-specific 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:2249-2260) was localized along cardiac muscle intercalated discs by immunogold labeling of ultrathin frozen sections. Analysis of this labeling indicated that the 135-kD protein, adherens junction-specific cell adhesion molecule (A-CAM), is tightly associated with the plasma membrane unlike vinculin labeling, which was present along the membrane-bound plaques of the fascia adherens. In cultured chick lens cells, A-CAM was associated with Ca2+-dependent junctions that were cleaved upon a decrease of extracellular Ca2+ concentrations to less than or equal to 0.5 mM. In the chelator-separated junction, A-CAM became exposed to exogenously added antibodies or to proteolytic enzymes. Upon addition of trypsin to EGTA-treated cells, A-CAM was cleaved into three major cell-bound antigenic peptides with apparent molecular masses of 78, 60, and 46 kD, suggesting that the extracellular domain of A-CAM has a size greater than or equal to kD. Incubation of electrophoretic gels with 125I-concanavalin A (Con A) indicated that one of the major Con A-binding proteins in chicken lens membranes is a integral of 135-kD glycoprotein that was partially purified on Con A-Sepharose column and identified as A-CAM by immunoblotting. Detergent partitioning assay using Triton X-114 biphasic system was carried out to determine whether A-CAM displays properties of an integral membrane protein. This assay indicated that the intact A-CAM molecule was recovered in the buffer phase but its cell-associated tryptic peptides, which presumably lost a great part of the A-CAM extracellular extension, readily partitioned into the detergent phase. The results obtained in this and in the following paper (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:1451-1464) strongly suggest that A-CAM is a Ca2+-dependent adherens junction-specific membrane glycoprotein that is involved in intercellular adhesion in these sites.

Concanavalin A and wheat germ agglutinin binding to sea urchin embryo basal laminae.

The basal laminae and inner extracellular matrices of Lytechinus pictus and Arbacia punctulata embryos were characterized on the basis of lectin binding. Developmental stage specific patterns of lectin binding were observed after microinjection of Con A-FITC and WGA-FITC. Lectin-specific patterns differed between control, sulfate free sea water (SFSW) and tunicamycin treated embryos. Con A injection resulted in the rounding-up of cells in the epithelium and was most pronounced in embryos cultured in the presence of tunicamycin. Basal laminae were isolated by Triton X-100 extraction of whole embryos. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose and incubated in biotinylated lectins. Lectin-binding glycoproteins were detected with avidin peroxidase. The electrophoretic pattern of Con A-binding proteins in early developmental stages of Arbacia was similar with several low molecular weight species appearing at gastrulation in control and SFSW embryos. WGA-binding in Arbacia and Lytechinus control embryos was limited to a 125,000 Mr glycoprotein (gp125). In addition to gp125, several high molecular weight WGA-binding glycoproteins were also detected in SFSW embryos. The evidence suggests that mesenchyme migration and gastrulation are correlated with changes in the molecular composition of the ECM.

Binding of the adhesive protein complex (LFA-1/Mac-1/p150,95) to concanavalin A.

At least 30 proteins from human PMNL plasma membranes capable of binding concanavalin A (Con A), can be identified after surface labeling with 125I and subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitation of the labeled proteins after solubilization in non-ionic detergents, with monoclonal antibodies (MAbs) directed against a family of leukocyte membrane proteins (LFA-1, Mac-1, p150,95, and the beta subunit these glycoproteins share), indicated that Mac-1 and p150,95 were bound by Con A. Dissociation of the alpha and beta subunits with sodium dodecyl sulfate, electrophoresis, transfer to nitrocellulose paper, and subsequent binding of these proteins by Con A demonstrated Con A retention by Mac-1-alpha, p150,95-alpha, and the common beta subunit. Affinity of Con A for LFA-1-alpha from human peripheral blood PMNL could not be confirmed by direct binding or electroblotting. Similar experiments in a patient deficient in LFA-1, Mac-1, p150,95, and the beta subunit confirmed that Mac-1-alpha and the beta subunit were important Con A-binding proteins.

The expression of concanavalin a binding glycoproteins during the development of cerebellar granule neurons in vitro

We present data on the expression of Concanavalin A (ConA) binding glycoproteins by granule cell enriched cultures derived from 8 day postnatal rat cerebellum. Time course studies were conducted over a 12 day culture period. ConA binding glycoproteins were localized on the cell bodies and fibres of the granule neurons using fluorescence microscopy. The fluorescence intensity increased between 4 and 12 days in vitro. Quantitative studies on the capacity of live cells to bind 125I-iodinated ConA showed that there was a significant increase in the amount of lectin bound between 4 and 8 days in vitro. However, in contrast to previous results on the developing cerebellum in vivo [Zanetta et al. (1978) Brain Res.142, 301–319.], there was no decline in binding capacity after 8 days in culture. Glycoproteins expressed by these cells were analysed by staining SDS polyacrylamide gels with [125I]ConA. A large number of lectin binding proteins were observed which spread over a wide range of molecular weights. Only minor changes were detected in the profile of [125I]ConA binding glycoproteins with the maturation of the cells in culture. The comparison of the findings on granule cells developing in culture and in vivo suggested that an interaction between granule cell axons and their normal target neurons is involved in the regulation of the ConA binding protein content in the cerebellum.

Increased rate of capping of concanavalin a receptors during early Xenopus development is related to changes in protein and lipid mobility

The mobility characteristics of plasma membrane constituents were studied in dissociated cells from embryos of Xenopus laevis at various stages of development from early blastula until neurulation. An increased rate of fluorescein isothiocyanate-concanavalin A induced patching and capping of Con A-binding proteins during this period of development was correlated with a threefold increase in the lateral mobility of the receptor molecules, as determined by the fluorescent photobleaching recovery (FPR) method, the major change occurring at the onset of gastrulation. Using the same method, it was demonstrated that the lateral mobility of plasma membrane lipids increases twofold during this period of development. The major change being detectable, however, at the late blastula stage. This is in coincidence with the initiation of cell motility in dissociated Xenopus embryo cells. It is concluded that the lateral mobility of membrane proteins and lipids increases significantly during early Xenopus development, but are at least in part subject to different control mechanisms. The results suggest that the initiation of morphogenetic movements is related to changes in the dynamic properties of plasma membrane constituents.

Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells.

Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.

Isolation and major components of core structure of postsynaptic density

The core structure of postsynaptic density (PSD-core) was prepared from rat cerebral synaptosomes by application of the isolation procedure of synaptic junctions (SJ) after trypsinization, which dissociated pre- and post-synaptic structures. The PSD-core was considered to consist mainly of cytoplasmic part of postsynaptic structure, and lack the proteins localized on the external surface of the synaptic plasma membrane, such as receptors for neurotransmitters, Con A-binding proteins and connecting molecule(s) between pre- and post-synaptic structures. The PSD-core proteins which increased greatly in their contents compared with those of SJ prepared from synaptosomes (Syn-SJ) were 120 k Mr Con A-binding protein (Con A-BP) and 30 k Mr protein. Electron microscopic histochemistry suggested that 120 k Con A-BP localized widely in the main structure of the PSD-core. Protein of 30 k Mr was not extracted from PSD-core with 6 M urea, whereas actin, major PSD protein, and tubulin were easily extractable. The 30 k Mr protein was the most resistant one to trypsinization in the SJ fraction. The results suggest that the 30 k Mr protein plays an important role in stabilization and integrity of the postsynaptic density.

Characterization of rabbit neutrophil membrane proteins. A 140K major membrane protein is the predominant Con A-binding protein.

The major plasma membrane proteins of rabbit neutrophils were characterized by SDS-PAGE, surface iodination, 125I-concanavalin A binding, and detergent extraction. Neutrophil membranes were prepared which lacked significant intracellular contamination with good retention of protease-sensitive proteins. The major protein and predominant Con A-binding protein was as surface exposed, 140,000 D (gp 140) protein which was solubilized by nonionic detergents but not low ionic strength. Actin and myosin but not other cytosol proteins were prominently associated with the isolated membrane particularly in a Triton-insoluble form. Membranes were also prepared from surface-iodinated neutrophils previously stimulated with a chemotactic peptide or degranulated. The granule membrane enzyme alkaline phosphatase was incorporated into the plasma membrane fraction of degranulated neutrophils. However, the membrane proteins in the different membrane preparations were identical on SDS-PAGE and autoradiography. Therefore, using these techniques, no major alterations in protein composition of the plasma membrane could be detected following stimulation or degranulation of rabbit neutrophils.

An analysis of protein synthesis, membrane proteins, and concanavalin A-binding proteins during conjugation in Tetrahymena thermophila

Conjugation in the ciliate Tetrahymena thermophila has been used as a system in which to analyze biochemical events associated with the execution of a complex cell-cell interaction. Two-dimensional electrophoretic analysis of [ 35S]methionine-labeled whole-cell proteins revealed major changes in protein synthesis correlated with costimulation and the onset of pairing; specifically, the major induced polypeptide was one of 80 kDa. A second change in the pattern of protein synthesis was associated with the onset of meiosis; the major induced product was another, perhaps related, 80-kDa polypeptide. An effort was made to detect changes in the patterns of membrane proteins and Con A-binding proteins during conjugation; no changes were found. These results are discussed in the context of earlier hypotheses regarding the distribution of Con A receptors on the surfaces of conjugating cells.

Characterisation of concanavalin A-binding glycoproteins from mouse splenic leukocytes by two-dimensional electrophoresis: Preferential binding of incompletely glycosylated forms of H-2 antigen to the lectin

Concanavalin A (Con A)§-binding proteins from mouse spleen leukocytes were characterised by two-dimensional electrophoresis of material precipitated, by Con A plus anti-Con A, from lysates of biosynthetically-labelled cells. Although most cell surface (iodinatable) proteins are known to bind Con A. some of the major Con A-binding proteins detected by immunoprecipitation. after a four-hour biosynthetic labelling period, are not iodinatable and arc probably intracellular. Thus the major biosynthetically labelled Con A-binding species are: (i) a non-iodinatable, high molecular weight glycoprotein (C-145); (ii) intracellular precursors of secretory immunoglobulins (IgM and, probably, IgAl; (iii) immature (not fully-sialylated) forms of H-2 D and K antigens; and (iv) Ia antigens, In the case of the H-2 antigens, (and possibly of other cell surface proteins) the selection of immature forms by Con A is not due to lack of biosynthetic labelling of mature products, but to preferential binding of Con A to incompletely glycosylated molecules.

Evidence for the association of two cell surface glycoproteins of 13762 mammary ascites tumor cells: Concanavalin A-induced redistribution of peanut agglutinin-binding proteins

The behavior of the cell surface concanavalin A (conA) receptors and of peanut agglutinin (PNA) receptors on the MAT-B1 ascites subline of the 13762 rat mammary adenocarcinoma was examined using fluorescein-labeled conA and PNA. ASGP-1, the major glucosamine-containing glycoprotein of these ascites cells, is the only PNA-binding protein observed by dodecyl sulfate electrophoresis. ASGP-2, the second most prominent component after glucosamine labeling, is the most abundant conA-binding protein. These two glycoproteins were previously shown to be associated as a complex in detergent extracts of the cells [20]. ConA-binding proteins, upon incubation with fluorescein-labeled conA (FITC-conA), redistribute on the cell surface into small and large aggregates similar, but not identical, to those seen in ‘patching’ and ‘capping’ experiments with lymphocytes. PNA-binding proteins failed to redistribute during incubation with fluorescein-labeled PNA (FITC-PNA) and appeared in a diffusely stained pattern around the circumference of the cells. However, when cells were treated with unlabeled conA followed by FITC-PNA, or with FITC-PNA followed by unlabeled conA, there was marked redistribution of the FITC-PNA. These results indicate that ASGP-1 redistributes in response to the movement of conAbinding proteins and supports our hypothesis that ASGP-1 and ASGP-2 are associated on the plasma membrane at the cell surface as well as in detergent extracts.

Characterization of concanavalin A precipitated proteins from early mouse embryos: A 2-dimensional gel electrophoresis study

Concanavalin A (Con A), a plant lectin which binds to mannose-containing components, has been used to precipitate a specific class of proteins synthesized during preimplantation mouse embryogenesis. Total cellular protein was extracted from labeled embryos with Nonidet P40, reacted with Con A, and then exposed to rabbit antiserum directed against Con A. Immune complexes were precipitated with protein A from Staphylococcus aureus. After washing, Con A-bound proteins were eluted with mannoside which released between 3 and 10% of the total radioactivity from unfertilized and fertilized eggs, 2-cell and 8-to 16-cell embryos, and late blastocysts. In control experiments, in which mannoside was added prior to the Con A, only 0.1–0.5% of the total radioactivity was eluted, indicating little release of proteins nonspecifically bound to the protein A-anti-Con A-Con A complex. When these precipitates were examined by two-dimensional gel electrophoresis, approximately 30–70 peptides in the defined periods of synthesis were detected in autoradiograms. To precipitate cell surface proteins, intact embryos were incubated in Con A and anti-Con A prior to extraction with Nonidet P40. Less than 1.0% of the total radioactivity was eluted with mannoside. A complex of three pairs of peptides was present in 2-cell and 8- to 16-cell embryos. The qualitative changes in two-dimensional gel patterns of Con A-binding proteins has provided a program of synthesis occurring during normal development for a specific class of proteins.

Inhibition of conjugation in Tetrahymena pyriformis by concanavalin A

A group of glycoproteins which form an insoluble complex with concanavalin A (ConA) are secreted during starvation of the mating types strain, as well as by the non-mating types of Tetrahymena pyriformis. These glycoproteins were isolated by Sepharose-ConA, characterized, and their relevance to the conjugation process studied. The isolated ConA binding proteins (CBM) contain about 26% of their total weight, a phenol sulfuric acid-positive material, presumably carbohydrates, and exhibit about 5–8 major bands by gel electrophoresis analysis. The possibility that ConA inhibits the conjugation process by precipitating with this material was tested. Addition of isolated CBM restored conjugation previously inhibited by ConA. However, incubation of isolated CBM with either of the mating types or with both did not have any effect on the kinetics of the conjugation process. Antibodies prepared against CBM-secreted by both mating types did not prevent conjugation when added to the conjugation medium. The data suggest that CBM does not directly participate in the conjugation process. Inhibition of conjugation by ConA is probably due to its interaction with specific membrane-bound glycoproteins.