Distribution and nature of saccharides of plasma membranes of rat ascites hepatoma cells as detected by ferritin-conjugated lectins and dialysed iron

Electron microscopic cytochemical studies were made on saccharides involved in the plasma membranes of rat ascites hepatoma cells (AH7974F) using ferritin-conjugated lectins and dialysed iron (DI). In the rat hepatoma cells, saccharide receptors for each of the three lectins used (concanavalin A (ConA), wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA)) were shown to be distributed homogeneously throughout the plasma membranes. When the cells were agglutinated, however, the saccharide receptors for each lectin appeared to form clusters on the plasma membranes. The cluster formation induced by one lectin was found to lead to a changed distribution of saccharide receptors for another lectin. None of the cluster formation types induced by lectins yield any noticeable effects upon the distribution of DI reactive acidic saccharides on the plasma membranes.     

Lipofuscin,Lipofuscin-Like Pigments and Autofluorescence

A brief overview is here provided on lipofuscin and lipofuscin-like substances, with particular reference to their biological significance as well as to their cellular origin and pathophysiological role. Special emphasis is also placed on the mutual relationships between lipofuscin and lipofuscin-like lipopigments on one side, and cell autofluorescence on the other.Key words: Lipofuscin, lipofuscin-like pigments, ceroid, ceroid lipofuscinosis, autofluorescence, fluorescence microscopy     

Comparative analysis of glycoprotein and glycolipid composition of virulent and avirulent strain membranes ofMycoplasma hyopneumoniae

The glycoproteins and glycolipids from membranes of virulent strain Z and avirulent strain M ofMycoplasma hyopneumoniae have been compared. The proteins and the glycoproteins were identified by SDS-polyacrylamide gel electrophoresis and concanavalin A-biotin labeling, respectively. The membrane preparation contained approximately 34 protein bands with molecular weights between 20 KD and 100 KD. The concanavalin A-biotin system reacted with a glycoprotein of a molecular weight of approximately 28,000 from avirulent strain M and did not react with the correspondent band from virulent strain Z. The membrane glycolipids of both strains consisted of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and the percentages of 160, 180, and 181 fatty acids comprised more than 80% of the total fatty acids of membrane glycolipids. The 180 fatty acid of MGDG in avirulent strain M was twofold higher than that of virulent strain Z.     

Effect of perfluidone on metabolism of lipids in maize (Zea mays L.) and sunflower (Helianthus annuus L.)

Treatment of germinating maize seedlings with 0.126 mM of the herbicide Perfluidone (Destun) (1,1,1-trifluoro-4-[phenylsulfonyl]-methanesulfona-o-toluidide) for 2 days in the dark, then 3 days in the light, at 25°C causes decreases in fresh weight, dry weight, shoot length, and in total chlorophyll and carotenoid contents; in contrast, sunflower seedlings seem not to be affected. Perfluidone causes marked decreases in total lipids and in glyco- and phospholipids of maize seedlings. In sunflower cotyledons, total lipids and pigments (chlorophyll, carotenoids) are not affected, but there is an increase in glycolipids at the expense of phospholipids. After Perfluidone treatment, a significant increase in the fatty acid mole ratio (180+181+182)/183 was found for the maize glycolipids, monogalactosyl diacylglycerol (MGD), digalactosyl diacylglycerol (DGD), and sterol glycoside (SG) + esterified sterol glycoside (ESG), and for the phospholipid, phosphatidylcholine (PC). In sunflower seedlings, however, only the fatty acid mole ratio of ESG + SG showed an increase and that of phosphatidylserine (PS) showed a large decrease. The differential response of the two plant species to Perfluidone suggests that the control of linolenic acid biosynthesis may vary depending on plant species and/or on plant tissues.On leave from the Department of Botany, University of London, Royal Holloway College, Egham, Surrey TW20 OEX England     

Water-soluble poly(acrylamide-allylamine) derivatives of saccharides for protein-saccharide binding studies

Water-soluble poly(acrylamide-allylamine) copolymers containing covalently bound amino groups, prepared by copolymerization of acrylamide and allylamine, can be used as general carriers for coupling of different types of saccharides or saccharide derivatives. The water-soluble macromolecular carbohydrate derivatives can be easily labelled and used in various solid-phase techniques to study protein-saccharide interaction. Two types of coupling reaction were used to prepare polyacrylamide derivatives of saccharides: reductive amination was applied to couple the reducing disaccharides and a carbodiimide reaction was used to couple heparin via its carboxyl groups to the amino groups of the poly(acrylamide-allylamine) derivative. Peroxidase labelled or biotinylated derivatives were shown to be useful in studies on the binding properties of lectins and proteins from boar seminal plasma.This is paper No. LXXXIV of the series Studies on Lectins by J.K.     

An ultrastructural study of the morphology and lectin-binding properties of human mast cell granules

Summary The morphological characteristics and lectin-binding properties of mast cell granules from four human neurofibromata are described. Ultrastructural examination of the granules revealed that some contained dense cores, others had membranous configurations and some forms were intermediate between the two. A round electron-lucent area was present in some granules.After treatment with biotinylated lectins (10 g ml^–1) followed by an avidin-peroxidase revealing system (5 g ml^–1 in 0.125m Tris-buffered saline with 0.347m NaCl, pH 7.6), mast cell granules strongly bound Concanavalin A, garden pea, lentil, wheatgerm, erythro- and leuco-kidney bean lectins. This indicated the presence of abundantN-linked complex-type saccharide sequences. Soybean and peanut lectins showed only weak binding, while the presence of sparse -l-fucosyl terminals was indicated by the weak binding of winged pea lectin. The staining intensity of wheatgerm lectin was considerably reduced when incubated in the presence of its specific competing sugar tri-N-acetylchitotriose.Despite a wide variety of morphological differences between granules, all showed similar staining patterns and all granules within a single cell shared the same binding characteristics.     

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.     

Cell surface receptors for lymphokines. II. Studies on the carbohydrate composition of the MIF receptor on macrophages using synthetic saccharides and plant lectins.

The carbohydrate composition of the surface receptor for macrophage migration inhibitory factor (MIF) on guinea pig macrophages has been studied by examining the interaction of MIF with different saccharides and by testing the ability of plant lectins with known saccharide binding affinities to bind to macrophages and block their response to MIF. Comparison of the effectiveness of a variety of natural and synthetic mono- and disaccharides in inhibiting MIF activity in lymphocyte supernatants revealed that inhibitory activity was confined to natural 5-methylpentose sugars (l-fucose > l-rhamnose = 6-deoxy-d-glucose) and synthetic saccharides containing α-fucosyl residues. Observations on the MIF inhibitory activity of synthetic fucosyl glycosides containing fucosyl residues of defined configuration at terminal and subterminal positions indicate that MIF interacts preferentially with terminal α-l-fucopyranosyl residues and does not recognize subterminal saccharides. Studies with disaccharides containing α-(1 → 2)-, α-(1 → 3), and α-(1 → 6)-linked l-fucosyl residues failed to reveal preferential interaction of MIF with any one linkage configuration. Incubation of macrophages before exposure to MIF with lectins that bind to terminal fucosyl residues (Lotus tetragonolobus and Ulex europaeus_I, agglutinins) rendered them unresponsive to MIF but lectins which bind to nonterminal fucosyl residues and to other saccharides had no effect. The role of fucosyl residues in the binding of MIF by macrophages is discussed with reference to the possible composition of the MIF receptor and the role of fucose-containing glycolipids as receptors for this lymphokine.     

Lectin cytochemistry of cell types in human and canine pituitary

Summary Labeled lectins specific for different sugars were employed to identify different cell types in pituitaries from six human autopsies and seven dogs. To determine the lectins bound by each cell type, fixed-paraffin embedded sections serial to those stained with lectins were immunostained for specific hormones and the serial pairs were examined in a comparison microscope. In human pituitaries corticotrophs stained selectively with lectins having affinity for -l-fucose and the core region of complex type N-glycosyl-proteins. Some corticotrophs also stained for the presence of terminal -galactose. Thyrotrophs stained selectively with a periodate oxidation-borohydride reduction-concanavalin A sequence. Some mammotrophs evidenced content of glycoconjugate with terminal -galactose. Dendritic cells stained selectively for abundant glycogen with the periodate-reduction-concanavalin A sequence and a lectin from Griffonia simplicifolia. Adenohypophyseal cells of dog pituitary differed in showing absence of terminal -galactose in corticotrophs, presence of terminal -galactose in thyrotrophs, presence of glycoconjugate with N-glycosidically bound oligosaccharide in thyrotrophs and gonadotrophs and presence of terminal -galactose with a different lectin affinity in mammotrophs. The main contributions of lectin histochemistry applied to the pituitary include providing an additional histologic method for identification of some cell types, and localizing glycosylated prohormone or other biochemically unrecognized non-hormone glycoconjugates whose function in pituitary cells remains to be explained.This research was supported by NIH Grants AM-10956 and HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant #79     

Lectin localization in human nerve by biochemically defined lectin-binding glycoproteins,neoglycoprotein and lectin-specific antibody

Summary Molecular recognition can be mediated by protein (lectin)-carbohydrate interaction, explaining the interest in this topic. Plant lectins and, more recently, chemically glycosylated neoglycoproteins principally allow to map the occurrence of components of this putative recognition system. Labelled endogenous lectins and the lectin-binding ligands can add to the panel of glycohistochemical tools. They may be helpful to derive physicologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant -galactoside-specific lectin of human nerves in affinity chromatography and in histochemistry to purify and to localize its specific glycoprotein ligands. In comparison to the -galactoside-specific plant lectins fromRicinus communis andErythrina cristagalli, notable similarities were especially detectable in the respective profiles of the mammalian and the Erythrina lectin. They appear to account for rather indistinguishable staining patterns in fixed tissue sections. Inhibitory controls within affinity chromatography, within solid-phase assays for each fraction of lectin-binding glycoproteins and within histochemistry as well as the demonstration of crossreactivity of the three fractions of lectin-binding glycoproteins with the biotinylated Erythrina lectin in blotting ascertained the specificity of the lectin-glycoprotein interaction. In addition to monitoring the accessible cellular ligand part by the endogenous lectin as probe, the comparison of immunohistochemical and glycohistochemical detection of the lectin in serial sections proved these methods for receptor analysis to be rather equally effective. The observation that the biotinylated lectin-binding glycoproteins are also appropriate ligands in glycohistochemical analysis warrants emphasis. Overall, the introduction of biotinylated mammalian lectins as well as the lectin-binding glycoproteins will aid to critically evaluate the physiological significance of the glycobiological interplay between endogenous lectins and distinct carbohydrate parts of cellular glycoconjugates.     

X-ray structure of a biantennary octasaccharide-lectin complex refined at 2.3-A resolution.

The structure and flexibility of saccharides have a profound and specific influence in several biological processes such as protein protection and the maintenance of conformational integrity, and in recognition events involving viruses, enzymes, and lectins. To establish the structural bases of these phenomena, we describe herein the extensively refined 2.3-A resolution x-ray structure of a biantennary octasaccharide of the N-acetyllactosamine type, complexed to isolectin I from Lathyrus ochrus. The two octasaccharides are located in clefts at each end of the long axis of the lectin. The conformations of both the lectin and the saccharide are slightly modified upon binding. The complex is stabilized by numerous hydrogen bonds, many of them involving water molecules. It is also stabilized by van der Waals interactions, including some with aromatic residues. A more general model of a possible lectin-glycoprotein interaction is also proposed.     

Capping of saccharides on the plasma membrane of lymphocytes as studied by fluorescein-labelled lectins

The capping of saccharides on the plasma membrane of rat splenic lymphocytes was studied by means of fluorescein-labelled lectins. Treatment of unfixed splenic lymphocytes with any one of the three lectins, concanavalin A (Con A), Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) led to the formation of caps of each saccharide receptor on the plasma membrane. Treatment of unfixed lymphocytes with Con A was found to result in the formation of caps of saccharide receptors for RCA, whereas cap formations were never noted in such double treatment of the cells with all other combined uses of two lectins. These results are taken to indicate that the saccharide receptors for Con A are associated with those for RCA in the plasma membrane of rat splenic lymphocytes.     

Diabetes mellitus associated changes in glomerular glycocompounds: A fluorescence microscopical study

Summary Renal biopsy specimens showing histological alterations typical of advanced diabetic glomerulopathy were studied for changes in glomerular glycoconjugates, using fluorochrome-coupled lectins as probes. All samples studied showed a marked reduction in the binding ofTriticum vulgaris (WGA) lectin in the glomerular basement membranes. On the other hand, new glomerular binding sites for the lectins ofDolichos biflorus (DBA),Helix pomatia (HPA) andArachis hypogaea (PNA), recognizing galactosyl moieties of glycoconjugates and giving no reaction in normal glomeruli, were seen in all samples studied. In addition,Wistaria floribunda lectin (WFA), recognizing galactosyl and.N-acetylgalactosaminyl configurations in glycoconjugates, gave a typical linear binding along the glomerular basement membranes, differing markedly from its reaction with normal kidney.Ulex europaeus (UEA I) showed reduced binding in the glomeruli of diabetic nephropathy.The results show that changes in glomerular glycoconjugates may appear in diabetic nephropathy, suggesting a disturbance in the turnover of the non-reducing terminal saccharide residues. In addition, the results show that lectins are useful probes for studying these changes further.     

Rat lung 29 kD β-galactoside-binding lectin is secreted by bronchiolar Clara cells into airways

Summary We isolated a mixture of -galactoside-binding lectins from rat lung and raised polyclonal antibody against 14 kD lectin purified from the mixture of lectins. Immunoblotting of the mixture of lectins, which was separated with SDS-PAGE under reducing condition and transferred onto a NC paper, showed that the antibody reacted with two bands at 14 and 29 kD, indicating that these two lectins have common antigenic determinant(s). Immunohistochemically, the antibody recognized only bronchiolar Clara cells with intense immunofluorescence in their apical cytoplasmic protrusions where the secretory granules of the cells are known to be stored. Thus, to determine if the lectin(s) might be secreted into airways, we next raised antibody against airway secretions free from serum as well as surfactant proteins. By immunoblot analysis, the resulting antibody stained 29,45 and 55 kD bands, but not 14 kD band, on a NC paper transferred with the mixture of lectins. These findings suggest that at least 29 kD lung lectin is located in bronchiolar Clara cells and secreted by these cells into airways.     

A colorimetric sensor array based on natural pigments for the discrimination of saccharides

A colorimetric sensor array based on natural pigments was developed to discriminate between various saccharides. Anthocyanins, pH‐sensitive natural pigments, were extracted from fruits and flowers and used as components of the sensor array. Variation in pH, due to the reaction between saccharides and boronic acids, caused obvious colour changes in the natural pigments. Only by observing the difference map with the naked eye could 11 common saccharides be divided into independent individuals. In conjunction with pattern recognition, the sensor array clearly differentiated between sugar and sugar alcohol with highly accuracy and allowed rapid quantification of different concentrations of maltitol and fructose. This sensor array for saccharides is expected to become a promising alternative tool for food monitoring. The link between anthocyanin and saccharide detection opened a new guiding direction for the application of anthocyanins in foods.     

Lectin binding by microvillous membranes and coated-pit regions of human syncytial trophoblast

Summary The distribution of saccharides on the microvillous membrane of the human syncytial trophoblast was investigated using ferritin conjugates of four lectins: concanavalin A (specific for -d-manno- and -d-glucopyranosyl residues), wheatgerm agglutinin (specific forN-acetylglucosamine),Limulus polyphemus lectin (specific forN-acetylneuraminic acid), andLotus tetragonolobus lectin (specific for -l-fucose). Concanavalin A and wheatgerm agglutinin (WGA) reacted strongly with the surface membrane and ferritin deposits were also observed in coated pit regions of the membrane. Lectins fromL. polyphemus andL. tetragonolobus, however, reacted only weakly with the microvillous border and neither reacted with coated pits.Enhanced agglutinability of trophoblast cells in comparison with other foetal cells from the same conceptus was seen with WGA. This agglutination was inhibited by addition of acetylglucosamine or by a solubilized membrane fraction which was bound by a column of WGA-Sepharose. The membrane fraction which did not bind to the column did not inhibit agglutination. Electrophoresis of the WGA-bound membrane proteins revealed six subunits, the major band having an apparent mol. wt. of 55 000. A protein of this mol. wt was also seen in coated vesicles isolated from equivalent human placentae.     

Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain

Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.     

Lectin receptors in Trypanosoma cruzi. An N-acetyl-D-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 131I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent Mr 85000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unaffected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since N-acetyl-D-glucosamine inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface N-acetyl-D-glucosamine-specific receptor activity.     

Differential response of the epidermal growth factor receptor tyrosine kinase activity to several plant and mammalian lectins

Biosignalling via lectins may involve modulation of protein kinase activities. This aspect of the biological action of mammalian and plant lectins has been investigated for their effect on the activity of the isolated epidermal growth factor receptor (EGFR). The constitutive tyrosine kinase activity of the epidermal growth factor receptor from rat liver, isolated by calmodulin-affinity chromatography, was activated by concanavalin A (ConA), and wheat germ agglutinin (WGA) to a similar extent as the measured enhancement induced by EGF. In contrast, two mannose-specific lectins, the mannan-binding protein (MBP) and serum amyloid P component (SAP), isolated from human serum, have inhibitory effects, both in the absence and presence of EGF. The differential effects of these lectins were tested using as phosphorylatable substrates a co-polymer of glutamic acid-tyrosine, as well as calmodulin. However, two galactoside-specific lectins, the laminin-binding -galactoside-binding 14 kDa lectin, isolated from bovine heart (14K-BHL), and the /-galactoside-binding lectin, isolated from mistletoe (Viscum album L.) leaves (VAA), do not inhibit the EGFR tyrosine kinase activity. The sugar dependence of the lectin-mediated action was studied by inhibition assays. Mannose and a mannose-containing neoglycoprotein prevent the activating effect of ConA, and N-acetyl-D-glucosamine partially prevents the activation produced by WGA. However, mannose and mannose-containing neoglycoprotein were ineffective to reduce the inhibitory effect of MBP or SAP. Although the response to binding of ConA and WGA was different to that of MBP or SAP with respect to the tyrosine kinase activity of the EGFR, it should be noted that the four lectins inhibited the binding of [¹²⁵I]EGF to its receptor with similar efficiency.Abbreviations EGF epidermal growth factor - EGFR epidermal growth factor receptor - ConA concanavalin A - MBP mannan-binding protein - SAP serum amyloid P component - WGA wheat germ agglutinin - 14K-BHL bovine heart 14 kDa lectin - VAA Viscum album L. (mistletoe) agglutinin - EGTA [ethylenebis(oxyethylenenitrilo)]-tetraacetic acid; poly(Glu:Tyr)-co-polymer of L-glutamic acid and L-tyrosine - Hepes 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - Tris tris(hydroxymethyl)-aminomethane - DSS suberic acid bis(N-hydroxy-succinimide ester) - PMSF phenylmethanesulfonyl fluoride - Man mannose - Gal galactose - BSA bovine serum albumin - Man-BSA neoglycoprotein containing -D-mannose - Lac-BSA neoglycoprotein containing -lactose - Gal-BSA neoglycoprotein containing galactose