Quantification of lectin receptors on B, T, T-gamma and T-mu lymphocytes. I. Concanavalin A, Pisum sativum, Lens culinaris and wheat-germ agglutinin

The immune system is formed of different lymphocyte subpopulations, each one having a defined role to defend the organism. Their plasma membranes present differences in the glycoproteinic or/and glycolipidic composition, as detected with labelled 125I-lectins. B lymphocytes have a greater number of receptors for the Pisum sativum, Lens culinaris and WGA lectins than T lymphocytes. On the other hand, T lymphocytes bind a greater number of Concanavalin A molecules than B lymphocytes. WGA lectin appeared to be more specific for T mu subpopulation, while Con A and Pisum sativum lectins were bound preferentially to T gamma lymphocytes while no significant differences were observed between both subpopulations for Lens culinaris lectin. From the affinity of each lectin to each lymphocyte population it could be deduced that the receptor structure, conformation and arrangement on the membrane was optimal in B lymphocytes for Con A and WGA binding, and T lymphocytes for Lens culinaris and Pisum sativum binding.     

Isolation and characterization of subunits from the predominant form of Dolichos biflorus lectin.

The subunits of the two molecular forms (A and B) of the Dolichos biflorus lectin were isolated by ion-exchange chromatography on DEAE-cellulose in 8.0 M urea. Subunits IA and IIA which comprise the predominant molecular form A of the lectin were found to have molecular weights of 27,700 and 27,300, respectively, as determined by sedimentation equilibrium studies in 8.0 M urea. These subunits have similar amino acid compositions and each have alanine at their amino-terminal ends. Comparison of the IA and IIA subunits by immunodiffusion against antisera to the seed extract as well as to subunits IA and IIA showed no antigenic differences between the two subunits. Carboxyl terminal analyses of subunits IA and IIA with carboxypeptidase A produced an essentially simultaneous release of both leucine and valine residues from subunit IA; no detectable amino acids were released from subunit IIA under identical conditions. The data suggest that the molecular form A of the lectin (molecular weight 113,000, Carter and Etzler, 1975) consists of four subunits with a possible stoichiometry of IA2IIA2. Other possible arrangements of the subunits are discussed.     

Isolation and characterization of a lectin from the roots of Dolichos biflorus

A lectin has been isolated from the roots of 7-day-old Dolichos biflorus plants and has been compared with the D. biflorus seed lectin. The root lectin differs from the seed lectin in molecular weight, subunit stoichiometry, amino acid composition, amino terminal amino acid sequence, and isoelectric focusing pattern. However, the root lectin has in common with the seed lectin a specificity for N-acetyl-D-galactosamine, and upon denaturation the root lectin will react weakly with antiserum made to denatured seed lectin. Distribution studies of this lectin in germinating seedlings show that the highest levels of lectin are found in 1-day-old roots. Upon dissection and analysis of 7-day-old roots, the highest levels of the lectin are in the uppermost segment. In addition, isoforms of this lectin also exist in the stems and leaves of the plant.     

cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin

The Dolichos biflorus seed lectin contains two structurally related subunits. A cDNA library was constructed using RNA isolated from D. biflorus seeds actively synthesizing the seed lectin. The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin-specific antiserum was used to isolate a seed lectin cDNA. Hybridization of the D. biflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single-size RNA of 1100 bases. An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA. Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin-specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit. This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA. These data support the existence of a single polypeptide precursor for both subunit types of the D. biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.     

Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development.

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number ((0.26 plus or minus 0.03)-10-6 sites/cell) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites fo Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by N-acetylgalactosamine, the determinant of Dolichos lectin, as well as by Dolichos antiserum.     

Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin.

The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine.     

Properties of the human erythrocyte membrane receptors for peanut and Dolichos biflorus lectins.

Neuraminidase treatment of blood type A and B human erythrocytes, which is required for the agglutination of these cells by peanut (Arachis hypogaea) lectin, increased the number of receptor sites for the lectin from about 5 × 10⁴ to 1.8 × 10⁶ sites/ cell for both blood types. The same treatment also increased the agglutinability of type A cells by the blood group A-specific Dolichos biflorus lectin, but the number of receptor sites for this lectin (~6 × 10⁵ sites/cell) did not change. D. biflorus lectin binding and agglutination of blood type B cells were negligible both before and after neuraminidase treatment. To isolate the peanut agglutinin receptor from the membrane of these cells, washed type A erythrocytes were incubated with neuraminidase and galactose oxidase and then treated with NaB³H₄, thus labeling the galactose residues on the membrane. For measuring peanut agglutinin receptor activity, a radioaffinity assay was developed based on the displacement of [¹⁴C]asialofetuin from peanut agglutinin by receptor and precipitation of the complex in the presence of polyethyleneglycol. Membranes were isolated by hypotonic lysis and were solubilized in 0.5% Empigen BB, a zwitterionic detergent, which was found to be superior to Triton X-100 for this purpose. The cell extract, after centrifugation, was subjected to affinity chromatography on peanut agglutinin-polyacrylhydrazido-Sepharose. Elution with lactose afforded a peak of radioactivity (32% yield) containing 70% of the applied receptor activity. The eluting sugar and the receptor were separated by chromatography on Bio-Gel P-2 with subsequent dialysis against 80% acetone to remove the detergent. The bulk of the isolated receptor radioactivity (91%) precipitated with peanut agglutinin. The amino acid composition, the glucosamine and galactosamine content and the electrophoretic mobility, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the peanut receptor were similar to those of asialoglycophorin. In addition, the peanut receptor coprecipitated with asialoglycophorin and with isolated erythrocyte T antigen on Ouchterlony double-diffusion plates against peanut agglutinin and the Ricinus communis lectin, but not with D. biflorus lectin, suggesting that the receptor for the latter lectin is distinct from the peanut agglutinin receptor.     

Dolichos biflorus agglutinin receptors in mouse muscle. II. Biochemical properties in relation to molecular forms of acetylcholinesterase

A biochemical analysis has been performed on the relationship between the receptors for Dolichos biflorus agglutinin (DBA) and collagen tailed acetylcholinesterase (16S AChE) in mouse skeletal muscle. The molecular forms of AChE were separated by differential salt extraction and by gradient centrifugation. DBA binding activity was measured using a microtiter plate binding assay and affinity chromatography. The 16S form of AChE was bound to DBA, whereas globular forms of AChE were not. However, only a small proportion of 16S AChE was capable of binding to DBA, and most of the DBA binding capacity in muscle extracts was not associated with the 16S AChE. The possible association with the neuromuscular synapse of DBA binding molecules other than 16S AChE is discussed with respect to our previous histochemical study on DBA binding sites in mouse muscle.     

Dolichins A and B,two pterocarpans from bacteria-treated leaves of Dolichos biflorus

Two minor isoflavonoids isolated from bacteria-inoculated leaves of Dolichos biflorus have been identified as the 6aR; 11aR; 2′S and 6aR; 11aR; 2′S isomers of 3,9-dihydroxy-10-2′-hydroxy-3′-methyl-3′-butenyl pterocarpan.     

cDNA cloning, primary structure, and in vitro biosynthesis of the DB58 lectin from Dolichos biflorus

Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.     

Fc receptors on human T lymphocytes. II. Cytotoxic capabilities of human T gamma, T mu, B, and L cells.

Subpopulations of human peripheral blood lymphocytes were prepared by rosetting techniques employing neuraminidase-treated sheep erythrocytes (SRBC_n), sheep erythrocytes coated with IgM and murine complement (EAC′), and bovine erythrocytes coated with IgG and IgM. The isolated subpopulations were tested in assays of natural cytotoxicity (NC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). B cells (SRBC_n?, EAC′+) did not mediate cytotoxicity. L cells (SRBC_n?, EAC′?) mediated NC and ADCC but not MICC. T cells (SRBC_n+) mediated NC, ADCC, and MICC. Separation of T cells into Fc-IgG (Tγ) and Fc-IgM (Tμ) subsets revealed that Tγ cells mediated NC, ADCC, and MICC while Tμ cells mediated only MICC. Thus MICC but not NC or ADCC was solely T-cell mediated. Tγ and L cells were functionally distinguishable in that Tγ cells but not L cells mediated MICC. Tγ cells and Tμ cells differed with regard to NC and ADCC effector function while both subsets mediated MICC.     

Production and characterization of a monoclonal antibody against the seed lectin of the Dolichos biflorus plant

Spleen cells from mice immunized with the Dolichos biflorus seed lectin were fused with cells from the mouse myeloma Sp2/O-Ag14 cell line to form hybridomas. Those hybridomas producing antibodies against the seed lectin were cloned at least four times and the monoclonal antibodies from clone C11/64-56.28 were characterized and found to be specific for Subunit I of the lectin; they do not react with the structurally similar Subunit II. In previous studies, we have shown that although these two subunits appear to differ only at their COOH-terminal ends, only Subunit I has carbohydrate binding activity. Using a solid phase enzyme immunoassay, the antigenic determinant fr the monoclonal antibody was found to be located on the COOH-terminal cyanogen bromide fragment of this subunit. The monoclonal antibody inhibits the ability of the lectin to agglutinate erythrocytes and N-acetyl-D-galactosamine, the specific hapten for the lectin, inhibits the ability of the antibody to combine with the lectin. These results suggest that the monoclonal antibody recognizes a determinant that is located either at or near the active site of the lectin or that is conformationally interdependent with the active site.     

Isolation of serine protease from granulated metrial gland cells of mice and rats with lectin from Dolichos biflorus.

Granulated metrial gland cells were the only cells in the endometria of pregnant mice and rats that reacted histochemically with fluoresceinated lectin (DBA) from Dolichos biflorus. Cell extracts of uteri of pregnant animals, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by lectin overlay blotting, contained DBA-reactive, 40-50 kDa, doublet glycoprotein bands. This glycoprotein was purified on a DBA agarose affinity column. It was identified by amino acid sequencing as a serine protease closely related to granzymes of T lymphocytes. We conclude that this granzyme accounts for the selective reactivity of granulated metrial gland cells with fluoresceinated DBA in histological sections of uteri of pregnant rodents and show that DBA affinity columns can be used for purification of granzyme derived from granulated metrial gland cells.     

Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

The studies described in this paper were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes.     

The stem and leaf lectin of Dolichos biflorus L., previously thought to be cell wall associated,is sequestered in vacuoles

Post-embedding immunogold labeling has shown that the DB58 lectin is sequestered in vacuoles. Previous evidence indicating that a significant fraction of the DB58 lectin is cell wall associated is shown to be in error.Abbreviations PBS 10 mM sodium phosphate, pH 7.1, 154 mM sodium chloride - TBS 20 mM Tris base, pH 7.6, 137 mM sodium chloride We thank Dr. Mary Alice Webb for sharing her electron-microscopy expertise. This work was supported by National Science Foundation grant DCB-9004967 (M.E.E.) and a University of California, Davis, Jastro-Shields fellowship (T.W.B.).     

Circulating T and B lymphocytes of the mouse. II. Lifespan

The average lifespan of circulating lymphocytes was investigated by determining the percentage of labeling of thoracic duct lymphocytes (TDL⁵) from mice injected with tritiated thymidine (³HT) for various periods. Percentage of labeling of TDL from normal CBA mice, which consist of approximately 85% T cells and 15% B cells, was found to be directly proportional to the time of ³HT administration. This technique thus failed to demonstrate the presence of more than one population of lymphocytes. Less than 50% of TDL were labeled after ³HT injection for 8 weeks.Percentage of labeling of TDL from nude mice (which consist solely of B cells) was likewise found to be directly proportional to the duration of ³HT injection but occurred at a rate three to four times faster than in non-T cell-depleted CBA mice. Further experiments, in which a marker for B cells was used, allowed the rate of ³HT labeling of B cells to be studied in normal CBA mice. These data corroborated the findings in nude mice and indicated that, with regard to lifespan, thoracic duct B cells consisted of a single population with an average lifespan of 5–7 weeks. Similarly it was calculated that the average lifespan of thoracic duct T cells was in the order of 4–6 months.Studies on the rate of formation of TDL during prolonged thoracic duct drainage of normal CBA mice indicated that the percentage of newly formed cells increased rapidly after 24-hr drainage. The total numbers of newly formed cells, however, were found to remain relatively constant throughout the period of drainage investigated (up to 9 days) except for a transient increase during the second and third day. Newly formed small lymphocytes were found to consist of approximately equal proportions of T cells, B cells, and other “mononuclear” cells which lacked surface markers for either T or B cells. The great majority of large lymphocytes, in contrast, were found to be neither T cells nor B cells and probably belonged to the plasma cell line. In nude mice, production of newly formed lymphocytes during prolonged thoracic duct drainage was found to be very low in comparison with normal CBA mice.     

Chemomodulatory effect of Dolichos biflorus Linn. on skin and forestomach papillomagenesis in Swiss albino mice

Effect of consumption of three different doses (2%, 4% and 6%, w/w) of Dolichos biflorus Linn. seeds on hepatic drug metabolizing enzymes, antioxidant enzymes, reduced glutathione content, lactate dehydrogenase and lipid peroxidation in Swiss albino mice has been reported. Anti-carcinogenic effect has been studied by 7,12-dimethylbenzanthracene (DMBA)-induced skin and benzo(a)pyrene[B(a)P]-induced forestomach papillomagenesis models. D. biflorus consumption resulted in a significant increase in hepatic carcinogen metabolizing enzyme systems especially at 4% and 6% doses. Significant increase in reduced glutathione content (GSH) and specific activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) in liver of mice, at 4% and 6% doses has been reported. Lactate dehydrogensae (LDH) activity and peroxidative damage has been significantly decreased at 4% and 6% doses. In skin papillomagenesis model, 4% and 6% dose in diet significantly reduced the tumor incidence (up to 25%), tumor multiplicity (up to 59%) and tumor volume per mouse (up to 70%) as compared to DMBA treated group. Importantly, significant reduction in tumor incidence (up to 33%) and tumor multiplicity (up to 61%) was evident for forestomach papillomagenesis model.