The binding of lectins to components of plasma membranes from porcine submaxillary lymph node lymphocytes

By sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis the plasma membranes from porcine lymphocytes contain at least 30--35 glycopolypeptides and one or more glycolipids to which one or more of 12 purified lectins bind. The specificities of binding generally followed the same pattern as those of the reaction of the lectin with intact pig lymphocytes. Some lectins (e.g., the isolectin pair, Agaricus bisporus lectins A and B and a group consisting of the Lens culinaris A and B isolectins and the closely related Pisum sativum lectins) bind to almost identical populations of plasma membrane components and compete with each other for all their binding sites. Others (e.g., Concanavalin A and the Lens culinaris-Pisum sativum group and a group consisting of phytohemagglutinin-L, Ricinus communis lectin-60 and Ricinus communis lectin-120 bind in a cross reactive manner to some common binding moieties but, in addition, to certain nonshared ones. Still others (e.g., soybean agglutinin, peanut agglutinin and wheat germ agglutinin) do not share any common binding moieties with the other lectins. The amount of lectin binding and the number of membrane components to which a lectin binds is directly related to the Ka of binding of the lectin to the intact lymphocyte. Those with high Ka (Cocanavalin A Lens culinaris lectins, Pisum sativum lectins, phytohemagglutinin-L), bind to 20-30 different components giving very complex binding patterns while those with lower Ka (Agaricus bisporus lectins, wheat germ agglutinin, peanut agglutinin, and soybean agglutinin) bind to 8--13 components with easily distinguishable patterns. Soybean agglutinin binds almost exclusively to a glycolipid fraction while for the others one or more glycopolypeptides served as the major lectin-binding molecule. The Ricinus lectins, two lymphocyte toxins, bind to essentially every plasma membrane component to which the mitogen phytohemagglutinin-L binds, in fact competing for most of those plasma membrane moieties which bind phytohemagglutinin-L.     

Plant lectins detect age and region specific differences in cell surface carbohydrates and cell reassociation behavior of embryonic mouse cerebellar cells

When plated at high cell density in a microwell culture system, freshly dissociated embryonic mouse cerebellar cells assemble into reproducible, 3-dimensional patterns. The addition of the dimeric lectin Succinyl Concanavalin A blocks reversibly the formation of the microwell pattern, suggesting that cell surface carbohydrates affect the reassociation behavior of embryonic mouse cerebellar cells. Agglutination studes of dissociated cell populations harvested from different regions of the embryonic brain reveal that different lectins agglutinate cell populations from different embryonic brain regions. Cells from E13 cerebellum are agglutinated with Concanavalin A, wheat germ agglutinin, Ricinus communis agglutinin, mol wt 60,000, Ricinus communis agglutinin, mol wt 120,000, and Lens culinaris, but not by soybean agglutinin or a fucose-binding protein. Cells from the midbrain are agglutinated only with Concanavalin A, Ricinus communis agglutinin, mol wt 60,000 and Ricinus communis agglutinin, mol wt 120,000; those from the cerebral cortex are agglutinated only with Lens culinaris; and those from the medulla are agglutinated only with Ricinus communis agglutinin, mol wt 60,000, and Ricinus communis agglutinin, mol wt 120,000. In addition, agglutination of cerebellar cells with Concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin is diminished over the course of development from embryonic day 13 to postnatal day 7. These studies suggest regional differences in the cell surfaces of the developling brain that are further modulated during the differentiation of the tissues. On a poly(D-lysine) treated substrate in microwell cultures, cell migration is unique to the cerebellum of the 4 brain regions studied. Surfaces treated with carbohydrate-derivatized poly(D-lysine) are currently being tested for their efficacy as substrates for differential cell migration.     

The identification of membrane glycoconjugates in Leishmania species

The membrane glycoconjugates of 8 different species of Leishmania were compared by lectin blotting. Five different lectins with various sugar specificities were examined: concanavalin A, Lens culinaris, Ricinus communis, soybean agglutinin, and peanut agglutinin. Concanavalin A and Lens culinaris reacted with every Leishmania tested. The patterns observed for these 2 lectins, as well as the various species of parasites, were different. However, a common 41,000-52,000 and a 160,000-185,000 Mr component was present in almost all the parasite isolates examined. Ricinus communis only recognized a nondiscrete galactose-containing glycoconjugate similar to Leishmania-excreted factor. Soybean and peanut agglutinins reacted with a few low molecular weight parasite components. Soybean agglutinin reacted with all the Leishmania species tested, whereas peanut lectin only recognized 3 isolates. The latter lectin bound to discrete components migrating with the dye front and with Mr's of 35,000 and 52,000. Increased glycosylation was noted on avirulent L. major promastigotes and was associated with the appearance of several new peanut agglutinin-binding glycoproteins.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

Differential expression of the laminin A and B chains in chimeric kidneys

The expression of laminin in embryonic kidneys growing in ovo is followed with mouse-specific, affinity-purified antibodies against the laminin A and B chains. In mouse kidneys growing on the chicken chorioallantoic membrane, the epithelium and nephrogenic mesenchyme are derived from mouse and the vasculature from chicken chorioallantoic vessels. Hence, with the mouse-specific antibodies, it is possible to analyze the deposition of laminin chains by the nephrogenic tissue, because laminin derived from the chicken vasculature remains unstained. In these chimeras, only the laminin B chain, but not the A chain, is expressed in the undifferentiated nephrogenic mesenchyme. The basement membrane around the ureter bud is labeled by the antibodies against both laminin A and B chains. In the mesenchyme, the laminin A chain appears when the mesenchyme converts into tubules. The results suggest that the laminin A and B chains are synthesized differentially in the embryonic nephrogenic tissue.     

Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both gamma-gamma and alpha-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Nearly 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

Quantification of lectin receptors on B, T, T-gamma and T-mu lymphocytes. I. Concanavalin A, Pisum sativum, Lens culinaris and wheat-germ agglutinin

The immune system is formed of different lymphocyte subpopulations, each one having a defined role to defend the organism. Their plasma membranes present differences in the glycoproteinic or/and glycolipidic composition, as detected with labelled 125I-lectins. B lymphocytes have a greater number of receptors for the Pisum sativum, Lens culinaris and WGA lectins than T lymphocytes. On the other hand, T lymphocytes bind a greater number of Concanavalin A molecules than B lymphocytes. WGA lectin appeared to be more specific for T mu subpopulation, while Con A and Pisum sativum lectins were bound preferentially to T gamma lymphocytes while no significant differences were observed between both subpopulations for Lens culinaris lectin. From the affinity of each lectin to each lymphocyte population it could be deduced that the receptor structure, conformation and arrangement on the membrane was optimal in B lymphocytes for Con A and WGA binding, and T lymphocytes for Lens culinaris and Pisum sativum binding.     

Selection and characterization of eight phenotypically distinct lines of lectin-resistant Chinese hamster ovary cell.

Clones resistant to the lectins phytohemagglutinin (PHA), wheat germ agglutinin (WGA), the agglutinin(s) from Lens culinaris (LCA), and ricin (RIC) have been selected from parental auxotrophic Chinese hamster ovary (CHO) cells. The sensitivity to other lectins of these cells and of CHO cells resistant to concanavalin A (ConA) has been determined, and their activity of UDP-N-acetyl-glucosamine glycoprotein N-acetyl-glucosaminyltransferase (GlcNAc-T) has been measured. At least 8 different phenotypes have been identified on the basis of this analysis, and complementation between 2 of them demonstrated.     

Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells

We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.     

Effect of certain lectins on platelet aggregation in healthy people

A study was made of the ability of some plant lectins, which bind specifically to different carbohydrate determinants of glycoproteins, to induce the platelet aggregation in healthy humans. It has been shown that phytohemagglutinin (PHA) and wheat germ agglutinin (MGA) induce a more marked platelet aggregation than concanvalin A (Con A). Lens culinaris agglutinin (LCA) had a slight aggregate activity. It was pointed at different roles played by carbohydrate determinants of platelet glycoproteins in fulfilling their aggregation function.     

Cuticular carbohydrates of three nematode species and chemoreception by Trichostrongylus colubriformis

The cuticle of Haemonchus contortus, Nippostrongylus brasiliensis and Trichostrongylus colubriformis contained N-acetylgalactosamine and N-acetylglucosamine, based on the binding of fluorescein-labelled lectins. Binding of the lectins varied between the sexes and body regions of the nematodes. Treatment of male T. colubriformis with the lectin Lens culinaris agglutinin (LcA) reduced the feeding by helminths that was stimulated by histamine and the male's response to their female's pheromone, based on in vitro assays. Mannose residues may be involved in the helminth's chemoreceptors for feeding and sexual attraction, based on the specific binding of LcA.     

Glycoconjugate profiles of adrenocorticotropic and melanotropic cells in the pituitary of adult sea lampreys (Petromyzon marinus): a lectin histochemical study

In the lamprey, adrenocorticotropin (ACTH) and melanotropins (MSHs) are produced from two distinct precursors, proopiocortin (POC) and proopiomelanotropin (POM). Both POC and POM have been suggested to be glycoproteins. The present study aimed to demonstrate glycoconjugates in ACTH and MSH cells in the pituitary of adult sea lampreys (Petromyzon marinus) by means of a lectin histochemistry. A total of 19 kinds of lectins were tested. ACTH cells were distributed in both the rostral pars distalis and the proximal pars distalis, and were stained positively with N-acetylglucosamine binding lectins (i.e., succinylated wheat germ agglutinin), N-acetylgalactosamine binding lectins (i.e., soybean agglutinin), D-mannose binding lectins (i.e., Lens culinaris agglutinin), and D-galactose binding lectins (i.e., Erythrina cristagall lectin). MSH cells were distributed in the pars intermedia, and were stained with N-acetylgalactosamine binding lectins (i.e., Dolichos biflorus agglutinin), D-mannose binding lectin (Pisum sativum agglutinin) and D-galactose binding lectins (i.e., peanut agglutinin). These results suggested that ACTH and MSH cells produce different types of glycoconjugates which may be attributed to the difference in glycoconjugate moieties between the precursor proteins, POC and POM.     

Lectin binding to cystic stages of Taenia taeniaeformis

Studies of membrane glycoconjugates of Taenia taeniaeformis were initiated by assays of the lectin binding characteristics of 35-day-old cysticerci. Parasites fixed in glutaraldehyde were incubated with one of the following FITC-labelled lectins: Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA), fucose binding protein (FBP) and wheat germ agglutinin (WGA) and either their specific or a nonspecific sugar. Ultraviolet microscopy revealed that only Con A and LCA bound in large amounts to the surface of cysticerci. This binding was partly inhibited by the specific sugar, but the nonspecific sugar had little effect. The lectin not removed by either of the sugars may have been bound nonspecifically to the charged glycocalyx. Lectins were primarily bound on the anterior third of the parasite around the scolex invagination. Kinetic studies of lectin interactions were carried out with LCA and RCA by spectrophotofluorometric analysis of the amount bound specifically or nonspecifically over a range of lectin concentrations. Lens culinaris lectin binding was found to be specific and involve 2 receptors which showed large differences in their affinity for lectin and prevalence on the surface. Ricinus communis lectin did not bind specifically but nonspecific interactions were observed. Adherence of small numbers of host cells was shown to have no measurable effect on the lectin binding characteristics. The results suggest that the major surface carbohydrates exposed are D-mannose and/or D-glucose residues with the other sugar groups poorly represented. This relatively homogeneous surface may have implications for the antigenicity of the parasite in its host.     

5''-Nucleotidase from Bovine Caudate Nucleus Synaptic Plasma Membranes: Specificity for Substrates and Cations; Study of the Carbohydrate Moiety by Glycosidases

We studied 5'-nucleotidase in preparations of synaptic plasma membranes from bovine caudate nucleus. The best substrates for this membrane-bound enzyme were purine nucleotides, particularly 5'AMP. Effects of metal cations and chelating agents suggest that 5'-nucleotidase is a metalloprotein. Optimal conditions for solubilization of the 5'-nucleotidase were found by using a low concentration of the zwitterionic detergent sulfobetaine 14. In contrast, another membrane-bound enzyme, acetylcholinesterase, was not solubilized under these conditions, but only in the presence of Triton X-100. The effects of lectins (concanavalin A, Lens culinaris agglutinin, wheat germ agglutinin, and Limulus polyphemus agglutinin) showed that both enzymes are glycoproteins. Sequential hydrolysis with specific glycosidases produced modifications of the effect of lectins on these enzymes. The results suggest the presence of a complex-type glycosylation, with a fucose residue on the internal N-acetyl-D-glucosamine of the pentasaccharide core.     

Lectin histochemistry of mammalian endothelium

Lectin-histochemical studies were performed on formalin-fixed, paraffin-embedded tissues from ten mammalian species to demonstrate the pattern of carbohydrate residues in vascular endothelium. Ten different biotinylated lectins were used as probes and avidin-biotin-peroxidase complex (ABC) was used as visualant. Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA) stained vascular endothelium in all species. Peanut agglutinin (PNA) stained vascular endothelium in all species only after preincubation with neuraminidase. Bandeirea simplicifolia agglutinin-I (BS-I) stained vascular endothelium in all species but human, while Ulex europeus agglutinin-I (UEA-I) stained only human endothelium. Individual differences in staining of human vascular endothelium were noted with BS-I and succinylated-WGA (SWGA). Similarly, individual differences in staining of animal vascular endothelium were noted with soybean agglutinin (SBA) after preincubation with neuraminidase. Finally, Concanavalia ensiformis agglutinin (Con A), Dolichos biflorus agglutinin (DBA) and Lens culinaris agglutinin (LCA) did not stain vascular endothelium in any of the species studied.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

Storage of glycoprotein in NCTR-Balb/C mouse. Lectin histochemistry, and biochemical studies.

A strain of Balb/C mice carrying a lysosomal storage disorder exhibits metabolic and phenotypic abnormalities similar to patients with sphingomyelin-cholesterol lipidoses type II (i.e., Niemann-Pick C and D). Their foamy cells, which belong to the reticuloendothelial system, stained intensely by periodate-Schiff (PAS) reagent and were resistant to predigestion with diastase. To identify the chemical nature of the PAS-positive storage material, we applied lectin histochemistry and biochemical methods. Paraffin embedded sections, and delipidated frozen tissue sections, were treated with biotinylated lectins and localized with avidin-biotin-peroxidase complex. Araldite-embedded semithin sections were incubated with biotinylated lectins followed by avidin-gold and were enhanced with silver. By both histochemical methods the affected foamy cells stained positively as follows: Concanavalia ensiformis agglutinin, Datura stramonium agglutinin, Griffonia simplicifolia-I, Lens culinaris agglutinin, peanut agglutinin, Ricinus communis agglutinin-I, wheat germ agglutinin (WGA), and succinylated-WGA. Biochemical analysis of liver extracts complemented the histochemical data and demonstrated accumulation of glycoproteins containing polylactosaminoglycans in affected mice. Our findings indicate that the storage material in NCTR-Balb/C mice is heterogeneous. The lipids that are extracted by organic solvents during the histologic preparations mask the occurrence of polylactosaminoglycan containing glycoproteins in native frozen sections.     

Ultrastructural localization of WGA, RCA I, LFA and SBA binding sites in the seven-day-old mouse embryo

In the present investigation we localized binding sites for the lectins WGA (wheat germ agglutinin), RCA I (Ricinus communis agglutinin), LFA (Limax flavus agglutinin) and SBA (soya bean agglutinin) in the 7-day-old mouse embryo at the ultrastructural level. Lectin binding sites were localized on formaldehyde fixed embryos, embedded in LR-Gold, using gold-labelled lectins. Binding sites for WGA and RCA I were observed at the surface of the embryonic ectoderm oriented towards the proamnion cavity and the outer surface of the extraembryonic and the embryonic endoderm. Staining for SBA and LFA binding sites was seen in the basement membrane of the ectoderm. Moreover, binding sites for LFA were observed in the nucleoli of cells of the ectodermal, the mesodermal and the endodermal layer and in free ribosomes located in the cytoplasm of these cells.     

Wheat germ agglutinin-binding glycoproteins are decreased in Alzheimer's disease cerebrospinal fluid

A number of biomarkers (e.g. Abeta, tau) has been identified in Alzheimer's disease CSF. However, none fulfils the criteria of sensitivity and specificity (> 80%) needed for the development of an accurate diagnostic test. The lack of a suitable marker has prompted the search for new CSF biomarkers. In this study, the glycosylation of CSF proteins was examined using lectin blotting. Lumbar CSF was collected ante mortem from 22 non-Alzheimer's disease and 12 probable Alzheimer's disease cases and ventricular CSF collected post mortem from 7 non-Alzheimer's disease and 16 Alzheimer's disease cases confirmed by pathologic examination. When CSF glycoproteins were stained with wheat germ agglutinin (WGA), the staining intensity was found to be significantly lower in the Alzheimer's disease group. No difference in staining was found using other lectins (Canavalia ensiformis agglutinin, Ricinus communis agglutinin, Lens culinaris agglutinin). The measurement of WGA-reactive glycoproteins in CSF may be a useful biomarker for diagnosis of Alzheimer's disease.