Lectin-like Substance Research Articles

Membrane glycoproteins and plant and animal proteins with lectin or lectin-like properties.

The fat cell, chiefly from porcine greater omentum, was treated with hypertonic sodium chloride and membranes left over from exuded fat were extracted with Triton X-100 and the extract subjected to gel electrophoresis. Of the 20 or more bands which showed on the gel, at least five were periodate-Schiff positive. By the treatment of the glycoproteins with Con A, followed by DEAE-cellulose, one with an apparent molecular weight of 74 kDA was obtained pure. The other components are not yet purified. However, two glycoproteins of apparent molecular weights of 89 and 112 kDa were obtained from the fat cell membrane by affinity chromatography. These were apparently lectins, since they possessed hemagglutinating activity and specificity towards maltose and caused adhesion among cells (erythrocytes) and aggregation of fat cells. The properties of lectins from various sources have also been studied: rice germ lectin, Allium sativum lectin, a lectin from a species of silkworm and one from loquat leaves. A crystalline protein called pinellin was isolated from Pinella ternata. Since it has been shown to exhibit cell aggregation and mitogenic activity, it is also a lectin, showing species as well as cell type specificity. Preliminary studies have also been made of the binding of pinellin to its receptor on fat cells. The protein has also been shown to possess an anti-implantation effect in rabbits. Studies have been made of membrane proteins on ram spermatozoa and Con A receptors on squamous epithelial cancer cells.

Separation of carbohydrates and lectins on carbohydrate-immobilized resins

A few carbohydrate-immobilized resins were prepared by coupling glycamines of mono- and disaccharides to an epoxy-activated methacrylate base, and the elution profiles of various carbohydrates and lectins from columns packed with the resultant resins were compared. The introduction of glucamine, maltamine or lactamine to the methacrylate base had little effect on the separation of stereoisomers of aldoses, though it enabled group separation of pentoses as well as 6-deoxyhexoses from hexoses. On the other hand, the maltamine-immobilized resin showed strong specific affinity for concanavalin A, as well as lentil and pea lectins. The lactamine-immobilized resin was specific to ricinus agglutinin, peanut agglutinin and soy bean agglutinin. These carbohydrate-immobilized resins were also useful for the isolation of a lectin-like substance from rat liver.

Mapping of the rat liver endothelial membrane with lectins and glycosylated ferritins.

We explored the luminal surface of liver sinus endothelium for the presence of lectin receptors and lectinlike substances capable of interacting with specific sugars. We used ferritin-conjugated lectins and glycosylated ferritins as probes. Incubation of small blocks of rat liver with these probes led to the binding of concanavalin A (on A), Ricinus communis (RCA), wheat germ agglutinin (WGA), phytohemagglutinin (PHA) and mannosyl ferritins to the luminal surface of endothelium. Ulex europaeus agglutinin I (UEA), fucosyl, galactosyl, and chitobiosyl-ferritins did not bind. The binding was patchy and sparse in the case of Con A and mannosyl-ferritins but uniform for others. Binding density did not correlate with hemagglutinability of lectins, suggesting that the difference in the hemagglutinability of these lectins did not account for the difference in their binding densities. Bindings were all completely inhibited in the presence of excess specific sugar inhibitors, indicating the specificity of binding. The distribution of binding was segregated on the endothelial membrane, being heaviest on luminal pits. To define the functional significance of this segregated distribution, sinus endothelium was compared to portal-vein endothelium in which endothelial fenestrations are also seen; and these fenestrations as well as pits may be covered by a thin diaphragm. Of interest was the total absence of binding to the diaphragm. The significance of these findings is discussed.

Morphological changes of rat small intestine after short-time exposure to concanavalin A or wheat germ agglutinin.

Dietary lectins of gluten origin have been suggested to play an important role in the mechanisms leading to the characteristic morphology of the intestine found in patients with celiac disease. To further explore this issue we have used Wheat Germ Agglutinin (WGA) or Concanavalin A (Con A) to challenge rat small intestine and study the ultrastructural changes of such a treatment. Both lectins affected the enterocytes at the base of the villi more than those at the top. The morphological findings included disarrangement of the cytoskeleton, increased endocytosis and shortening of the microvilli. The interrelationship between the observed changes, and their relevance for similar morphological alterations found in patients with celiac disease are discussed. In conclusion, the morphological findings in our rat model resemble early changes in patients with celiac disease, thus supporting the idea that lectins or lectin-like substances are involved in the pathogenesis of this disease.

Expression of lectin receptors on the surface of granulocyte‐macrophage progenitor cells (cfu‐gm)

It has been emphasized that specific bindings between membrane glycoproteins and membrane lectin-like substances are important in cell-to-cell interactions. We explored the surface of granulocyte-macrophage precursor cells (CFU-gm) by the differential agglutination technique. Enrichment of CFU-gm in the agglutinated fraction, containing the cells which have lectin receptors, from marrow treated with soybean agglutinin (SBA), peanut agglutinin (PNA) and concanavalin A (Con A), suggests the presence of reactive galactosyl and mannosyl residues on the surface of CFU-gm. On the other hand, wheat germ agglutinin (WGA), phytohemagglutinin (PHA) and ulex europaeus agglutinin (UEA), which bind to reactive N-acetylglucosamine, N-acetylgalactosamine and fucose, respectively, did not specifically agglutinate CFU-gm. Thus, reactive groups containing galactosyl and mannosyl structures on the surface of CFU-gm may possibly play a role in the process of cell-to-cell interactions between CFU-gm and marrow stromal cells.

Expression of lectin receptors on the surface of granulocyte-macrophage progenitor cells (CFU-gm).

It has been emphasized that specific bindings between membrane glycoproteins and membrane lectin-like substances are important in cell-to-cell interactions. We explored the surface of granulocyte-macrophage precursor cells (CFU-gm) by the differential agglutination technique. Enrichment of CFU-gm in the agglutinated fraction, containing the cells which have lectin receptors, from marrow treated with soybean agglutinin (SBA), peanut agglutinin (PNA) and concanavalin A (Con A), suggests the presence of reactive galactosyl and mannosyl residues on the surface of CFU-gm. On the other hand, wheat germ agglutinin (WGA), phytohemagglutinin (PHA) and ulex europaeus agglutinin (UEA), which bind to reactive N-acetylglucosamine, N-acetylgalactosamine and fucose, respectively, did not specifically agglutinate CFU-gm. Thus, reactive groups containing galactosyl and mannosyl structures on the surface of CFU-gm may possibly play a role in the process of cell-to-cell interactions between CFU-gm and marrow stromal cells.

Identification of Lectin-like Substances Recognizing Galactosyl Residues of Glycoconjugates on the Plasma Membrane of Marrow Sinus Endothelium

Plasma membrane components capable of specific binding to the sugar residues of glycoproteins have recently been identified in several cell systems and implicated in the recognition and specific uptake of soluble or membrane-bound glycoproteins. Because the endothelium of bone marrow sinuses is the site of massive cellular and molecular traffic that is often specific, we studied the surface of sinus endothelium for the presence of sugar-recognizing systems. Neoglycoprotein probes were synthesized by covalently binding bovine serum albumin (BSA) to activated pyrannose form of galactose, fucose, or mannose. The probe was then labeled with colloidal gold. Galactosyl-BSA gold bound to endothelial membrane at 4 degrees C and was internalized at 37 degrees C. Both the binding and the internalization were inhibited in the presence of excess unlabeled galactosyl-BSA. Gold-labeled mannosyl-BSA and fucosyl-BSA did not bind to the endothelium. Nor did BSA-gold without galactosyl residues bind to endothelial membrane, confirming that galactosyl moiety was responsible for the binding. The uptake of galactosyl-BSA was further confirmed by perfusion of 125I-galactosyl-BSA into the abdominal aorta. Small but highly specific uptake was noted in tibias and femurs. These data provide evidence for the presence of a lectin-like substance on the luminal surface of marrow endothelial membrane capable of specific interaction with galactosyl residues of circulating and cell-bound glycoproteins and may provide a mechanism for specific recognition of these glycoproteins.

Identification of lectin-like substances recognizing galactosyl residues of glycoconjugates on the plasma membrane of marrow sinus endothelium

Plasma membrane components capable of specific binding to the sugar residues of glycoproteins have recently been identified in several cell systems and implicated in the recognition and specific uptake of soluble or membrane-bound glycoproteins. Because the endothelium of bone marrow sinuses is the site of massive cellular and molecular traffic that is often specific, we studied the surface of sinus endothelium for the presence of sugar-recognizing systems. Neoglycoprotein probes were synthesized by covalently binding bovine serum albumin (BSA) to activated pyrannose form of galactose, fucose, or mannose. The probe was then labeled with colloidal gold. Galactosyl- BSA gold bound to endothelial membrane at 4 degrees C and was internalized at 37 degrees C. Both the binding and the internalization were inhibited in the presence of excess unlabeled galactosyl-BSA. Gold- labeled mannosyl-BSA and fucosyl-BSA did not bind to the endothelium. Nor did BSA-gold without galactosyl residues bind to endothelial membrane, confirming that galactosyl moiety was responsible for the binding. The uptake of galactosyl-BSA was further confirmed by perfusion of 125I-galactosyl-BSA into the abdominal aorta. Small but highly specific uptake was noted in tibias and femurs. These data provide evidence for the presence of a lectin-like substance on the luminal surface of marrow endothelial membrane capable of specific interaction with galactosyl residues of circulating and cell-bound glycoproteins and may provide a mechanism for specific recognition of these glycoproteins.

Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c3 from various Desulfovibrio.

Stage‐Dependent Toxicity of N‐Acetyl‐Glucosamine to Plasmodium falciparum1

The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of 3H-hypoxanthine. Inhibition of 3H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.

Synthetic neoglycoproteins: a class of regents for detection of sugar-recognizing substances.

Specific interactions between proteins and sugars have recently been emphasized in many biological systems. To detect sugar-recognizing substances, known as lectin-like substances or endogenous lectins, we describe a method in which various sugars were covalently bound to a carrier protein such as albumin. This neoglycoprotein was stable at -20 degrees C for a period of 6 months. It was conjugated to various cytochemical markers (125I, fluorescein isothiocyanate, colloidal gold, or latex minibead). Detection of the marker then indicates the presence of the sugar-binding protein. Control experiments in the presence of unlabeled neoglycoprotein or specific sugar indicated the specificity of the reaction. This method was used to analyze the kinetics of binding for a mannose-recognition system in the mouse peritoneal macrophages. The data obtained were in agreement with those previously reported. The method can be used for detection of other sugar-recognizing systems as virtually every simple sugar can be bound to a carrier protein to produce these neoglycoproteins. Some of the consideration required for successful production of these reagents are discussed. These synthetic neoglycoproteins are useful in studying the distribution and kinetics of sugar-recognizing systems and may help to further our understanding of this rapidly developing area.

A possible mechanism for the cellular coaggregation between Actinomyces viscosus ATCC 19246 and Streptococcus sanguis ATCC 10557.

The cells of Actinomyces viscosus ATCC 19246 (Av19246) and Streptococcus sanguis ATCC 10557 (Ss10557) coaggregated immediately after mixing in 40 mM-Tris/HCl buffer. Optimal conditions were pH 7.5 in the presence of Ca2+ at 0.1 mM or higher. Na2 EDTA and its analogues, Na2MgEDTA and Na2MnEDTA at 7.5 mM inhibited the coaggregation. Trypsin and heat treatment impaired the reactive site on Av19246 cells, but not on Ss10557 cells. The coaggregates, once formed, dissociated gradually during extended incubation at 37 degrees C; this was prevented by addition of sufficient Ca2+. The disaggregation appears to be a spontaneous denaturation of the proteinaceous reactive site on Av19246 cell surface. Thus, the coaggregation involves the interaction of a lectin-like substance on the surface of Av19246 with a carbohydrate site on Ss10557. Native Ss10557 cell walls possessed reactivity with Av19246 cells but 5% (w/v) TCA-extracted cell wall residues did not. A carbohydrate moiety extracted from Ss10557 exhibited a high potency in blocking coaggregation, and coaggregates were dissociated upon addition of the carbohydrate. Lactose, galactose and N-acetyl-D-galactosamine (the latter two are major constituents of the antigen extract) also significantly inhibited the coaggregation, but the other antigen components, glucose and rhamnose, did not. Relative inhibitory activity, expressed as molar potency, of carbohydrate antigen, lactose, galactose and N-acetyl-D-galactosamine respectively, was approximately 26 X 10(3):16:4:1. Ss10557 cells and cell walls reacted only with a Ricinus communis (castor bean) agglutinin-120 but not with Glycine max (soybean) agglutinin, Arachis hypogaea (peanut) agglutinin or Phaseolus vulgaris agglutinin (phytohaemagglutinin).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of Eikenella corrodens adherence to human buccal epithelial cells and partial purification of the bacterial lectin-like substance from bacterial cells which participate in adherence

Eikenella corrodens のヒト頬粘膜上皮細胞への付着機構を in vitro で解析した結果, E. corrodens 1073は, その菌体成分である細菌レクチン様物質を介して頬粘膜上皮細胞表層のN-アセチル-D-ガラクトサミンあるいはD-ガラクトース類似レセプターに付着することが示唆された。この付着に関与する細菌レクチン様物質をE. corrodens 1073の細胞エンベロープから Triton X-100抽出, アフィニティクロマトグラフィー, およびゲル濾過により1つの強固に結合した複合体として部分精製した。そしてこのレクチン様物質は, E. corrodens 1073の頬粘膜上皮細胞への付着特性と同一様式をとることから, これがヒト頬粘膜上皮細胞への付着に関与する決定的な菌体側因子であることを明確にした。

Mapping of the bone marrow sinus endothelium with lectins and glycosylated ferritins: Identification of differentiated microdomains and their functional significance

The distribution of lectin binding sites and sugar-recognizing systems (lectin-like substances) was studied on the luminal side of the bone marrow sinus endothelium in rats. Ferritin-conjugated lectins (Con A, PHA, RCA, WGA, UEA) and glycosylated ferritins (mannosyl, fucosyl, chitobiosyl) were used as probes. With the exception of UEA, all lectins bound to the endothelial surface. The binding was heavier on the plasmalemma proper of the nuclear region (PP-N) compared to that of the tapered region (PP-T). Lectin mapping also identified differentiated microdomains with less or no bindings. These domains related to the transport organelles in the endothelium: luminal vesicles (LV) and diaphragmmed fenestrae (DF). The relative density of binding for these four domains demonstrated the following spectrum PP-N greater than PP-T greater than LV greater than DF. The relation of this binding pattern to the transport function of marrow sinus endothelium for various cells and molecules has been discussed. Lectin mapping also identified heavily labeled microvilli and microprojections from the membrane. These organelles may have a function in recognizing cells and molecules for the transport. The absence of binding for UEA, also absent in liver endothelium, may differentiate sinus endothelia from other endothelia. This lectin is thought to be endothelium-specific in other systems. No binding was observed with glycosylated ferritins suggesting the absence of lectin-like substances on marrow sinus endothelium.

Inhibition of growth of hair follicles by a lectin-like substance from rat skin.

A small molecular weight (5 000-10 000) substance has been isolated from rat skin by affinity chromatography on a column of acid-hydrolysed Sepharose. The substance agglutinates rabbit red blood cells, inhibits DNA synthesis in rat hair follicles, and causes the appearance of autophagic vacuoles in the epithelial cells of the lower follicle bulb.

Interaction of bacteria and fungi with lectins and lectin-like substances.

INTRODUCTION . Background .. What Are Lectins? . Areas of Study . Scope of This Review ..

Effects of aloe extracts on human normal and tumor cells in vitro

Fractions of leaf extracts from 2 local types, labeledAloe vera (subsequently identified asAloe barbadensis Mill, andA. saponaria Haw.), were prepared by differential centrifugation and tested by in vitro assays for the presence of lectinlike activities and for effects on the attachment and growth of human normal and tumor cells. Fractions of extracts of fresh leaves and commercially “stabilized”Aloe vera gel had high levels of lectin-like substances measured by immunodiffusion and hemagglutination assays. Substances in fluid fractions from both fresh leaf sources were found to markedly promote attachment and growth of human normal, but not tumor, cells and to enhance healing of wounded cell monolayers. In contrast, fractions of “stabilized”Aloe vera gel were equally cytotoxic for human normal and tumor cells in vitro. Results from cell assays suggested that the observed growth promotion and wound healing effects of aloe substances in vitro may be analogous to what has been observed in vivo during healing of wounds and burns.

Interaction of parotid saliva basic glycoprotein with Streptococcus sanguis ATCC 10557.

It is postulated that an initial step in dental plaque formation is the adherence of oral bacteria to the salivary pellicle. Recently, we have found that a proline-rich and basic glycoprotein (MGP) from human parotid saliva, which is successfully purified by Concanavalin A-Sepharose affinity chromatography, binds to some oral streptococci such as S. mitis and S. sanguis. This paper deals with some inhibitors which affect the binding of the MGP to S. sanguis ATCC 10557. The assay for the binding ability of the radioactive MGP to the bacterial cells was performed by incubation of the reaction mixture containing 10 microgram of [3H]MGP (6000 dpm) and about 4.5 mg of bacterial cells (dry weight) in 0.5 ml of 0.05 M Tris-HCl buffer containing 0.05 M NaCl, pH 8.0 with a final volume of 0.51 ml. After 1 hour standing at 4 degrees C, the cells were washed five times with the same buffer. The resulting sediment was solubilized in 0.5 ml of NCS tissue solubilizer and the radioactivity was measured. The binding of the radioactive MGP to bacterial cells was specifically inhibited by galactose, lactose and N-acetyllactosamine. Applications of heat and trypsin on the cell surface, strikingly reduced the binding ability. These findings strongly suggested that a lectin-like substance may be present on the bacterial cell surface.

Lectin-like activity of components extracted from human glomerular basement membrane.

Different fractions of human glomerular basement membranes have been isolated by enzymatic and chemical methods. These fractions were analyzed for their chemical structure and biological activity. The hypothesis that human glomerular basement membrane glycopeptide fractions related to collagenous sequences could act as a lectin-like substance was explored; in fact, extracellular glycoprotein factors play a role in cell-cell interactions and cell adhesion. These collagenous glycopeptides agglutinate human transformed or embryonic cells within 2 hr. The cell agglutination is inhibited by the following sugars: N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. In addition, the rapid cytoagglutination is followed by a cell spreading effect after a further 20-hr incubation. These data led to the postulate that the maintenance of tissue differentiation is governed by the interaction of cells with peculiar sites of the basement membrane consisting of a proteolysis-resistant association between collagen and matrix glycoprotein.

Laser nephelometry of lectin-glycoprotein complexes: A new method for their quantitation and differentiation

The quantitative determination of glycoproteins and lectins by laser nephelometry is based on the fact that these two compounds form a complex comparable to the antigen-antibody precipitate. Due to the high sensitivity and reliability of this technique less than 25 pmol of glycoprotein or 4.5 pmol of lectin can be determined, thus providing a suitable method (a) to investigate the specificity of different lectins for certain carbohydrate residues, (b) to study the mode of interaction, and (c) to explore mammalian tissue for still-unknown lectin-like substances.