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 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.

Echinostoma caproni and E. trivolvis alter the binding of glycoconjugates in the intestinal mucosa of C3H mice as determined by lectin histochemistry

1993 ◽  
Vol 67 (3) ◽  
pp. 179-188 ◽  
Author(s):  
T. Fujino ◽  
B. Fried
Keyword(s):  
Ricinus Communis ◽  
Villous Atrophy ◽  
Lectin Binding ◽  
Goblet Cells ◽  
Lectin Histochemistry ◽  
Dolichos Biflorus ◽  
Echinostoma Caproni ◽  
Ulex Europaeus ◽  
C3h Mice ◽  
Small Intestines

AbstractMouse (C3H) mucosal glycoconjugates were examined in normal small intestines and intestines infected with Echinostoma caproni, or E. trivolvis using six different fluorescein-conjugated lectins: Triticum, vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin I (RCA-I). Glycine max soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and Arachis hypogaeu peanut agglutinin (PNA). The expression of lectin-binding sites and the intensity of the binding of lectins in the mouse small intestines were changed by infection with the echinostomes. Specific differences in the reaction to glycoproteins were clearly observed between the mouse intestines infected with E. caproni and those infected with E. trivolvis. In E. caproni infection, binding of most of the lectins to the villi was remarkably reduced in accord with the villous atrophy and loss of goblet cells. In contrast, in E. trivolvis infection, the binding of WGA, RCA-I and DBA was reduced in the microvillar surfaces, but binding of UEA-I and SBA were unchanged compared to the control intestines. The lectin binding to goblet cells in E. trivolvis-infected mice mostly increased. These observations may reflect the marked increase in goblet cells and the less severe damage in the villi of E. trivolvis infection compared to E. caproni infection. Most of the glycoconjugates were slightly reduced in the hyperplastic crypts except for N-acetyl glucosamine. It is possible that glucose metabolism in the host intestines infected with E. trivolvis was activated. resulting in an increase in the rate of mucin synthesis as well as qualitative changes in mucus, thereby mediating the expulsion of the worms.

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.

Changing surface antigen and carbohydrate patterns during the development of Oesophagostomum dentatum

Parasitology ◽  
1999 ◽  
Vol 119 (5) ◽  
pp. 491-501 ◽  
Author(s):  
A. JOACHIM ◽  
B. RUTTKOWSKI ◽  
A. DAUGSCHIES
Keyword(s):  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Soybean Agglutinin ◽  
Peanut Agglutinin ◽  
Oesophagostomum Dentatum ◽  
Periodate Treatment

Living and fixed specimen of Oesophagostomum dentatum were labelled in situ with serum antibodies or a panel of biotin- labelled lectins. Specific binding of antibodies was observed in all parasitic stages – freshly exsheathed 3rd-stage larvae (L3), 3rd- and 4th-stage (L4) larvae cultured in vitro and L3 and L4 and adults isolated from pig intestines. The shedding of the stained layer by motile larvae was inhibited by levamisole-induced paralysis. Larvae cultured in vitro exposed serum-derived proteins on their surface which could be labelled with secondary antibody directed against the respective serum donor species. While freshly exsheathed larvae were recognized by O. dentatum-positive serum only, older larvae and adults cross-reacted with serum from pigs infected with O. quadrispinulatum, a closely related species. Lectin binding varied considerably between stages. While binding was not observed in pre-parasitic stages, Concanavalin A, Soybean Agglutinin, Wheat Germ Agglutinin, Ricinus communis Agglutinin and Peanut Agglutinin bound to developing larvae in varying degrees. Dolichos biflorus Agglutinin only bound to advanced (luminal) larval stages, while adults generally displayed only weak or partial lectin binding (except with Concanavalin A and Wheat Germ Agglutinin). Ulex europaeus Agglutinin only labelled larvae derived from cultures containing 10% pig serum. Cleavage of the carbohydrate residues by sodium periodate treatment resulted in reduction of antibody binding to cultured larvae, but not to freshly exsheathed L3. Concanavalin A, Soybean Agglutinin, and Peanut Agglutinin binding was also reduced by periodate treatment, while binding of Wheat Germ Agglutinin and Ricinus communis Agglutinin was inhibited only in early L3, but not in older stages. The different lectin labelling patterns are related to the different stages of the nematode – infective, invasive, histotropic, and luminal – and may serve as a mode of adaptation for the parasite against the host's immune attack by surface glycoprotein variation, together with antigen shedding (as demonstrated by labelling of motile larvae) and a possible acquisition of host molecules at the parasite's surface. Furthermore, a possible role of this developmental variation in surface carbohydrates in parasite–parasite interactions is discussed.

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.

scholarly journals Localization of carbohydrate components in rat colon with fluoresceinated lectins.

1978 ◽  
Vol 26 (6) ◽  
pp. 452-458 ◽  
Author(s):  
E Essner ◽  
J Schreiber ◽  
R A Griewski
Keyword(s):  
Epithelial Cells ◽  
Concanavalin A ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Rat Colon ◽  
Dolichos Biflorus ◽  
Ulex Europaeus ◽  
Descending Colon ◽  
Carbohydrate Components ◽  
Cryostat Sections

Cryostat sections of rat descending colon were studied by fluorescence microscopy after exposure to conjugates of fluorescein isothicoyanate with lectins from Glycine max (soybean), Triticum vulgaris (wheat germ), Ricinus communis (castor bean), Ulex europaeus, (gorse), Dolichos biflorus (horse gram) and Canavalia ensiformis (concanavalin A) (Jack bean). No two lectins showed identical patterns of fluorescence. FITC-conjugates of soybean and D. biflorus lectins reacted strongly with the mucus present in the crypt lumens and with the surface (as well as cytoplasm) of the epithelial cells suggesting that these sites are rich in terminal, non-reducing, N-acetylgalactosamine residues. Wheat germ, R. communis, U. europaeus and concanavalin A-FITC conjugates did not stain mucus but showed fluorescence in the cytoplasm of absorptive cells as well as in the lamina propria and submucosa. The FITC-R. communis conjugate also reacted with structures in the apical portion of epithelial cells that may correspond to the Golgi apparatus.

scholarly journals A comparison of lectin binding in rat and human peripheral nerve.

1986 ◽  
Vol 34 (11) ◽  
pp. 1487-1493 ◽  
Author(s):  
A K Gulati ◽  
A A Zalewski ◽  
K B Sharma ◽  
D Ogrowsky ◽  
G S Sohal
Keyword(s):  
Peripheral Nerves ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Nerve Degeneration ◽  
Binding Affinities ◽  
Light Microscopic Level ◽  
Dolichos Biflorus ◽  
Ulex Europaeus ◽  
Maclura Pomifera ◽  
Subsequent Regeneration

Eleven different fluorescein- or peroxidase-conjugated lectins with different sugar-binding affinities were employed to analyze and compare glycoconjugates of rat and human peripheral nerves at the light microscopic level. A majority of lectins showed a distinct binding pattern in different structures of the nerve. Lectin binding was similar but not identical in rat and human nerves. Limulus polyhemus agglutinin did not stain any structures in rat or human nerves. In both species, all other lectins bound to the perineurium. Perineurial staining was intense with Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Maclura pomifera (MPA); moderate with Glycine max (SBA), Griffonia simplicifolia-I (GS-I) and GS-II; weak with Ulex europaeus (UEA), Dolichos biflorus (DBA), and Ricinus communis (RCA). In the endoneurium of both species, ConA staining was intense, MPA and WGA moderate, SBA, GS-II, PNA, and RCA weak, and UEA and DBA absent. Interestingly, GS-I stained rat but not human endoneurium. Most lectins bound to blood vessels. GS-I bound to rat but not human, whereas UEA bound to human but not rat vessels. The results show that lectins can be used to reveal heterogeneity in sugar residues of glycoconjugates within neural and vascular components of nerves. They may therefore be potentially useful in detecting changes in glycoconjugates during nerve degeneration and subsequent regeneration after trauma or in pathological states.

Lack of binding of serum glycoprotein hormone α subunit to Concanavalin A–Sepharose reflects increased branching of the oligosaccharide chains

1984 ◽  
Vol 103 (1) ◽  
pp. 111-116 ◽  
Author(s):  
A. J. Chapman ◽  
J. T. Gallagher ◽  
C. G. Beardwell ◽  
S. M. Shalet
Keyword(s):  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Pituitary Tumours ◽  
Con A ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Binding Forms

ABSTRACT The lectin-binding properties of serum α subunit were studied by lectin affinity chromatography. Normal individuals and most patients with pituitary tumours produced α subunit which bound specifically to Concanavalin A–Sepharose (Con A). Some patients with pituitary tumours produced both Con A-reactive α subunit and α subunit which did not bind to Con A. Concanavalin A–Sepharose-binding α subunit from all sources bound strongly to Ricinus communis agglutinin–Sepharose after treatment with neuraminidase. Serum α subunit from those patients with pituitary tumours, which did not bind to Con A, bound to wheat germ agglutinin–Sepharose, exhibiting both weakly binding and strongly binding forms. Serum α subunit from both patients and controls, which did bind to Con A, showed only weak affinity for wheat germ agglutinin–Sepharose. Neither the low affinity nor the high affinity of serum α subunit from any source for wheat germ agglutinin–Sepharose was affected by neuraminidase. These findings show that (a) the predominant pattern of glycosylation of serum α subunit from normal controls is a Con A-reactive, biantennate complex oligosaccharide and (b) that the structural alteration which results in serum α subunit which does not bind to Con A in some patients with pituitary tumours is not an absence of carbohydrate, rather the α subunit contains highly branched, either complex or hybrid oligosaccharides. J. Endocr. (1984) 103, 111–116

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