Limax Flavus Agglutinin Research Articles

Lectin-gold cytochemistry of mucin oligosaccharide biosynthesis in Golgi apparatus of airway secretory cells of the hamster.

To elucidate the mechanism for the biosynthesis of O-linked mucin oligosaccharides, airway secretory cells of the hamster trachea were embedded in Lowicryl K4M resin, and sections were examined by lectin-gold cytochemistry with special attention focused on the Golgi apparatus. The interrelations between the Golgi cisternae stained with five different lectins were determined by double-staining procedures using various combinations of lectins conjugated with 14-nm and 8-nm colloidal gold. Several cis cisternae were stained only with HPA (Helix pomatia agglutinin specific for terminal alpha-N-acetylgalactosamine). The next medial cisternae were not stained with HPA, but reacted positively with two lectins, GSII (Griffonia simplicifolia agglutinin II specific for terminal alpha- or beta-N-acetylglucosamine) and RCAI (Ricinus communis agglutinin I specific for beta-galactose). The trans cisternae as well as condensing and mature secretory granules were labeled with four lectins, UEAI (Ulex europaeus agglutinin I specific for terminal alpha-L-fucose) and LFA (Limax flavus agglutinin specific for terminal N-acetyl or N-glycolyl neuraminic acid) in addition to HPA and RCAI. The same number of trans cisternae were positive to HPA and UEAI, whereas LFA bound to a few transmost cisternae but fewer than were stained with HPA or UEAI. The observed sequential appearance of different sugar residues in different levels of Golgi cisternae (from cis to trans cisternae) coincides quite well with the sugar sequence of airway mucin oligosaccharide (from reducing to nonreducing ends) proposed by biochemical analysis. It is suggested that airway mucin oligosaccharides elongate during a vectorial movement through the Golgi stack from cis toward trans and that the stack consists of at least three functionally distinct segments, cis, medial, and trans; in these three segments there take place, respectively, the initial O-glycosylation of mucin core peptide, the formation of a core region of oligosaccharide chain, and the completion of chain growth by addition of terminal sugar moieties.

Ultrastructural demonstration of lectin binding sites in the Golgi apparatus of rat epiphyseal chondrocytes.

Binding sites for wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ricinus communis I agglutinin (RCA I) and Limax flavus agglutinin (LFA) have been ultrastructurally detected in rat epiphyseal chondrocytes by a post-embedding cytochemical technique using colloidal gold as marker. The four lectins labelled exclusively the Golgi apparatus of chondrocytes embedded in Lowicryl K4M resin by two different methods. WGA binding sites were localized in medial and trans cisternae as well as in immature secretory vesicles, whereas those for DBA were seen concentrated in cis and medial cisternae. Labelling with both RCA I and LFA lectins was distributed throughout all the cisternae of the Golgi stack, and the latter also in vesicles and tubules at the trans face. Neuraminidase pretreatment of the sections abolished LFA staining, decreased reaction with WGA and increased that with RCA I, while it did not affect DBA staining. After chondroitinase ABC treatment only the RCA I reaction was modified, revealing new binding sites in the trans Golgi face, secretory granules and extracellular matrix. These results indicate that the distribution of subcompartments in the Golgi apparatus of chondrocytes is different from that in cells secreting glycoproteins as major products.

Lectin binding of subunits of placental and serum oxytocinase after electrophoresis and transblotting to nitrocellulose

1. 1. Oxytocinase enzymes were purified from maternal serum and human placenta, run by SDS-PAGE and transferred onto nitrocellulose. Both enzymes were homogeneous in protein staining with M r of 145,000. 2. 2. Both serum and placental oxytocinases bound concanavalin A (Con A), limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA). The WGA-binding of the placental enzyme was more strongly inhibited by 0.2 M N-acetylglucosamine than that of the serum enzyme which may indicate a higher sialic acid content in the serum enzyme. 3. 3. Neuraminidase treatment did not affect the binding of Con A but decreased the bincling of WGA to serum and placental enzymes. Serum enzyme showed a pl 4.7 on isoelectric focusing.

The function of the human factor V carbohydrate moiety in blood coagulation.

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5-2-fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin-induced activation, whereas deglycosylated factor V activation was impaired. Lectin-blot experiments with sialic-acid-specific Limax flavus agglutinin (LFA), galactose-specific Ricinus communis agglutinin (RCA-II) and mannose-specific concanavalin A on thrombin-induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1F2 contained terminal galactose residues. LFA and RCA-II inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation.

Ethylnitrosourea treatment increases lectin binding to mouse germ cells

Germ cell toxicity was assessed by investigating the binding of FITC-labeled lectins to mouse testis cells before and 18 days after treatment with ethylnitrosourea (ENU). Flow cytometry of testis cells dual-labeled with FITC-lectin plus the DNA stain, propidium iodide, allowed analysis of haploid (1C), diploid (2C), and dividing (4C) cell populations. Soybean agglutinin, wheat germ agglutinin, concanavalin A and Limax flavus agglutinin bound to normal mouse testis cells containing 1C, 2C or 4C DNA. Asparagus pea lectin and Bandeireae simplicifolia I isolectin B 4 did not. ENU treatment reduced the number of testis cells and increased lectin binding, particularly of those lectins which bound to untreated cells.

Effect of trypsinization on lectin binding to germ cells from ICR and T/t6 mice.

Flow cytometry was used to quantify the binding of fluorescein isothiocyanate (FITC)-labeled lectins to testis cells from ICR and T/t6 mice before and after trypsin treatment. Soybean agglutinin, wheat germ agglutinin, and concanavalin A bound well to testis cells of both mouse strains. Limax flavus agglutinin (LFA) bound very slightly and Ulex europeas agglutinin (UEA) did not bind at all. Trypsinization increased binding of soybean agglutinin and decreased binding of wheat germ agglutinin in both mouse strains, providing evidence for masked carbohydrate-binding sites on the surface of germ cells. It did not affect binding of the other lectins. Trypsin treatment was an attempt to increase lectin binding, particularly the binding of LFA and UEA to the reported T/t-specific carbohydrates, sialic acid, and L-fucose, respectively. These studies indicate that the T/t6 locus alleles do not alter the surface carbohydrate content of testis cells sufficiently to be detected by lectin-binding differences.

Lectin histochemistry of plaques and tangles in Alzheimer's disease.

Biotinyl derivatives of several lectins and avidin-horseradish peroxidase were used to study the localization of glycoconjugates in amyloid plaques and in neuritic tangles in brains of patients with Alzheimer's disease (AD), Downs syndrome (DS) and Gerstmann-Sträussler syndrome (GSS). The lectins tested recognize the following residues: beta-D-galactosyl [Ricinus communis agglutinin 120, (RCA-1) and peanut agglutinin, (PNA)]; alpha-D-galactosyl [Griffonia simplicifolia agglutinin (GSA)]; alpha-D-mannosyl greater than alpha-D-glucosyl [concanavalin A (Con A) and Lens culinaris agglutinin (LcH)]; N-acetyl- and N-glycolyl-neuraminic acid [Limax flavus agglutinin (LFA) and Limulus polyphemus agglutinin (LPA)]; N-acetyl-glucosaminyl and sialyl [wheat germ agglutinin (WGA)]; N-acetyl-D-galactosaminyl [Helix pomatia agglutinin (HPA) and Dolichos biflorus agglutinin (DBA)] and alpha-L-fucosyl [Ulex europeus agglutinin (UEA-1)]. The majority of lectins listed above bind preferentially to the peripheral area of AD plaques, whereas in plaques of DS they are mainly bound to central amyloid core. In neurofibrillary tangles of AD brains only residues recognized by WGA and HPA or DBA were found, whereas in DS brains, in addition to above mentioned, beta-D-galactose (RCA-1) and sialic acid (LFA) were also present. In brain microblood vessels the strongest reaction in endothelia appeared with UEA-1 and RCA-1, indicating the abundance of alpha-L-fucosyl and beta-D-galactosyl residues. In AD brains deposits of amyloid were noted in the wall of some blood vessels, where monosaccharide residues recognized by RCA-1, GSA, UEA and WGA but not by Con A and LFA were present. However, our studies of some organs (liver, kidney, heart and testes) of patients with generalized amyloidosis revealed a lack of these sugar residues. It indicates, that the composition of amyloid present in brains of AD is different to that in other organs in generalized amyloidosis.

Phagocytic interactions of sialated glycoprotein, sugar, and lectin coated beads with rat retinal pigment epithelium.

Latex beads coated with mucin (a sialic acid containing glycoconjugate), N-acetylglucosamine (GlcNAc), or with the lectins, succinylated wheat germ agglutinin (sWGA) (binds to GlcNAc) or Limax flavus agglutinin (LFA) (binds to sialic acid residues) were used as phagocytic particles. Phagocytosis of these coated beads by retinal pigment epithelial (RPE) explants was determined by bead uptake in normal (Long Evans) and dystrophic (Royal College of Surgeons, RCS/p+) rat retinas. Electron microscopy showed that beads coated with mucin or LFA lectin were not phagocytized by either dystrophic or normal RPE. sWGA-coated beads were phagocytized by both dystrophic and normal RPE, while GlcNAc-coated beads were taken up by dystrophic RPE only. Specificity of uptake for sWGA and GlcNAc bead coatings was shown by the reduction in the number of beads phagocytized in the presence of the appropriate competing sugar or lectin. The lack of phagocytic uptake of beads coated with a sialated glycoprotein or a sialic acid binding lectin suggests that sialic acid residues are not recognized as particulate ligands in this phagocytic assay. The data which show the uptake of beads coated with sWGA (binds only GlcNAc) together with results which showed WGA (binds both GlcNAc and sialic acid)-coated beads were not taken up, further suggest that GlcNAc residues may be involved in bead phagocytosis. The most striking difference between normal and dystrophic RPE engulfment of coated beads is the uptake of GlcNAc-coated beads by dystrophic RPE only. These results suggest that the receptor molecules on dystrophic RPE cell surface membranes may be different from those on normal RPE membranes.

45] Properties of a sialic acid-specific lectin from the slug Limax flavus

Publisher Summary Lectins are a group of nonimmunoglobulin-like proteins that exhibit a high binding specificity toward carbohydrate residues of glycoproteins and glycolipids. They have found wide application in studies on glycoproteins because they interact with carbohydrates in a highly specific manner. In spite of the ubiquitous presence of lectins in nature, only a few have been characterized to be specific for sialic acid. It has been recently demonstrated that this slug contains a lectin that is highly specific for sialic acid residues of glycoproteins. This lectin, limax flavus agglutinin (LFA), has been recently purified to homogeneity. The chapter discusses the hemagglutination assay process and the purification procedure. The macromolecular properties of LFA have been presented in the chapter in a tabular form. There is a discussion about the use of LFA–Sepharose 4B in affinity chromatography.

Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina.

The pigmented epithelium of the vertebrate retina phagocytizes the discarded tips of photoreceptors and it is likely that a specific cellular recognition process is involved in this phenomenon. The apical surface of retinal pigmented epithelium (RPE) contains microvilli which interdigitate with the outer segment regions of photoreceptor cells and it is this apical microvillous surface that is of particular interest with respect to phagocytosis. The present study is a report of a method to isolate a fraction that is enriched in microvilli from the apical surface of this highly polarized epithelial cell. Wheat germ agglutinin (WGA) conjugated sepharose beads are used to remove the microvillous membranes which are then observed with scanning and transmission electron microscopy. The proteins of this RPE-subfraction are separated through use of SDS-polyacrylamide gel electrophoresis. The relative molecular weights (Mr) and lectin binding properties of glycoproteins are examined in Western blots through the use of lectin-peroxidase conjugates as probes for carbohydrate residues. A preliminary comparison of membranes isolated from Long Evans (normal) and Royal College of Surgeons (dystrophic) rat retina RPE shows that the glycoproteins in these two preparations are different with respect to the binding of Concanavalin-A (Con-A) and WGA. In particular a glycoprotein in the normal RPE preparation with a Mr of 175K binds Con-A and WGA, but in the dystrophic RPE preparation binds little or no WGA. A glycoprotein present in the normal RPE preparation with a Mr of 86K binds Con-A and WGA, but both lectins have reduced binding sites in the dystrophic preparation. Limax flavus agglutinin (specific for sialic acid residues) binds to a high molecular weight glycoprotein with a Mr of 195K-196K which is present in both normal and dystrophic RPE membrane preparations and which also binds Con-A and WGA.

Examination of sialic acid binding on dystrophic and normal retinal pigment epithelium

In order to study differences in cell-surface sugars which may be involved in the phagocytic defect in Royal College of Surgeons (RCS) retinas, we have examined the presence or absence of lectin binding to carbohydrates on retinal pigment epithelial (RPE) plasma membranes of dystrophic (RCS-p+) and normal (Long-Evans) rats. A lectin which binds to both sialic acid and N-acetylglucosamine sugar residues, wheat germ agglutinin-ferritin (WGA-fe), was used. The specificity of WGA-fe binding to each sugar was studied by either pre-treating the tissue with neuraminidase enzyme which removes sialic acid residues, or by incubating the WGA-fe lectin with one of the haptens, N-acetylglucosamine. In non-enzyme-treated tissue, RPE cell-surface membranes from RCS retinas were densely labeled with WGA-fe as compared with the labeling on normal RPE, which appeared less dense and patchy in distribution. Wheat germ agglutinin-ferritin labeling in the presence of N-acetylglucosamine was blocked on both RCS and normal RPE surface membranes. After pre-treatment with neuraminidase, WGA-fe labeling on dystrophic RPE membranes was similar to non-enzyme-treated tissue but was enhanced on normal RPE. Labeling was blocked when N-acetylglucosamine was present with the lectin after enzyme pre-treatment. Other lectins which specifically bind to sialic acid, Limulus polyphemus agglutinin-ferritin (LPA-fe) and Limax flavus agglutinin (LFA) demonstrated sparse or no labeling on both RCS and normal RPE membranes. Our data suggests that N-acetylglucosamine residues predominate on RCS and normal RPE cell-surface membranes and that sialic acid binding sites are either not accessible to the lectins or may not be present.

The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin.

The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.

Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels.

Lectin- or glycoprotein-colloidal gold complexes were used for detection of specific monosaccharide residues in mouse brain micro-blood vessels (MBVs). The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin-120, RCA-1), alpha-N-acetylgalactosaminyl (Helix pomatia agglutinin, HPA), alpha-D-mannosyl and alpha-D-glucosyl (Concanavalin A, Con A), sialoglycoconjugates (Limax flavus agglutinin, LFA), N-acetylglucosaminyl and sialyl (wheat germ agglutinin, WGA), and alpha-L-fucosyl (Ulex europeus agglutinin, UEA-1). Use of these lectin-gold complexes and ultrathin sections of Lowicryl K4M-embedded tissue makes it possible to gain insights into localization of lectin receptors in the entire cross-section of MBV walls. Receptors for all lectins, except UEA-1, were found on both luminal and abluminal fronts of the endothelial cells (ECs). Differential labeling of luminal and abluminal fronts of ECs with some lectins (Con A, HPL) is considered to reflect the polarity of the endothelium. Some differences noted in the distribution of lectin receptors in the wall of representatives of three types of MBVs (capillaries, arterioles, and venules) are thought to be associated with different functions performed by the above-mentioned segments of the microvasculature in maintenance of the blood-brain barrier.

Ultracytochemistry of glycosylation sites of rat colonic mucous cells with labeled lectins.

The mucous glycoproteins of mucous cells in the lower half of the rat proximal colon were examined ultracytochemically to study the intracellular sites of glycosylation in relation to the cell differentiation and the functional polarity of the cell organellae using a combination of a hydrophilic resin embedment and a postembedding staining technique with lectin gold com plexes.In the immature mucous cells located near the branching point of the glandular crypt, the Golgi stacks and mucous granules were stained with soybean agglutinin (SBA), Ricinus communis agglutinin-I (RCA-I) and Helix pomatia agglutinin (HPA). In the mature mucous cells at the bottom of the crypt, the cis region of the Golgi stack was stained with SBA, a few intermediate saccules were stained with RCA-I, and both the cis and trans sides of the cisternae and mucous granules, but not the intermediate lamellae, were stained with HPA. After neuraminidase digestion of ultrathin sections, the RCA-I binding sites were greatly increased. Ulex europeus agglutinin-I (UEA-I) and Limax flavus agglutinin (LFA) positively stained the trans side lamellae and mucous granules of the mucous cell at the bottom of the glandular crypt.The processing of the glycosylation of the mucous glycoprotein in the lower half crypt of the rat proximal colon was partly clarified electron microscopically.

Changes in the distribution of endothelial surface glycoconjugates associated with altered permeability of brain micro-blood vessels.

Lectin-binding sites located on the endothelial cell (EC) surfaces in unaltered, leaking and resorbing micro-blood vessels (MBVs) in cryo-injured cat brain were studied. Lectin or glycoprotein-gold complexes and brain samples embedded in hydrophilic resin Lowicryl K4M were used. The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA and peanut agglutinin, PNA), sialyl (Limax flavus agglutinin), N-acetyl-D-galactosaminyl (Helix pomatia agglutinin and soybean agglutinin, SBA), alpha-D-glucosyl and alpha-D-mannosyl (concanavalin A). The luminal front was labeled with SBA, and both fronts of the EC were labeled with PNA only after neuraminidase digestion. The most abundant and regularly distributed on both fronts of the EC were beta-D-galactosyl residues (RCA). These residues were also most affected in altered MBVs. The labeling of sialic acid residues was less pronounced on both sides of the EC. Following alteration of the function of the blood-brain barrier by cold-lesion injury, in leaking MBVs which represent increased luminal transport, we observed a conspicuous diminution of the labeling of the luminal surface of the EC with some lectins. On the other hand, in resorbing blood vessels located in the area of edema, where a presumably reverse (abluminal) transport occurs, major changes in the distribution of lectin-binding sites occurred on the abluminal front of the EC and in the basement membrane. The results reported here indicate that luminal and abluminal fronts of the EC change their properties in various functional conditions of MBVs, and that these changes can also be a reflection of functional polarity of brain endothelium.

Characterization of antigenic determinants of human apolipoprotein B. Distribution on tryptic fragments of low density lipoprotein.

In the hope of obtaining useful probes to study the structure of human apolipoprotein B (apo B), we characterized monoclonal antibodies against low density lipoprotein (LDL). We examined the distribution of their corresponding antigenic determinants on tryptic fragments of LDL separated by monodimensional (SDS) or two-dimensional electrophoresis. Each antibody reacted with several different fragments even when the proteolysis was apparently complete. A peptide of 125,000 daltons was the smallest fragment recognized by all the antibodies. The antibody, 2D8, which cross-reacts with apo B-48 and 3A8 which blocks the LDL pathway both reacted with the same 43,000 dalton fragment. Two other antibodies, 3F5 and 4G3, previously shown to be close together in LDL, also appeared close together in the primary structure of apo B. A determinant present on apo B-26 (1D1) was dissociated from all others examined on fragments of less than 125,000. Similarities in the patterns of reactivities with LDL-tryptic fragments between certain monoclonal antibodies and the lectins Concanavalin A and Limax flavus agglutinin indicated the proximity of the corresponding antigenic determinants to carbohydrate moieties. Competition studies suggested that the two major carbohydrate chains of LDL do not participate in the determinants themselves.