scholarly journals Lectin-binding components of normal granulocytes and leukaemic myeloid cells

1983 ◽  
Vol 213 (3) ◽  
pp. 661-670 ◽  
Author(s):  
F A Spring ◽  
D J Anstee
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Helix Pomatia ◽  
Sodium Dodecyl ◽  
Myeloid Cells ◽  
Neuraminidase Treatment ◽  
Leukaemic Cells

A panel of lectins was used to analyse glycoproteins of normal granulocytes and leukaemic myeloid cells. The glycoproteins of detergent-solubilized whole cells were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their lectin-binding properties determined by incubation of the fixed gels with radioiodinated lectins. Normal granulocytes and leukaemic myeloid cells in different stages of maturation possess a cell-surface sialic acid-rich glycoprotein of apparent mol.wt. 115 000 (GP115), that can be labelled by both the lactoperoxidase and periodate/NaB3H4 cell-surface labelling techniques. The sialoglycoprotein of leukaemic myeloblasts has a slightly lower apparent mol.wt., 112000 (GP112). After neuraminidase treatment before cell solubilization, both GP115 and GP112 bind the lectins from Arachis hypogaea (peanut) and Helix pomatia (snail) and have an increased apparent molecular weight of 125000. Two concanavalin A-binding glycoproteins of apparent mol.wts. 98000 and 90000 are present in leukaemic myeloblasts. Concanavalin A binding to these glycoproteins is decreased in more mature leukaemic cells and absent in granulocytes. As concanavalin A binding decreases in the maturer forms, there is a concomitant increase in the binding of Ricinus communis (castor bean) and Maclura aurantiaca (osage orange) lectins to these glycoproteins. Whole granulocytes, but not leukaemic myeloblasts, contain a major cell-surface concanavalin A binding glycoprotein of apparent mol.wt. 130000, which is labelled by the periodate/NaB3H4 technique. Concanavalin A binding to this glycoprotein increases as the morphology of leukaemic cells approaches that of mature granulocytes.

Glycosylation of developing human glomeruli: lectin binding sites during cell induction and maturation.

1987 ◽  
Vol 35 (1) ◽  
pp. 33-37 ◽  
Author(s):  
H Holthöfer ◽  
I Virtanen
Keyword(s):  
Binding Sites ◽  
Mesangial Cells ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Helix Pomatia ◽  
Cell Types ◽  
Basement Membranes ◽  
Phenotypic Change ◽  
Neuraminidase Treatment ◽  
Nephron Development

Expression of cellular glycoconjugates during differentiation of human fetal kidney was studied using fluorochrome-labeled lectins. Each lectin revealed a characteristic binding pattern during the phenotypic change of the nephrogenic mesenchyme and during distinct stages of nephron development. The uninduced mesenchymal cells were positive for Pisum sativum (PSA), Concanavalin A (ConA), Wistaria floribunda (WGA), and Ricinus communis (RCA-I) lectins. However, these lectins failed to react with the uninduced cells of the S-shaped bodies, whereas Maclura pomifera (MPA), Triticum vulgaris (WGA) and, after neuraminidase treatment, Arachis hypogaea (PNA) agglutinins bound intensely to the presumptive podocytes. During later stages of nephrogenesis, MPA positively on the podocytes weakened and could not be observed in adult kidney glomeruli. Binding sites for Helix pomatia (HPA) agglutinin in glomeruli were also expressed only transiently during nephrogenesis. During further development PSA, ConA, WFA, and RCA-I reacted with mesangial cells in addition to the glomerular basement membranes. The segment-specific lectin binding patterns of the tubuli emerged in parallel with the appearance of brush border and Tamm-Horsfall antigens of the proximal and distal tubuli. The results show that nephron site-specific saccharides appear in a developmentally regulated manner and in parallel with morphologic maturation of the nephron. Lectins therefore appear to be useful tools for study of induction and maturation of various nephron cell types.

Further Characterization Of Platelet Membrane Glycoproteins

1981 ◽  
Author(s):  
P Clezardin ◽  
J L McGregor ◽  
K J Clemetson ◽  
M Dechavanne ◽  
E F Lüscher
Keyword(s):  
Ricinus Communis ◽  
Lectin Binding ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Membrane Glycoproteins ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Reducing Conditions ◽  
Surface Labelling

The binding of 125I-labelled lectins to major and minor platelet glycoproteins (GP) and their subunits has been investigated. Human platelets were isolated, washed, solubilized in sodium dodecyl sulphate (SDS) under non-reducing conditions and separated on 5, 7.5 and 10 % non-reduced/reduced 2-D polyacrylamide gels. The gels were incubated with 125I-labelled lectins; Lens culinaris lectin (LCL), concanavalin A (ConA) wheat germ agglutinin (WGA) or Ricinus communis agglutinin (RCA-120), then washed extensively dried and exposed to X-ray film by indirect autoradiography. Surface-labelled platelets were similarly separated. WGA and RCA bound predominantly to GPIbα but also to two minor bands above and below it which were affected by neuraminidase treatment. One of them bound two 125I-lectins (LCL and ConA) while GPIbα did not. Additional GP bands were detected by lectin binding and by surface-labelling beneath GPIIIβ (IV). With platelets labelled by the neuraminidase/galactose oxidase/NaB3H4 method a GP was detected between Ila and Ilia which was not found with periodate/ NaB3H4 labelling (not affected by reduction). Two spots on the diagonal bound LCL and ConA. GP Ibβ bound LCL more strongly than IIbp. GPIbp also bound WGA and RCA. GPIcβ apparently bound only ConA. GPIbβ and IIbβ were labelled equally strongly by surface labelling techniques, Icβ was apparently not labelled. Further GP subunits were detected one below Ibβ and IIbβ and another which originated in the GPVII region. These techniques demonstrate that the platelet surface is even more complex than previously thought.

scholarly journals A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane

1976 ◽  
Vol 153 (2) ◽  
pp. 265-270 ◽  
Author(s):  
M J A Tanner ◽  
D J Anstee
Keyword(s):  
Ricinus Communis ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Erythrocyte Membrane ◽  
Individual Pattern ◽  
Direct Demonstration ◽  
Staining Techniques ◽  
Membrane Components

1. A method which allows the characterization of lectin-binding components is described. This method should be useful in defining the nature and heterogeneity of these components in cell membranes. 2. The method, which we have used on erythrocyte “ghosts”, involves the fixation of “ghost” components after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and incubation with purified 125I-labelled lectins. 3. Each of the four lectins used shows an individual pattern of reactivity towards “ghosts” components. Band 3, the major membrane-penetrating glycoprotein, is bound by the lectins from Ricinus communis and Phaseolus vulgaris (phytohaemagglutinin) and by concanavalin A. The major erythrocyte sialoglycoprotein is bound by the lectins from R. communis, P. vulgaris and Maclura aurantiaca. 4. Three of the lectins displays binding for other membrane components, some of which are not demonstratable by conventional protein- and carbohydrate-staining techniques.

scholarly journals Lectin-binding proteins in central-nervous-system myelin. Binding of glycoproteins in purified myelin to immobilized lectins

1979 ◽  
Vol 183 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Richard H. Quarles ◽  
Laurence J. McIntyre ◽  
Carol F. Pasnak
Keyword(s):  
Rat Brain ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Preceding Paper ◽  
The Other ◽  
Double Labelling ◽  
Myelin Associated Glycoprotein

The capacities of immature and mature rat brain myelin, bovine myelin and human myelin to be agglutinated by soya-bean agglutinin, Ricinus communis agglutinin, wheatgerm agglutinin, and Lotus tetragonolobus agglutinin were examined. The first two lectins, which are specific for galactose and N-acetylgalactosamine, strongly agglutinated immature and mature rat myelin, weakly agglutinated bovine myelin, but did not affect human myelin. The other myelin and lectin combinations resulted in very weak or no agglutination. [3H]Fucose-labelled glycoproteins of purified adult rat brain myelin were solubilized with sodium dodecyl sulphate and allowed to bind to concanavalin A–Sepharose and each of the other lectins mentioned above, which had been immobilized on agarose. About 60% of the radioactive fucose was in glycoproteins that bound to concanavalin A–Sepharose and these glycoproteins could be eluted with solutions containing methyl α-d-mannoside and sodium dodecyl sulphate. Periodate/Schiff staining or radioactive counting of analytical gels showed that most of the major myelin-associated glycoprotein (apparent mol.wt. approx. 100000) bound to the concanavalin A, whereas the glycoproteins that did not bind were mostly of lower molecular weight. Preparative polyacrylamide-gel electrophoresis of the glycoprotein fraction that was eluted with methyl α-d-mannoside yielded a relatively pure preparation of the myelin-associated glycoprotein. Similar results were obtained with each of the other lectins, i.e. the myelin-associated glycoprotein was in the fraction that bound to the immobilized lectin. Double-labelling experiments utilizing [3H]fucose-labelled glycoproteins from adult myelin and [14C]fucose-labelled glycoproteins from 14-day-old rat brain myelin did not reveal any difference in the binding of the mature and immature glycoproteins to any of the immobilized lectins. The results in this and the preceding paper [McIntyre, Quarles & Brady (1979) Biochem. J.183, 205–212] suggest that the myelin-associated glycoprotein is one of the principal receptors for concanavalin A and other lectins in myelin, and that this property can be utilized for the purification of this glycoprotein.

Specificity of mycoparasite attachment to the host cell surface

1985 ◽  
Vol 63 (4) ◽  
pp. 772-778 ◽  
Author(s):  
M. S. Manocha
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Recognition ◽  
Binding Reaction ◽  
Choanephora Cucurbitarum ◽  
Host Cell Surface ◽  
Germ Tubes

The use of isolated cell wall fragments of Choanephora cucurbitarum (Berk. & Rav.) Thaxter (a host), and of Linderina pennispora Raper and Fennell (a nonhost), has provided not only a convenient method to quantify attachment of the parasite, Piptocephalis virginiana Leadbeater and Mercer, by the artificial inoculation and washing-off procedure, but also an excellent material for investigations on the molecular basis of specificity and host recognition. The parasite germ tubes are attached to the cell wall fragments of the host but not of the nonhost. Attachment was inhibited by the addition of sugars, chitobiose and chitotriose, and by treatment with acid or alkali indicating the involvement of proteins or glycoproteins in recognizing sugar residues at the cell surface. Both host and nonhost showed a positive binding reaction with fluorescent lectins specific for N-acetyl-D-glucosamine oligomer. The cell surface of the nonhost also contains D-galactose and N-acetyl-D-galactosamine residues as lectin binding sites. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of cell wall extracts of host and nonhost revealed four bands of glycoproteins common to both fungi and two were specific to the host.

scholarly journals A lectin-binding glycoprotein of Mr 135,000 associated with basal keratinocytes in pig epidermis

1986 ◽  
Vol 237 (2) ◽  
pp. 405-414 ◽  
Author(s):  
I A King ◽  
A Tabiowo ◽  
F M Pope
Keyword(s):  
Basal Cell ◽  
Concanavalin A ◽  
High Speed ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Basal Layer ◽  
Particulate Fraction ◽  
Ulex Europaeus ◽  
Stratified Epithelia

Pig epidermis separated by 1 M-CaCl2 treatment was homogenized and separated into three fractions by filtration through nylon mesh and high-speed centrifugation. Lectin-binding glycoproteins were isolated from urea/deoxycholate/mercaptoethanol extracts of the residue fraction that resisted filtration, from deoxycholate extracts of the particulate material in the filtrate and from the soluble fraction. Concanavalin A, Ricinus communis (castor bean) agglutinin 1, peanut (Arachis hypogaea) agglutinin and Ulex europaeus (gorse) agglutinin-binding glycoproteins in the three epidermal fractions were analysed by SDS/polyacrylamide-gel electrophoresis. A major neuraminidase-sensitive glycoprotein component of the particulate fraction of Mr 135,000 was strongly bound by concanavalin A and Ricinus communis agglutinin 1, but only weakly by peanut and Ulex europaeus agglutinins. This glycoprotein was not detected in the residue or soluble fractions of the epidermis, indicating that it had only a limited distribution within the tissue. The 135,000-Mr glycoprotein was one of two major glycoprotein antigens in the particulate fraction. Rabbits immunized with total particulate glycoproteins produced antibodies directed mainly against 135,000- and 110,000-Mr components. Monospecific antibodies were obtained from guinea pigs immunized with the 135,000-Mr glycoprotein band excised from polyacrylamide gels. Indirect immunofluorescence with the use of affinity-purified antibodies showed that the 135,000-Mr glycoprotein was present at the surface of cells in the basal layer of the epidermis as well as at that of other stratified epithelia. It was not present on differentiating cells in the suprabasal layers of the epithelium, suggesting an important role in the attachment or proliferative functions of basal cells in stratified epithelia. Metabolic labelling studies with skin explants cultured in the presence of D-[3H]glucosamine showed that this basal-cell glycoprotein was synthesized by cultured tissue. The major D-[3H]glucosamine-labelled glycoprotein component in the residue and particulate fractions of cultured epidermis had an Mr of 135,000, was immunoprecipitated by rabbit antisera raised against particulate epidermal glycoproteins and was bound by concanavalin A. The labelling of this glycoprotein with D-[3H]glucosamine was sensitive to tunicamycin, indicating that the basal-cell glycoprotein contained N-glycosidically linked oligosaccharides.

The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides

1988 ◽  
Vol 66 (4) ◽  
pp. 273-278 ◽  
Author(s):  
C. Anthony Rupar ◽  
Jeffery D. Whitehall
Keyword(s):  
Membrane Proteins ◽  
Rat Liver ◽  
Concanavalin A ◽  
Ricinus Communis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peanut Agglutinin ◽  
Ulex Europaeus ◽  
Ricinus Communis Agglutinin ◽  
Lysosome Membrane

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze–thaw shock and membranes were isolated. The release of β-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.

scholarly journals Altered Cell Surface N-Glycosylation of Resting and Activated T Cells in Systemic Lupus Erythematosus

2019 ◽  
Vol 20 (18) ◽  
pp. 4455 ◽  
Author(s):  
Enikő Szabó ◽  
Ákos Hornung ◽  
Éva Monostori ◽  
Márta Bocskai ◽  
Ágnes Czibula ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Lupus Erythematosus ◽  
Lectin Binding ◽  
Systemic Lupus ◽  
Activated T Cells ◽  
Neuraminidase Treatment ◽  
Altered Cell

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE.

scholarly journals Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield

1981 ◽  
Vol 197 (2) ◽  
pp. 519-522 ◽  
Author(s):  
E G Afting ◽  
M L Recker
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Acid Proteinase ◽  
Chromatographic Purification

Cathepsin D was purified by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin-Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.

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