Biomedical subjects
Publications and source records attributed to F Uehara.
peanut agglutinin, a lectin with specific affinity for galactose beta-(1----3)-N-acetyl-galactosamine disaccharides, showed binding to the slender cones in the rod-free fovea as well as to the conical cones in the periphery of the monkey (Macaca fuscata) retina, but not to the rods, as revealed by light-microscopic cytochemistry with horseradish-peroxidase-conjugated lectin.
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The binding sites of two lectins, peanut agglutinin (PNA) and wheat germ agglutinin (WGA), in the interphotoreceptor matrix (IPM) and photoreceptor plasma membranes of the Japanese monkey (Macaca fuscata) retina were localized using a pre-embedding staining method with ferritin-conjugated (Fer) lectins as well as a postembedding staining method with fluorescence-labeled (FITC) lectins. FITC-PNA, but not WGA, stained cylindrical domains of the IPM around cone outer and inner segments, while the IPM around rods stained with FITC-WGA but not PNA. When the intact (not detached) retinal tissues were incubated with Fer-lectin, the lectin generally labeled neither the IPM nor photoreceptor plasma membranes, but labeled only those structures in detached portions occurring at the edges of occasional retinal tissue blocks. Thus, the neural retinas physically isolated from the retinal pigment epithelium (RPE) were utilized principally here. Ultrastructurally, the IPM in the intact retina consisted of granular and filamentous materials; the IPM in the isolated neutral retina also retained those components, although somewhat loosely organized, and the IPM around cones appeared to be preserved better than did the IPM around rods. Fer-PNA bound to the IPM associated with cones, but not rods; Fer-WGA bound to the rod- but not cone-associated IPM. The ferritin particles were found to lie close to the granular and filamentous materials. Those photoreceptor-associated IPMs extended to the apical surface of the RPE in detached portions or to the apical villi of the RPE which were frequently found in the isolated neural retinas. Also, Fer-PNA labeled the cone, but not rod, plasma membranes; Fer-WGA bound heavily to the plasma membranes of rod and cone outer segments, but sparsely to those of their inner segments. These results suggest that the IPM comprises chemically and physically differential domains specialized for cone and rod photoreceptor cells, and that these specialized IPM are structurally so stable that may be involved in isolating photoreceptor cells physicochemically from each other and in the interactions between the photoreceptors and the RPE, such as retinal adhesion.
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Proteins and glycoproteins of the bovine interphotoreceptor matrix (IPM) with or without neuraminidase treatment was analysed by two-dimensional gel electrophoresis combined with Western blotting and staining with seven horseradish peroxidase-labeled lectins. More than 80 spots of proteins and glycoproteins were revealed on the gel. Nineteen spots (or groups of spots) were revealed by staining with five lectins [concanavalin A, wheat germ agglutinin (WGA), peanut agglutinin (PNA), Ricinus communis agglutinin-1 (RCA-1) and soybean agglutinin (SBA)]; some of those spots were specific for one lectin and others reacted with several lectins. We could not detect distinct spots reacting with Dolichos biflorus agglutinin or Ulex europaeus agglutinin-1. Neuraminidase digestions of the IPM increased and unmasked the binding spots for PNA, RCA-1 and SBA. The spots of WGA-receptors without neuraminidase treatment were mostly identical to the receptors for PNA, RCA-1 and SBA, which became prominent after the digestion. Spots reacting with RCA-1 were mostly identical to the spots of SBA-receptors. The spots reacting with PNA coincided only partially with the spots reacting with RCA-1 and SBA.
From rabbit antiserum against peanut agglutinin (PNA)-receptors of the bovine interphotoreceptor matrix, an antibody was isolated by immunoaffinity chromatography on immobilized PNA-receptors. The antibody reacted with the bovine interphotoreceptor matrix at the molecular weight of 130,000 daltons. Immunohistochemical studies revealed that the antibody was bound not only to the surfaces of the cone photoreceptor cells but also to the surfaces of the rods, suggesting that the antibody purified here may have been formed by the major structure of glycoprotein that is common in the interphotoreceptor matrix around the cones and rods.
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Binding of sugar-specific lectins to the monkey retina was investigated with particular reference to the effects of pretreatment with neuraminidase on the photoreceptor cells. Neuraminidase-treated or untreated retinal sections were examined with a fluorescence microscope after incubation with the following fluorescein-labeled lectins: peanut agglutinin (PNA), wheat germ agglutinin (WGA), Ricinus communis agglutinin-1 (RCA-1) and Dolichos biflorus agglutinin (DBA). In the neuraminidase-untreated tissue, PNA, which is specific for D-galactose beta 1----3 N-acetyl-D-galactosamine (Gal beta 1----3GalNAc), was bound preferentially to the cones, and after treatment of sections with neuraminidase, PNA could bind to both cones and rods. WGA, which is specific for sialic acid and N-acetyl-D-glucosamine, was strongly bound to the rods but moderately to the cones without neuraminidase pretreatment, whereas neuraminidase-treated sections showed a weak binding. RCA-1, which is specific for D-galactose, showed patchy fluorescence on the basal and distal portions of the outer segments of the cones and rods, whereas neuraminidase-treated sections had uniform fluorescence throughout the tissues. DBA, which is specific for N-acetyl-D-galactosamine, did not bind to either cones or rods before and after neuraminidase treatment. These results suggest that there is a difference in the carbohydrate chains of glycoconjugates in the cones and rods: in the cones Gal beta 1----3GalNAc may be the terminal sugar as recognized by PNA, whereas in the rods this sugar is contained in the carbohydrate chain but masked by terminal sialyl residues so that those glycoconjugates are inaccessible to PNA without neuraminidase treatment.
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Paraffin-embedded and frozen biopsies from the human conjunctival epithelium were examined by fluorescence microscopy after labeling with eight fluorescein-conjugated lectins: wheat germ agglutinin; soybean agglutinin; peanut agglutinin; Ricinus communis agglutinin-1; Limulus polyphemus agglutinin; Ulex europaeus agglutinin-1; Dolichos biflorus agglutinin; concanavalin A. The lectins were used as specific molecular probes to detect carbohydrate composition of glycoproteins secreted from the conjunctival goblet cells. The labeling pattern of goblet cells and conjunctival epithelial surfaces with various lectins suggested that N-acetyl-glucosamine, galactose, N-acetyl-galactosamine and sialic acid are contained in goblet cells and contribute to the formation of tear mucus glycoprotein. Fucose and mannose, which are present in the tear mucus, were not detectable in goblet cells.
Histochemical studies were carried out of the binding sites of a lectin, peanut agglutinin (PNA), in the mammalian retina, using lectin labeled with either fluorescein or horseradish peroxidase. In all mammalian species examined, monkey, pig, cat and rabbit, the cones were intensely labeled with the lectin but the rods were unlabeled. The cone synaptic pedicles, the inner synaptic layer, the internal limiting membrane and the retinal vessels were also labeled. It is suggested from the specificity of the PNA binding that galactosyl and/or galactosaminyl residues are present in the mammalian cones, but not in the rods.
The binding of eight fluorescence-labeled lectins to the photoreceptors of the monkey retina was investigated using a post-embedding staining method. Concanavalin A (specific for mannosyl and glucosyl residues) bound to the outer and inner segments of both rods and cones, while the degree of staining was more intense in the rods. The rod outer segments showed patchy fluorescence and the proximal portions of the inner segments were diffusely stained. Wheat germ agglutinin (specific for sialyl and N-acetylglucosaminyl residues) bound preferentially to the surface of the outer and inner segments of both rods and cones. Ricinum communis agglutinin-1 (specific for galactosyl residues) stained the rod outer segments in patches, particularly strongly in the region dividing the outer and inner segments. The cones were also stained, although faintly, in the same pattern as the rods. The distal halves of the rods and cones showed diffuse weak staining and their proximal halves stained spotty. Peanut agglutinin (specific for Gal beta 1 leads to 3GalNAc sequence) bound preferentially to the cones and only scarcely to the rods. The external surface of both outer and inner segments of cones were uniformly stained; the interior of the cone outer segments was also stained, while the interior of the inner segments was not. Two lectins specific for fucosyl residues, namely, Ulexeuropaeus agglutinin-1 and Lotustetragonolobus agglutinin, bound diffusely to the distal halves of the inner segments of both rods and cones. Lectins reacting with N-acetyl-galactosamine residue, i.e. Dolichos biflorus agglutinin and soybean agglutinin, bound weakly to the distal portions of rods and cones.
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