Fractionation of lymphocytes using affinity chromatography with 9 lectins

Lymphocyte subclasses from normal peripheral blood have been fractionated by affinity chromatography with lectins. Concanavalin A (Con A), Lens culinaris lectin (LC), Pisum sativum lectin (PS), Phaseolus vulgaris lectin (PHA), Dolichos biflours lectin (DB), Glicine max lectin (SBA), Ricinus communis lectin (RCA II), Tetragonolobus purpureus lectin (TP) and Triticum vulgaris lectin (WGA), were coupled to Sepharose 6MB, and lymphocytes labelled with 125I were eluted through the chromatographic columns. The binding of lymphocytes to WGA and SBA lectins was 32% and 13% respectively. The binding to the other lectins tested were found to be between 32% and 13%. When solutions of increasing concentrations of specific sugar were added to the columns a progressive elution of bound lymphocytes was observed. These results indicate the existence of a large range of lymphocyte subclasses, with different binding capacity to lectins, which was a function of the receptor number or/and receptor affinity to each lectin. Furthermore, these two parameters were found to vary in each functional population. Even though all the lymphocytes had lectin receptors, T lymphocytes showed higher affinity for Con A, PHA and TP lectins, while B lymphocytes appeared to be more specific for LC, PS, SBA, DB, RCAII and WGA lectins.     

Quantification of lectin receptors on B, T, T-gamma, and T-mu lymphocytes. II. PHA, SBA, Dolichos biflorus, and Tetragonolobus purpureus

Plasma membranes composition and structure of B, T, T gamma and T mu lymphocytes from peripheral blood have been studied using four 125I-labelled lectins. The results showed that B lymphocytes have a greater number of receptors for SBA and Dolichos biflorus lectins than T lymphocytes, whilst the opposite was observed for PHA-lectin. However, no significant differences between the two lymphocyte populations were found regarding the receptors for Tetragonolobus purpureus lectin. Among T lymphocyte subpopulations, SBA, Dolichos biflorus and tetragonolobus purpureus lectins appeared to have higher specificity for T gamma lymphocytes and PHA for T mu. PHA-lectin had greater affinity for B lymphocytes, while the rest showed higher affinity for T lymphocytes. The T gamma subpopulation had lower association constant for PHA-lectin than T mu lymphocytes, whilst the contrary was found to be true for the other three lectins.     

Separation of human lymphocyte subclasses by rosettes with latex-lectin particles

The specificity and affinity of eight lectins (concanavalin A, L. culinaris, P. sativum, phytohemagglutinin P, D. biflorus, soybean agglutinin, T. purpureus, and T. vulgaris) to B, T, T gamma, and T mu lymphocytes from the blood of normal subjects were determined. Lectins attached to latex particles were used to evaluate the binding of each lectin to individual cells. The rosette percentage found in each lymphocyte population expresses the specificity index and the specific sugar concentrations needed to decrease the rosette percentage by 50% is taken as the affinity index. B Lymphocytes showed a major subclass, with respect to T lymphocytes, with receptors for WGA, SBA, D. biflorus, L. culinaris, and P. sativum lectins. In contrast, T lymphocytes exhibit a greater number of cells with specific receptors for Con A, T. purpureus, and PHA lectins than B lymphocytes, the T gamma subpopulation being responsible for the specificity of the first two lectins and the T mu subpopulation for the PHA lectin.     

Lectin binding ability of B-chronic lymphocytic leukaemia cells.

The binding of five fluorescein-labelled lectins: peanut agglutinin (PNA), lentil agglutinin (LEN), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and asparagus pea agglutinin (ASP) to human B-cell chronic lymphocytic leukaemia (B-CLL) and B lymphocytes of normal donors was studied. The specificity of the fluorescence was demonstrated by inhibition with appropriate saccharides. The proportion of B cells was estimated using anti-B cell monoclonal antibody. Both leukaemic and normal B cells showed the binding ability of all except of one (ASP) studied lectins. We have found the differences in surface carbohydrate patterns between B-CLL and normal B lymphocytes. B-CLL cells showed the considerably lower ability to bind SBA and slightly higher expression of PNA and LEN receptors in comparison to normal B cells. The analysis of WGA binding allowed for recognizing two groups of CLL patients: one with high and the second one with low WGA receptor expression. The double marker studies revealed that B cells could simultaneously react with anti-B cell monoclonal antibody and fluorochrome labelled lectins.     

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.     

Cell surface changes in transformed human lymphocytes : I. ConA and E-PHA induced unique changes in surface topography

Binding studies with six purified plant lectins were used to investigate membrane alterations that occur in lymphocyte transformation. Normal human peripheral blood lymphocytes transformed with E-Phytohemagglutinin (E-PHA) or concanavalin-A (Con-A) generally possessed increased numbers of lectin receptors. When this increase was corrected for the expanded surface area of transformed lymphocytes, it appeared that E-PHA and ConA each produced a unique and complex reorganization of cell surface topography. Surface alterations occurred independently of DNA synthesis, cell proliferation, and microtubule or microfilament function. Puromycin inhibited emergence of new lectin receptors on cells transformed with E-PHA, but not with ConA. Lymphocytes incubated with either lectin showed increased incorporation of [¹⁴C]galactose into trypsin-sensitive cell surface glycoproteins. This incorporation was abolished by puromycin in cells stimulated by E-PHA but not by ConA. These studies demonstrate that although both lectins induce similar morphological alterations in human lymphocytes, at the molecular level the structural changes induced in the cell membrane by these two lectins differ considerably. Furthermore, these structural alterations are mediated via different mechanisms in the two groups of cells. De novo protein synthesis is required for cell surface reorganization in PHA-stimulated cells, but not in cells stimulated by ConA. The effect of ConA appears to be to enhance attachment of saccharide structures to pre-synthesized membrane proteins.     

Comparison of different markers on blood lymphocytes of chronic lymphocytic leukemia

Mononuclear cells (MNC) of 17 patients suffering from B chronic lymphocytic leukemia (B-CLL) were analysed by various immunological methods. The B cell nature of CLL cell was determined by classical tests (MRBC-rosette-test, immunofluorescence test for detection of membrane bound immunoglobulins). The cytochemical detection of the new T-cell marker dipeptidyl peptidase IV (DP IV) was found to be suitable for the characterization of B-CLL. The B-CLL cells showed granular pattern of alpha-naphthylacetate esterase (ANAE) reaction and binding of the monoclonal pan T antibody BL-T2. These non typical reactions for normal B lymphocytes can be used for differential diagnosis of B-CLL in combination with other reliable T cell markers. Avoiding the separation of T cells, the mixed rosette assay was used to enumerate Fc-IgG-receptor bearing T(TG) and non T cells. Both cell populations were found to be significantly elevated in MNC of B-CLL.     

Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.     

Distribution of saccharides in pig lymph-node high-endothelial venules and associated lymphocytes visualized using fluorescent lectins and confocal microscopy

Summary The distribution of saccharides in pig lymph nodes, particularly on high-endothelial venule (HEV) endothelium and on lymphocytes in these vessels, was studied by examining the binding of fluorescent conjugates of 18 different lectins. Eight of the lectins, particularly with glycan specificity restricted to mannose and polyacetyllactosamine determinants, were found to bind with a high affinity to these structures. Competitive inhibition experiments revealed that polylactosamine-containing glycans were present on endothelia and lymphocytes using lectins from Lycopersicon esculentum and Solanum tuberosum, the latter lectin reacting with lymphocytes only when apparently adherent to the luminal endothelium. The absence on pig endothelium of the Ulex europaeus binding, shown by human endothelia due to the presence of certain fucose epitopes, was confirmed. Pig lymph-node endothelium, however, bound the fucose-specific lectin of Tetragonolobus purpureas, indicating the presence of fucose on pig endothelia in a different conformation to that seen on human endothelia. The results suggested that pig lymph-node HEV endothelium expressed a core fucosylated tri- or tetra-antennary complex glycan with polylactosamine extensions and expressing an Le^y determinant.Abbreviations used BS-I Bandeiraea simplicifolia BS-I - BS-I-B B. simplicifolia isolectin B₄ - BS-II B. simplicifolia, lectin II - FACS fluorescence-activated cell sorter - FITC fluorescein isothiocyanate - HEV high-endothelial venule - LN lymph node - MLR mixed lymphocyte reaction - PBS phosphate-buffered saline - PPME phosphomannan - UEA-I Ulex europeaus lectin I     

Detection of anomalies in cell-surface carbohydrates on thrombasthenic platelets using 125I-labeled lectins

The composition of carbohydrates on the surface of platelets from a patient with Glanzmann's thrombasthenia and from seven normal donors were determined and compared. To this end, binding studies were performed using nine different purified 125I-labeled lectins; Concanavalin A, P-Phytohaemagglutinin, Wheat Germ Agglutinin, Dolichos biflorus, Pisum sativum, Ricinus communis II Agglutinin, Tetragonolobus purpureus, Lens culinaris and Soybean Agglutinin. These studies show that thrombasthenic platelets bear significantly decreased numbers of receptors for Concanavalin A and Lens culinaris, both with a specificity for D-mannose, and Ricinus communis II, with specificity for D-galactose. There were no detectable differences in the numbers of other lectin receptors. These results provide further evidence of molecular defects in thrombasthenic platelets. Moreover, the use of 125I-labeled lectins, as shown here, provides a fast and reliable technique for identifying abnormalities in the carbohydrate composition on the surface of platelets in various thrombopathies.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

Effect of Low-Frequency Low-Energy Pulsing Electromagnetic Fields on the Response to Lectin Stimulation of Human Normal and Chronic Lymphocytic Leukemia Lymphocytes

Many literature reports suggest that at least one of the mechanisms of action of low-frequency pulsing electromagnetic fields (PEMFs) is to favor Ca++ movement into the cell. Ca¹ influx Is a fundamental step In the activation process of lymphocytes by lectins. We report here the results of the exposure of human normal and chronic lymphocytic leukemia (CLL) lymphocytes to lectins and PEMFs. Simultaneous exposure to PEMFs and mitogens significantly increased the number of normal responsive lymphocytes compared to those exposed to the mitogens alone. Almost all the normal lymphocytes entered into the mitotic cycle. The number of CLL lymphocytes stimulated by simultaneous exposure to PEMFs and lectins was doubled compared with lectin exposure alone. Ca^?1 influx was increased when the cultures were exposed to PEMFs. The stimulatory effect of PEMFs was mediated by an increased release of some B-cell growth factor by the T-Zymphocytes.     

B 细胞膜CD20 抗原的分布与单分子力谱探测

CD20抗原分子在B细胞上表达下降是慢性B淋巴细胞白血病 (B-CLL) 的标志性特征。采用激光扫描共聚焦显微镜 (LSCM) 和量子点标记相结合的方法对正常和B-CLL外周血CD20+B淋巴细胞膜表面CD20抗原分子的表达及分布进行了荧光成像。同时,采用原子力显微镜 (AFM) 对CD20+B细胞的形貌及超微结构特征进行了表征,并且将AFM针尖用生物素化的单克隆抗体进行修饰,对CD20+B细胞表面的CD20抗原-抗体之间的单分子力谱进行了探测。LSCM荧光图像显示,B-CLL CD20+B淋巴细胞上CD20分子的表达量比正常CD20+B淋巴细胞显著降低。AFM结果显示,B-CLL CD20+B淋巴细胞超微结构比正常的粗糙。力谱结果显示,CD20抗原-抗体的相互作用力大约是非特异性黏附力的5倍,CD20分子在正常CD20+B淋巴细胞膜上分布比较均匀,小部分有聚集现象,反之,在B-CLL CD20+B淋巴细胞膜表面分布稀疏。利用以上两种方法能进一步观察到B-CLL外周血B淋巴细胞的异常,并在一定程度上解释临床上B-CLL病人对利妥昔的低反应现象,为针对抗原CD20的治疗用药选择提供参考。     

Lectins as Markers of Human Epidermal Cell Differentiation

The expression of sugar residues on human epidermal cells was investigated by means of lectin binding, as a way of determining membrane structural changes occurring during the differentiation of the epidermis. Fourteen lectins of different sugar specificity were conjugated with fluorescein isothiocyanate (FITC-lectins) and tested in fluorescence microscopy on frozen sections of normal human epidermis. In parallel, FITC-lectins were tested on psoriatic-involved epidermis to visualize differences in the expression of sugar residues that might occur during abnormal epidermal differentiation. No labelling could be obtained with lectins from Bandeira simplicifolia I, Dolichos biflorus, Limulus polyhemus, Tetragonolobus purpureas, Ulex europeus I , and Triticum vulgaris (group 1 lectins). A "pemphigus-like" intercellular labelling of the whole epidermis, except the stratum corneum, was obtained with lectins from Canavalia ensiformis, Maclura pomifera, Phaseolus vulgaris , and Ricinus communis I (group 2 lectins). A selective intercellular labelling of the stratum spinosum and the stratum granulosum was seen in normal epidermis with lectins from Arachis hypogaea, Glycine max, Helix pomatia , and Sophora japonica (group 3 lectins). In psoriatic epidermis, not only the basal cell layer, but also cells from the adjacent lower stratum spinosum were found to be negative, using FITC-lectins of group 3. These data indicate that the expression of lectin binding sites in normal epidermis differs according to the maturation of the cell from the basal cell to the more mature keratinocyte in the stratum granulosum. They suggest that lectins may be used as markers of epidermal cells in various stages of normal and abnormal differentiation.     

Mucin-specific bark lectin from elderberry Sambucus sieboldiana and its applications to the affinity chromatography of mucin

Three bark lectins were isolated from elderberry Sambucus sieboldiana using fetuin-Sepharose 4B and mucin-Sepharose 4B, and were studied comparatively for their binding to glycoprotein and to clarify various physicochemical features. For each, a unique pattern on isoelectric focusing was noted and their affinity toward various glycoproteins differed, indicating the structures of their carbohydrate binding sites possibly differ. One bark lectin showed specific binding toward porcine mucin. The purity of mucin from a crude porcine stomach mucin or an extract of porcine submaxillary glands could be improved by affinity chromatography on immobilized lectin having binding specificity toward mucin.     

FRACTIONATION OF GLYCOPROTEINS ASSOCIATED TO ADULT RAT BRAIN MYELIN FRACTIONS

Abstract— The chloroform-methanol insoluble residue of adult rat brain myelin fractions (My-CMI) contains only 20% of protein but all myelin-associated glycoproteins (Z anetta et al ., 1977a). After solubilization in sodium dodecyl sulphate, these glycoproteins were separated by sequential affinity chromatography on 4 immobilized lectins. Ten fractions (9 of which contained only glycoproteins) were obtained. Glycoproteins added up to at least 25% of My-CMI proteins. Many minor glycoproteins were detected in the different fractions. However most of them appeared not to be intrinsic to myelin. On the contrary a major glycoprotein electrophoretic band, component A, appeared to be intrinsic to myelin although its presence also on oligodendrocyte plasma membrane cannot be excluded. Component A was tentatively identified with the'major myelin associated glycoprotein'described by QUARLES (1972, 1973 a, b ). It accounted for less than 0.4% of proteins and 8% of glycoproteins of myelin fractions and consisted of at least two'glycopolypeptides'which, both, bind to concanavalin A and to the Ulex europeus lectin. The other major glycoprotein, component B, did not bind to any of the lectins used and, thus, must have N -acetyl neuraminic acid as only terminal sugar. The separation of myelin-associated glycoproteins according to their affinity for lectins allowed a tentative identification of the lectin binding sites of myelin sheath.     

Unusual lectin-binding properties of a herpes simplex virus type 1-specific glycoprotein

Lysates from herpes simplex virus type 1-infected cells were subjected to affinity chromatography on soybean and Helix pomatia lectins. One of the virus-specified glycoproteins, probably the herpes simplex virus type 1-specific gC glycoprotein, bound to the lectins and was eluted with N-acetylgalactosamine. The affinity chromatography permitted a high degree of purification of the type-specific glycoprotein with respect to both host cell components and other viral glycoproteins. The lectin affinity pattern of this glycoprotein indicates the presence of a terminal alpha-N-acetylgalactosamine in an oligosaccharide, a finding not reported previously for glycoproteins of enveloped viruses.     

Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin

An indirect affinity chromatography procedure utilizing biotinylated lectins and designed for the specific isolation of surface glycoproteins is described. The method is illustrated with intact acute leukemic lymphoblastic cells (ALL cells) with biotin-epsilon-aminocaproyl-concanavalin A (biocap-Con A) and streptavidin-Sepharose 4B. Biocap-Con A, containing on average 27 biotin residues per tetrameric lectin molecule, is used to isolate Con A-binding glycoproteins from the surface of [35S]methionine-radiolabeled intact cells. The biocap-Con A/glycoprotein complexes, after solubilization in detergent, are retrieved on immobilized streptavidin. The surface glycoproteins isolated from intact ALL cells by this method are subjected to two-dimensional gel electrophoresis and detected by autoradiography. More than fifty Con A-binding glycoproteins can be separated from the ALL cells. These glycoproteins retrievable from the cell surface were compared to those retrieved by the indirect affinity chromatography procedure from isolated plasma membrane fractions. Certain groups of glycoproteins present in the fraction isolated from intact cells were not detected in that from the plasma membrane preparations. The advantage of using the biocap-con A/streptavidin system with intact cells rather than isolated plasma membranes for the detection of surface glycoproteins is discussed.     

Interaction of concanavalin A and a divalent derivative with lymphocytes and reconstituted lymphocyte membrane glycoproteins

Both concanavalin A (con A) and its divalent derivative, succinyl-concanavalin A (S-con A) are mitogenic for porcine lymph node lymphocytes. We have compared the binding of these two lectins to intact porcine lymphocytes and phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins. Both con A and S-con A showed high- and low-affinity binding to intact cells, as indicated by LIGAND analysis of Scatchard plots of binding data. Despite the apparently identical saccharide specificities of the two lectins, high-affinity binding sites for S-con A were only one-third as numerous as high-affinity sites for the parent lectin. Large numbers of low-affinity binding sites existed for con A, while many fewer were present for S-con A. It is suggested that these sites result from hydrophobic association. Con A bound to lymphocytes in a positively cooperative fashion, while S-con A showed noncooperative behavior. Lectin binding to large unilamellar phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins was measured using a rapid filtration assay, and was linear with the glycoprotein content of the vesicles. Almost all of the outward-facing glycoprotein was functional in terms of lectin binding. Reconstituted glycoproteins showed only a single class of high-affinity binding sites for both con A and S-con A, with association constants similar to those measured for intact cells. Con A, but not S-con A, showed positively cooperative binding to reconstituted vesicles. Cooperativity was observed in both gel phase and liquid crystalline phase lipid, and was thus not dependent on long-range lateral rearrangement of glycoprotein receptors. Results suggested that con A induces a microredistribution of receptors on the lymphocyte membrane surface, leading to the exposure of glycoproteins that were previously inaccessible to the lectin. S-Con A does not cause glycoprotein redistribution, and a large fraction of the receptors remain cryptic.     

Interaction of Concanavalin A and a Divalent Derivative with Lymphocytes and Reconstituted Lymphocyte Membrane Glycoproteins

Both concanavalin A (con A) and its divalent derivative, succinyl-concanavalin A (S-con A) are mitogenic for porcine lymph node lymphocytes. We have compared the binding of these two lectins to intact porcine lymphocytes and phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins. Both con A and S-con A showed high- and low-affinity binding to intact cells, as indicated by LIGAND analysis of Scatchard plots of binding data. Despite the apparently identical saccharide specificities of the two lectins, high-affinity binding sites for S-con A were only one-third as numerous as high-affinity sites for the parent lectin. Large numbers of low-affinity binding sites existed for con A, while many fewer were present for S-con A. It is suggested that these sites result from hydrophobic association. Con A bound to lymphocytes in a positively cooperative fashion, while S-con A showed non-cooperative behavior. Lectin binding to large unilamellar phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins was measured using a rapid filtration assay, and was linear with the glycoprotein content of the vesicles. Almost all of the outward-facing glycoprotein was functional in terms of lectin binding. Reconstituted glycoproteins showed only a single class of high-affinity binding sites for both con A and S-con A, with association constants similar to those measured for intact cells. Con A, but not S-con A, showed positively cooperative binding to reconstituted vesicles. Cooperativity was observed in both gel phase and liquid crystalline phase lipid, and was thus not dependent on long-range lateral rearrangement of glycoprotein receptors. Results suggested that con A induces a microre-distribution of receptors on the lymphocyte membrane surface, leading to the exposure of glycoproteins that were previously inaccessible to the lectin. S-Con A does not cause glycoprotein redistribution, and a large fraction of the receptors remain cryptic.