Serum i antigen: A new human blood-group glycoprotein

A glycoprotein has been found in normal serum which inhibits the hemagglutination caused by human anti-i cold agglutinins. This glycoprotein binds to a Sepharose anti-i affinity column at 4°C and can be quantitatively eluted at 37°C. The eluted glycoprotein is specific in that it has potent hemagglutination-inhibition activity against anti-i antibodies but does not inhibit the hemagglutination by anti-I, -A, -B, -H or by the lectins Con A, PHA or Vicia graminea. The anti-i affinity method should provide a simple procedure for large scale purification and subsequent characterization of the i-antigen.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

Triiodothyronine affects the phytohemagglutinin to concanavalin A proliferative response ratio in sex-linked dwarf chickens

Euthyroid Cornell K strain and sex-linked dwarf (SLD) strain cockerels (which have abnormally low serum triiodothyronine concentrations) were supplemented with either 0, 0.01, 0.1, or 1.0 ppm of triiodothyronine (T3) in the diet. Peripheral blood lymphocytes (PBL) from these cockerels were obtained by slow-speed centrifugation (slow-spin-prepared PBL). The proliferative response of these PBL to phytohemagglutinin (PHA) and concanavalin A (Con A) was determined when the chicks were 6, 9, and 12 weeks of age. Con A responsiveness was also determined in 12-week-old cockerels using PBL which were separated on Ficoll (Ficoll-prepared PBL). Using slow-spin-prepared PBL, PHA, and Con A responsiveness increased in both strains with increasing levels of T3 supplementation. This enhancing effect of T3 was particularly evident in older cockerels. In 6- and 12-week-old SLD strain cockerels, the PHA:Con A response ratio was significantly (P less than 0.05) lower than in K strain cockerels. At 12 weeks of age the PHA:Con A response ratio of the SLD strain was elevated to K strain control levels by T3 supplementation. Therefore, the lower PHA:Con A response ratio in the SLD strain appears to be partially due to the existing peripheral hypothyroidism in this strain. Using Ficoll-prepared PBL, the effects of T3 on Con A responsiveness differed from those observed when slow-spin-prepared PBL were used. From this study we conclude that T3 supplementation affects mitogen responsiveness and the PHA:Con A response ratio. However, the effects of T3 on mitogen responsiveness depend on the age of the chicken, the level of T3 supplemented, the T cell population stimulated, and the method of lymphocyte enrichment.     

Detection of a Concanavalin A binding protein in the mollicute Spiroplasma citri and purification from the plasma membrane

A protein that binds Concanavalin A (Con A) was detected on Western blots of Spiroplasma citri proteins. Its apparent molecular weight was 84000. It was localized in the plasma membrane. Affinity chromatography on Con A-agarose was used to isolate this protein. The glycosylation inhibitor, tunicamycin, inhibits S. citri growth and seems to block the glycosylation of the Con A-binding protein.     

Purification of the glycoprotein allergen Ag7 from mugwort pollen by concanavalin A affinity chromatography.

Two affinity columns comprising immobilized concanavalin A (Con A), Con A-Sepharose and Con A-XP3507, were evaluated for their purifying ability for the glycoprotein allergen Ag7 from a partially purified extract of mugwort pollen. The most pronounced difference between the two columns was the nature of their nonspecific interactions; hydrophobic interactions were dominant with Con A-XP3507, whereas ionic interactions were dominant with Con A-Sepharose. Both Con A-columns were effective for purifying Ag7 with a recovery of 50% after specific elution with displacing sugars. The inclusion of 1.0 M NaCl and 20% ethylene glycol in the elution medium was useful for desorbing nonspecifically bound material, prior to specific elution of adsorbed Ag7 in the presence of the displacing sugars, alpha-methyl glucoside and alpha-methyl mannoside. The most efficient purification of Ag7 was achieved with Con A-Sepharose at room temperature rather than at 4 degrees C. Affinity chromatography with Con A-XP3507 resulted in a slightly more contaminated product (purity 54%) than with Con A-Sepharose (purity 64%).     

Interactions of lyophilised and rehydrated mouse spleen lymphocytes with phytohaemagglutinin (PHA) and concanavalin A (Con A)

Rehydrated lymphocytes freeze-dried in the presence of polyvinyl-pyrrolidone (PVP) and foetal calf serum (FCS) have been shown to retain plaque forming activity (5) but were unable to respond to plant mitogens (6) or to bind to immobilised phytohaemagglutinin (PHA) or Concanavalin A (Con A) (16).Studies with fluorescein conjugated and radio iodinated antibody to PHA and Con A showed that the ability of the cells to bind the lectins was undiminished. However, agglutination by lectin and capping of lectin-anti-lectin were impaired by freeze-drying, suggesting a defect in membrane fluidity, perhaps resulting from damage to the microfilament and microtubule apparatus of the cell.     

Affinity differences between some commercially available immobilized Con A with rat alpha-fetoprotein

Rat alpha-fetoprotein contains three Con A-affinity molecular variants evidenced by affino-immuno-electrophoresis, (a) Con A-non reactive (52 %), (b) Con A-weakly reactive (31 %), (c) Con A-reactive (17 %). Affinity chromatography on several commercially available Con A-linked agarose displays different alpha-fetoprotein affinity patterns. The Con A-weakly reactive variant can be either bound and eluted with the Con A-reactive fraction or unbound and eluted with the Con A-non reactive one. These differences of chromatographic behaviour should be taken into account in structural, biochemical and biological studies dealing with the Con A-affinity molecular variants of alpha-fetoprotein or of other glycoproteins.     

Characterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases

Glycoproteins which bind concanavalin A (Con A) can be located on nitrocellulose sheets after electrophoretic transfer from slab gels, by sequential incubation of the sheets with Con A and peroxidase, and visualization of the peroxidase by an insoluble reaction product. We refer to this method as affinoblotting. Differential elution of Con A from the blots by washing the sheets with different concentrations of alpha-methylglycosides is used to demonstrate the affinity of Con A for the oligosaccharide side chains, and to differentiate between proteins with weak and those with high affinity for Con A. Concanavalin A has a high affinity for the four plant glycoproteins (phaseolin, phytohemagglutinin, jackbean alpha-mannosidase, and the glycosylated precursor of Con A) studied here. Incubation of the blots with alpha-mannosidase and endoglycosidase H (endo H) is used to demonstrate that the oligosaccharide chains can be degraded by glycosidases while the proteins are immobilized on the nitrocellulose. With this approach we show here that the four plant glycoproteins used as models in this study interact with Con A through high-mannose oligosaccharide side chains sensitive to alpha-mannosidase and endo H degradation.     

Computational analysis of Concanavalin A binding glycoproteins of human seminal plasma

Glycoproteins have immense clinical importance and comparative glycoproteomics has become a powerful tool for biomarker discovery and disease diagnosis. Seminal plasma glycoproteins participate in fertility related processes including sperm-egg recognition, modulation of capacitation and acrosome reaction inhibition. Affinity chromatography using broad specificity lectin such as Con A is widely applied for glycoproteins enrichment. More notably, Con A-interacting fraction of human seminal plasma has decapacitating activity which makes this fraction critically important. In our previous study, we isolated Con A-interacting glycoproteins from human seminal plasma and subsequently identified them by mass spectrometry. Here, we report the computational analysis of these proteins using bioinformatics tools. The analysis includes: prediction of glycosylation sites using sequence information (NetNGlyc 1.0), functional annotations to cluster these proteins into various functional groups (InterProScan and Blast2GO) and identification of protein interaction networks (STRING database). The results indicate that these proteins are involved in various biological processes including transport, morphogenesis, metabolic processes, cell differentiation and homeostasis. The clusters illustrate two major molecular functions - hydrolase activity (6) and protein (4)/carbohydrate (1)/lipid binding (1). The large interactomes of proteins point towards their versatile roles in wide range of biological processes.     

Role of A cells in the mitogenic stimulation of lymphocytes by Mycoplasma

The dependence of the blast transformation of lymphocytes in response to phytohemagglutinin (PHA), concanavalin A (Con A) and Mycoplasma arthritidis on the concentration of A-cells and the time of the introduction of M. arthritidis into the culture was studied. The level of blast transformation in response to PHA, Con A and M. arthritidis increased with the decrease of the concentration of A-cells in the culture. After the combined inoculation of the culture with M. arthritidis and PHA the resulting effect was higher than that induced by PHA alone and lower than the level of blast transformation in response to M. arthritidis at all A-cell concentrations under study. After the combined inoculation of M. arthritidis and Con A the summation of response was observed in cultures with a high concentration of A-cells, while in cultures with a low concentration of A-cells the resulting response was lower than that induced by any of these mitogens alone. The inoculation of the culture with M. arthritidis 24-48 hours after the cultivation of splenocytes with PHA and Con A was started led to the suppression of response to the mitogens. The suppression of response to PHA was most pronounced at the maximum concentration of A-cells, while the suppression of response to Con A reached its highest level when the concentration of A-cells was low. These data are in accord with the suggestion that M. arthritidis and PHA, as well as M. arthritidis and Con A, stimulated the overlapping subpopulations of lymphocytes in rats, the adhesive properties being most pronounced in the subpopulation of PHA- and M. arthritidis-positive lymphocytes.     

Changes of glycosylation of serum proteins in Sjögren's syndrome: correlation with interleukin-6 and soluble interleukin-2 receptor

A strong increase of the affinity for concanavalin A (Con A) of serum alpha 2-macroglobulin, a non-acute-phase protein, was observed by lectin blotting in patients with Sj?gren's syndrome (SS). On the contrary, the total Con A reactivity of serum proteins, measured by enzyme-linked lectin assay, was not augmented in SS, compared with normal donors, probably because positive changes of certain proteins were balanced by negative changes of others, as suggested by lectin blotting analysis. However, a significant increase of total Con A reactivity occurred in subjects with increased serum concentrations of soluble interleukin (IL)-2 receptor, compared with patients with normal concentrations of this marker of disease activity. On the other hand, the same parameter did not appear to be different in patients with normal or increased serum concentrations of IL-6, indicating that this cytokine was not probably responsible for the changes of glycosylation described here.     

Tandem affinity monolithic microcolumns with immobilized protein A, protein G', and antibodies for depletion of high abundance proteins from serum samples: integrated microcolumn-based fluidic system for simultaneous depletion and tryptic digestion

Affinity monolithic microcolumns with immobilized affinity ligands including protein A, protein G' and polyclonal antibodies were developed for the microscale depletion of the top eight most abundant proteins in human serum. These various affinity microcolumns were evaluated for their sample loading capacities with the standard protein substrates. In general, the sample loading capacity of protein A and protein G' was about 7-25 fold higher than that of the antibody-based affinity columns. The macroporous nature of the monolithic columns, which offers high permeability in pressure-driven flow, allowed the design of long tandem affinity columns for the simultaneous depletion of the top eight most abundant proteins in a single run. The tandem format could be extended to include additional affinity monolithic columns to deplete other proteins for which specific antibodies are available without running into high inlet pressure. Furthermore, the tandem affinity columns were integrated with immobilized trypsin monolithic columns to achieve the simultaneous depletion and digestion of proteins. The various formats investigated in this study could be down scaled to achieve nanoLC or up scaled to perform conventional HPLC depending on the size of the proteomic samples.     

A study of the sites of glycosylation of zein proteins using the lectin concanavalin A

The sites of glycosylation of zeins, the maize (Zea mays L.) storage proteins, were studied using the affinity of the lectin Concanavalin A (Con A) for certain glycosides. Zeins which were extracted from kernels of Illinois High Protein (IHP), W22, W64A and Oh43 were separated by isoelectric focusing and analyzed with a radiolabeled Con A binding technique. Certain sub-groups of the zein proteins contained carbohydrate moities which bound Con A while others did not. Zeins extracted from Oh43 kernels had a higher relative affinity for Con A than those of other maize lines. Further analyses of the zeins of Oh43 by gas chromatography demonstrated the presence of fucose, mannose and glucose.     

Alpha-C-mannosyltryptophan is not recognized by conventional mannose-binding lectins

Alpha-C-mannosyltryptophan (C-Man-Trp) is a novel, naturally occurring C-linked carbohydrate-protein linkage first found in 1994 from human ribonuclease 2. Since then, a number of C-Man-Trp residue have been found from several important proteins such as interleukin 12 beta, components of complement system, thrombospondin-1, and erythropoietin receptor, however, the biological functions have remained unknown even though its biosynthetic pathway has been revealed. In order to find a clue as to the biological functions, we examined the affinity of C-Man-Trp with conventional mannose lectin such as concanavarin A (Con A) and mannose-binding lectin (MBL). The affinity of C-Man-Trp with Con A, a typical mannose-binding lectin from plant was examined using a Con A-Sepharose column. Unlike p-nitrophenyl-alpha-O-Man, C-Man-Trp was not retained on the column. MBL-C, a major mannose-binding lectin purified from mouse serum, did not bind with N-biotinylated C-Man-Trp, judging from ELISA based assay. These results imply that C-Man-Trp may be recognized with the other specific proteins associated with its unknown biological functions.     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of cell-mediated immunity.

To assess the effects of the selective T-cell mitogens concanavalin A (Con A) and phytohemagglutinin P (PHA) on cell-mediated immunity (CMI), the mitogens were injected before, with, or after intravenous (iv) challenge of mice with Listeria monocytogenes. Mitogenic treatment differentially influenced the CMI response to Listeria. Con A enhanced listericidal activity when given before or with Listeria challenge, but Con A suppressed the CMI response when given after infection with Listeria. In contrast, PHA enhanced listericidal activity at all intervals. Since Con A, but not PHA, affected the growth of Listeria in the spleens of mice 24 and 48 hr after infection, Con A was shown to have an immediate effect on the development of listericidal activity and PHA was shown to have a delayed effect. In addition, Con A induction of immediate nonspecific listericidal activity was short-lived, while PHA induced a longer-lasting effect on resistance to Listeria. The mitogen-induced effects in the CMI response to Listeria were shown to be dependent upon the activities of activated T cells. The enhancement and suppression of listericidal activity appears to result from the activation of different T-cell subpopulations, known to be stimulated preferentially by Con A or PHA.     

Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Thymocyte-stimulating factor from peripheral blood cells.

Human peripheral blood leukocytes (HPBL) produce a thymocyte-stimulating factor (TSF-HPBL) that enhances the phytohemagglutinin (PHA) and concanavalin A (Con A) responsiveness of murine thymocytes. This activity is considerably specific for thymocytes. TSF-HPBL is not mitogenic by itself. Experiments with cell cultures pretreated with carbonyl iron particles showed that phagocytic cells are not involved in the production of mouse and rat TSF but are involved in the production of TSF-HPBL. The dose-response profile to PHA of murine thymocytes cultured in the presence of TSF-containing supernatants is similar to that of mature, immunocompetent spleen cells. TSF-HPBL, however, does not enhance the PHA responsiveness of murine thymocytes at low (<0.25 μg/microwell) concentrations of mitogen. TSF enhances the PHA and Con A responsiveness of the high-density subpopulations of thymocytes isolated on a Ficoll-Hypaque gradient. In general, the enhancing effect of TSF-HPBL on these subpopulations of thymocytes is smaller than that exerted by TSF. While supernatants containing TSF confer to thymocytes the ability to participate in a mixed lymphocyte reaction, this effect is not exerted by supernatants containing TSF-HPBL. A factor enhancing the PHA and Con A responsiveness of murine thymocytes is also produced by murine peripheral blood leukocytes (TSF-MPBL). This factor, similarly to TSF-HPBL, is produced by phagocytic cells and does not confer to murine thymocytes the ability to participate in a mixed lymphocyte reaction. Human T-cell lines do not enhance the PHA or Con A responsiveness of murine thymocytes. TSF-HPBL has a molecular weight of about 30,000 daltons, as measured by Sephadex filtration. Its half-time of inactivation as 56 °C is 162 ± 8 min.     

Immunologically specific cytolytic activity induced in long-term mixed leukocyte culture cells by concanavalin A.

Stimulation of cells from long-term primary MLC with Con A resulted in the generation of CTL activity comparable in magnitude to that induced by reexposure of the cells to the original stimulating cellular antigen. CTL generated by stimulation of long-term MLC cells with ConA had lytic activity specific for the original stimulating alloantigen used in primary MLC. The pattern of stimulation of long-term MLC cells with Con A differed from that of restimulation with alloantigen in that there was no detectable CTL activity the first 24 hr after Con A stimulation and the peak lytic activity occurred later. Unlike restimulation with alloantigen early lytic activity after Con A stimulation was dependent on DNA synthesis. PHA also proved to be an effective agent for stimulating cytolytic activity in long-term MLC cells. The response to PHA was comparable in magnitude to that generated by Con A. Stimulation of long-term MLC cells with T cell mitogens gave decreased cell recoveries relative to restimulation with alloantigen, however, the lytic activity per cells recovered was generally greater in the mitogen-stimulated cultures.     

Selective binding of concanavalin A to target cell major histocompatibility antigens is required to induce nonspecific conjugation and lysis by cytolytic T lymphocytes in lectin-dependent cytotoxicity

The exquisite immunological specificity of cytotoxic T lymphocytes-target cell (CTL-TC) conjugation and lysis is overridden in the presence of certain plant lectins. The role of concanavalin A (Con A) in lectin-dependent, CTL-mediated cytolysis (LDCC) has been investigated. Papain-treated TC are refractory to LDCC, but regain susceptibility following a 3-hr incubation without the enzyme. Papain-treated TC allowed to recover in the presence of tunicamycin (TM; an inhibitor of N-linked glycosylation), are totally refractory to LDCC. Refractoriness of TM-treated TC to LDCC is not due to an overall resistance to lysis or to lack of Con A binding, as these cells can be lysed by specifically sensitized CTL or by H-2 antibody and complement and display a sufficiently high Con A-binding capacity, indistinguishable from intact TC, probably through O-linked, cell-surface glycosyl residues. The finding that TC (TM-treated) capable of binding normal Con A quantities cannot, however, engage in lectin-dependent CTL-TC conjugation and lysis indicates that Con A must react selectively with a specific TC-surface component(s), thereby rendering the TC recognizable by effector CTL, rather than by simply bridging ("glueing") CTL and TC. Affinity absorption and elution from Sepharose-Con A beads as well as specific immunoprecipitations by antibodies against cell surface determinants, have shown effective Con A binding to TC surface components of molecular weights corresponding to 45-kDa product of the H-2K and D MHC genes and, possibly, to a 30-kDa component. Antibodies against MHC proteins but not against non-MHC surface proteins of the TC have produced effective inhibition of LDCC. This and previous investigations show that in nonspecific LDCC as in specific CTL-mediated lysis, TC-MHC determinants are involved in signaling TC recognition and lysis.