Distribution of concanavalin A and wheat germ agglutinin binding sites in the rat peripheral nerve fibres revealed by lectin/glycoprotein-gold histochemistry

Summary The affinity of rat peripheral nerve fibres for concanavalin A (Con A) and wheat germ agglutinin (WGA) was tested in semithin sections of Epon-embedded material. A two-step post-embedding technique was used. As a first step, Con A and WGA were used in pure form. As a second step, peroxidase-gold (for Con A) and ovomucoid-gold (for WGA) complexes were applied. The lectin-binding sites, visualized by means of signal amplification with the photochemical silver reaction, were associated mainly with the myelin sheaths and the surfaces of Schwann cells.     

Changes in lectin binding of lumbar dorsal root ganglia neurons and peripheral axons after sciatic and spinal nerve injury in the rat

Summary The effects of chronic lesions of rat lumbar spinal or sciatic nerves on the binding of Glycine max (soybean) agglutinin to galacto-conjugates, in small-and medium-size primary sensory neurons of the L₄ and L₅ dorsal root ganglia, were examined over a 580-day period. Spinal nerve section resulted in a marked decrease in the population of stained neurons within 7 days. However, despite some retrograde morphological changes triggered by axonal injury, the proportion of stained nerve cells was normalized 180 days postoperatively. This temporary decrease in perikaryal lectin reactivity was initially associated with a marked accumulation of stained material in the nerve, proximal and distal to the site of section, with similar accumulations also being noticeable at each level of injury in sciatic nerves subjected to double ligature. This may reflect the presence of glycocompounds linked to the autolysis of nerve fibers during the phase of retrograde dying-back and Wallerian degeneration. At later stages, stained deposits could be seen scattered along central and peripheral axonal processes of the dorsal root ganglion neurons in the vicinity of the cell body. They may indicate a disturbance in the peripheral turnover of glycoproteins in chronically-transected nerves, with piling up of neuronal products. Sciatic nerve injury caused similar but less severe effects which, except for the L₄ ganglion cells, were rapidly reversible.     

A comparison of the binding of Alcian Blue and inorganic cations to polyanions in peripheral nerve

Synopsis Polyanions at the nodes of Ranvier of peripheral nerve fibres have been studied with cationic stais including colloidal iron, inorganic salts and Alcian Blue. The various cations were found to differ in their interactions with tissue macromolecules. Such differences have been examined critically in an attempt to understand the underlying physico-chemical principles involved in the histochemical demonstration of acidic macromolecules. In addition, certain phenomena associated with polyanions, such as ion-exchange, demonstrated in the present study, are discussed in relation to the possible physiological function of such macromolecules at nodes of Ranvier in the conduction of the nerve impulse.     

A binding protein from rat nerve

Leucine has been found to bind competitively to a soluble protein (molecular weight 97,000 daltons) from rat sciatic nerve under certain experimental conditions to form a high molecular weight aggregate (MW greater than 302,000). Kinetic study showed that the equilibrium constant for leucine-binding is 1.33 X 10(4) l/m and the rate constants for binding and unbinding are k1 = 0.424 l/m/sec and k-1 = 3.18 X 10(5) sec-1 respectively. The binding reaction is accompanied by an endothermic enthalpy change of 5,000 cal/mole and the favorable equilibrium appears to be due to the large positive (35.3 eu) entropy of binding. L-Proline, thymidine, and succinic acid were also found to bind, non-competitively with leucine, to proteins in the same fraction. Binding of those compounds and leucine was enhanced by the presence of Mg2+. Rat muscle and plasma proteins did not significantly bind leucine under these experimental conditions. The presence of this binding protein in rat nerve suggests an additional mechanism in the metabolism and in the transport of amino acids for incorporation into a protein structure in nerve.     

Identification of lectin binding sites in the rat brain

Lectins belong to a class of proteins or glycoproteins able to bind carbohydrates. The study reported here describes the identification of lectin-binding sites in the adult rat brain. The results indicate that among the 31 lectins utilized, eight show a specific positive reaction with neurons. Staining was also observed with other cerebral structures such as myelin, leptomeninges, choroid plexus and capillaries. Lectins are, therefore, an important histochemical tool and can be easily and reliably used for the identification of cells and cerebral structures in the adult rat brain.Abbreviations Gal galactose - Fuc fucose - Man mannose - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - NeuNAc sialic acid     

A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces

Summary A quantitative evaluation of lectin binding to adult rat hepatocyte cell surfaces was done using cells isolated by two different collagenase perfusion methodologies and cultured as monolayers with two different tissue culture media formulations (protocol I vs. protocol II). The presence of α-D-mannosyl and α-D-glucosyl groups was detected by the binding of Concanavalin A (Con A), Lens culinaris agglutinin (LCA), and Pisum sativum agglutinin (PSA) to freshly isolated cells. Furthermore, β-D-galactose [Ricinus communis agglutinin (RCA)] and sialic acid residues [wheat germ (WGA)] were also found. Protocols I and II served as models for evaluation of: a) the stripping effect of collagenase separation procedures, b) the restoration in culture of collagenase-stripped sugar residues, c) the effect of the culture environment on cell viability [as measured by lactic acid dehydrogenase (LDH) leakage] and the protein content of hepatocytes, and d) the presence of cell surface sugar residues as a function of culture duration. The ultrastructural morphology of freshly isolated and cultured hepatocytes was also evaluated. These studies indicated that a decline in lectin binding invariably occurred earlier than a massive leakage of LDH and a decrease in the protein content of the cells in culture. Ultrastructurally, autophagocytosis was an early phenomenon in cells isolated and cultured by protocol I, which was also inferior to protocol II regarding the preservation of hepatocyte glycocalyces. Sugar residues lost due to the collagenase-stripping effect were restored, as shown by lectin binding, within the first 24 h of culture. This stripping effect was confirmed by quantitative evaluations of lectin binding to hepatocytes in culture after an incubation with collagenase. This study shows that the binding of peroxidase-labeled lectins is a useful tool for quantitative evaluation of the sugar composition of hepatocyte cultures. This study was supported by grant I-ROI-AM 26520 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Bethesda, MD, and by W. R. Grace Corporation.     

PA-II,the L-fucose and D-mannose binding lectin of Pseudomonas aeruginosa stimulates human peripheral lymphocytes and murine splenocytes

Pseudomonas aeruginosa lectin PA-II agglutinates human peripheral lymphocytes and stimulates mitogenesis (predominantly in T cells), like the plant lectins PHA and Con A. Murine splenocytes are also agglutinated and stimulated by PA-II as by Con A. Sialidase treatment of the human and murine cells enhances their agglutination and augments the stimulation of human lymphocytes at low PA-II concentrations. The PA-II agglutinating and mitogenic effects are specifically inhibited by L-fucose. The bacterial source and the specificity of PA-II for L-fucose are both rare features among the hitherto described mitogenic lectins. However, since this lectin also binds mannose, a mannose-bearing receptor might be involved in its mitogenicity.     

A survey of lectin binding in Paramecium

To better understand the general distribution of glycoproteins and the distribution of specific glycoprotein-bound sugar residues in Paramecium, a survey of the binding pattern of selected lectins was carried out in P. tetraurelia, P. caudatum, and P. multimicronucleatum. Lectins studied were concanavalin A (Con A), Griffonia simplicifolia agglutinins I and II (GS I and GS II), wheat germ agglutinin (WGA), Ulex europaeus (UEA I), peanut agglutinin (PNA), Ricinis communis toxin (RCA60) and agglutinin (RCA120), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), Dolichos biflorus agglutinin (DBA), and Maclura pomifera agglutinin (MPA). Those giving the most distinctive patterns were Con A, GS II, WGA, UEA I, and PNA. No significant differences were found between the three species. Concanavalin A, a mannose/glucose-binding lectin, diffusely labeled the cell surface and cytoplasm and, unexpectedly, the nuclear envelopes. Events of nuclear division, and nuclear size and number were thus revealed. Both WGA and GS II, which are N-acetylglucosamine-binding lectins, labeled trichocyst tips, the cell surface, and the oral region, revealing stages of stomatogenesis. The lectin WGA, in addition, labeled the compartments of the phagosome-lysosome system. The lectin PNA, an N-acetyl galactosamine/galactose-binding protein, was very specific for digestive vacuoles. Finally, UEA I, a fucose-binding lectin, brightly labeled trichocysts, both their tips and body outlines. We conclude that a judicious choice of lectins can be used to localize glycoproteins and specific sugar residues as well as to study certain events of nuclear division, cellular morphogenesis, trichocyst discharge, and events in the digestive cycle of Paramecium.     

Light and electron microscopical localization of concanavalin A lectin binding sites in rat epiphyseal chondrocytes

Summary Concanavalin A lectin binding sites have been detected within the cytoplasm of epiphyseal chondrocytes. Correlative light and electron microscopic results were obtained, indicating the presence of-d-mannose and/or -D-glucose residues detected by the lectin in the rough endoplasmic reticulum region. Quantitation of the electron microscopic cytochemical reaction also showed that the specific labelling was almost exclusively localized in the lumen of endoplasmic reticulum cisternae. No significant staining was found in other membrane compartments or extracellular matrix. This labelling pattern could be considered as the cytochemical evidence ofN-glycosylation processes occurring during the biosynthesis of cartilage extracellular matrix components by chondrocytes.     

Immunochemical comparison of vitamin A binding proteins of rat

Specific antibodies against cellular retinol-binding protein (CRBP), raised in rabbit, were detected by sucrose gradient centrifugation and gel filtration using tritium-labeled CRBP, prepared by reductive methylation. By means of radioimmunoassay, pure CRBP from liver and testis as well as CRBP present in a crude extract of liver were compared. All preparations showed identical immunoreactivity, suggesting CRBP is not tissue-specific. In contrast, two other vitamin A-binding proteins, cellular retinoic acid-binding protein, and serum retinol-binding protein, showed no cross-reaction in the radioimmunoassay.     

Postnatal development of lectin binding sites in the rat ventral prostate

Summary Five Fluorescein-isothiocyanate (FITC)-labelled lectins were used to study the postnatal development of carbohydrate constituents in the rat ventral prostate: Concanavalin A (Con A), wheat germ agglutinin (WGA), peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andRicinus communis agglutinin I (RCA-I) With all the lectins, tested, except RCA-I, specific binding sites could be shown for every stage of differentiation in the glandular epithelium. Binding sites for Con A, WGA, PNA and DBA were found from day 10 to 13 post partum onwards. Each lectin showed a characteristic localization. Binding sites for the lectins used changed to different extents during the following two weeks. After the 24th day post partum no further changes in the lectin binding pattern could be found. The development of the lectin binding properties showed that the changes in carbohydrate-containing constituents of the prostate correlate with the beginning of prostatic secretion and to prostatic epithelial differentiation. In the periacinar stroma the development of the lectin binding pattern was similar to that in the glandular epithelium. The changes of stromal binding sites for Con A and WGA during epithelial differentiation may reflect the changes of epithelial-stromal interactions in the prostate.     

A close structural relationship of rat liver Z-protein to cellular retinoid binding proteins and peripheral nerve myelin P2 protein

Striking sequence homologies were found among the major cytosolic lipid and dye binding protein (Z-protein), cellular retinoid binding proteins and peripheral nerve myelin P2 protein. In addition, the presence of a number of cytosolic lipid binding (or exchange) proteins having similar molecular weights and amino acid compositions suggests a new family of intracellular proteins with high affinities to lipids, possibly diverged from a common ancestor. In view of regulatory roles of these proteins in lipid metabolism and transport, myelin P2 protein may also possess a similar physiological function(s).     

A novel inositol-containing glycosphingolipid isolated from human peripheral nerve

An inositol-phosphate-containing glycosphingolipid, not reported earlier, was isolated from human cauda equina. Structural characterization showed the glycosphingolipid to be inosital-phosphoryl-2(3) galactosylceramide. The concentration varied between 25 and 30 nmol/g fresh tissue.     

Flow cytometric analyses of lectin binding to rat alveolar macrophages

The binding characteristics of ten FITC-labeled plant lectins (Con-A, MPA, BPA, PNA, WGA, SBA, UEA-I, DBA, GS-I, GS-II) to lavaged rat alveolar macrophages were assessed by flow cytometry. The alveolar macrophages (AM) were incubated with varying concentrations of each lectin in a pinocytosis-inhibiting buffer. In addition to measuring lectin-associated green fluorescence, the electronic cell volumes and axial light loss characteristics of the AM were also measured flow cytometrically. These latter parameters were found to be good indicators of cell agglutination caused by some of the lectins, and, in conjunction with green fluorescence measurements, usefully serve to determine optimal or nonagglutinating lectin concentrations for flow cytometric studies. With the exception of UEA-I, all of the lectins examined bound to AM, although a wide range of binding was observed among the lectins. At subagglutinating concentrations, Con A, MPA, BPA, PNA, WGA, SBA, and GS-I bound to the AM with unimodal patterns. Histograms of lectin-associated fluorescence intensity obtained with DBA clearly presented a pattern consistent with a more complex, bimodal distribution of labeled AM, suggesting the presence of at least two subset populations. The low-intensity distribution of AM represented congruent to 70% of the cells, while the more strongly labeled subset represented congruent to 15% of the parent AM population. The remaining balance of the AM was identified as another subpopulation by the failure to detectably bind to the DBA. While GS-II bound to all of the AM, this lectin labeled about 5% of the cells much more intensely than the bulk of the population. Thus, two subset populations of AM could be resolved according to their differing avidities for the GS-II lectin.     

Structural basis of sympathetic-sensory coupling in rat and human dorsal root ganglia following peripheral nerve injury

Tyrosine hydroxylase immunocytochemistry was used to reveal the sympathetic postganglionic axons that sprout to form basket-like skeins around the somata of some primary sensory neurons in dorsal root ganglia (DRGs) following sciatic nerve injury. Ultrastructural observations in rats revealed that these sprouts grow on the surface of glial lamellae that form on the neurons. Sciatic nerve injury triggers glial cell proliferation in the DRG, and the formation of multilamellar pericellular onion bulb sheaths, primarily around large diameter DRG neurons. We infer that these glia participate in the sprouting process by releasing neurotrophins and expressing growth supportive cell surface molecules. Many DRG cell somata, and their axons in intact nerves and nerve end neuromas, express α2A adrenoreceptors intracytoplasmically and on their membrane surface. However, sympathetic axons never make direct contacts with the soma membrane. The functional coupling known to occur between sympathetic efferents and DRG neurons must therefore be mediated by the diffusion of neurotransmitter molecules in the extracellular space. Sympathetic basket-skeins were observed in DRGs removed from human neuropathic pain patients, but the possibility of a functional relation between these structures and sensory symptoms remains speculative.     

Changes in lectin binding sites during early human liver development

 In this study we investigated whether changes in glycosylation during liver morphogenesis correlate with the early development of individual structures in the human liver. Therefore, we localized the binding of the lectins from Sambucus nigra (SNA; specific for sialic acid), Triticum vulgare (WGA; specific for N-acetylglucosamine and sialic acid), Ricinus communis (RCA I; specific for β-galactose), Lotus tetragonolobus (LTA; specific for α-fucose) and Concanavalia ensiformis (Con A; specific for α-mannose) in the human liver between the 4th and the 12th gestational week (GW). Cell membranes of early hepatocytes (5th–6th GW) showed strong staining for RCA I, which decreased noticeably from the 8th–9th GW onward. Early intrahepatic capillaries (4th–5th GW) showed reactions only for WGA and RCA I. Reactions for SNA occurred later (6th–9th GW). At this time a fine granular staining for SNA was visible at the sinusoidal sides of hepatocytes. The hepatocytes of the outer limiting plate were specifically stained by WGA, Con A, and SNA in the 9th GW and the staining remained visible in developing bile ducts up to the 12th GW. The possible biological significance of the appearance or disappearance of carbohydrate moieties during early human liver development is discussed. Accepted: 21 October 1996