Summary
The lectin from the elderberry (Sambucus nigra L.) bark, shown to recognize the sequence neuraminic acid (α2,6) galactose/N-acetylgalactosamine, was applied for detecting binding sites in Lowicryl K4M sections by light and electron microscopy. The lectin was used either directly complexed to colloidal gold or in a two-step cytochemical affinity technique. The lectin-gold complex proved to be superior and thus was extensively tested on rat liver, kidney and hepatoma cells as well as on sheep and bovine submandibular glands. Controls to establish specificity of lectin-gold binding included sugar and glycoprotein inhibition tests and enzymic removal of sialic acid. In agreement with biochemical data demonstrating the potentiating effect of sialic acid on the binding of the lectin to oligosaccharides, enzymic removal of sialic acid from liver sections resulted in abolition of lectin staining. However, in the submandibular glands, neuraminidase pretreatment of the sections had no effect on the subsequent lectin-gold binding. In rat kidney some structures became negative while others retained the lectin-gold staining due to binding to penultimate.N-acetylgalactosamine exposed after sialic acid removal. In line with this, spot blot analysis demonstrated that the lectin-gold complex reacted with both fetuin and asialofetuin. Taken together, these results suggest that, for cytochemical staining, the sialic acid and the galactose/N-acetylgalactosamine lectin combining subsites ofSambucus nigra L. lectin are equally reactive with cellular glycoconjugates and that neuraminidase predigestion of tissue sections is of utmost importance to ensure specificity of staining for the sequence neuraminic acid (α2,6) galactose/N-acetylgalactosamine.
Similar content being viewed by others
References
Bhavanandan, V. P. &Katlic, A. W. (1979) The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid.J. Biol. Chem. 254, 4000–8.
Broekaert, W. F., Nsimba-Lubaki, M., Peeters, B. &Peumans, W. J. (1984) A lectin from elder (Sambucus nigra L.) bark.Biochem. J. 221, 163–9.
Charest, P. &Roth, J. (1985) Localization of sialic acid in kidney glomeruli: regionalization in the podocyte plasma membrane and loss in experimental nephrosis.Proc. Natl. Acad. Sci. USA 82, 8508–12.
Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., Levine, J. &Reisfeld, R. A. (1984) A monoclonal antibody recognizes anO-acetylated sialic acid in a human melanoma-associated ganglioside.J. Biol. Chem. 259, 7453–9.
Ellinger, A. &Pavelka, M. (1985) Post-embedding localization of glycoconjugates by means of lectins on thin sections of tissues embedded in LR White.Histochem. J. 17, 1321–36.
Frosch, M., Görgen, I., Boulnqis, G. J., Timmis, K. N. &Bitter-Suermann, D. (1985) NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules ofE. coli K1 and group B meningococci.Proc. Natl. Acad. Sci. USA 82, 1194–8.
Goldstein, I. J. &Poretz, R. D. (1986) Isolation, physiochemical characterization, and carbohydrate-bindinding specificity of lectins. InThe Lectins. Properties, Functions, and Applications in Biology and Medicine (edited byLiener, I. E., Sharon, N. &Goldstein, I. J.), pp. 35–247. London: Academic Press.
Gottschalk, A. &Graham, E. R. B. (1959) 6-α-D-sialyl-N-acetylgalactosamine: the neuraminidase-susceptible prosthetic group of bovine salivary mucoprotein.Biochim. Biophys. Acta 34, 380–91.
Hakomori, S., Patterson, C. M., Nudelman, E. &Sekiguchi, K. (1983) A monoclonal antibody directed toN-acetylneuraminosyl-α2,6-galactosyl residue in gangliosides and glycoproteins.J. Biol. Chem. 258, 11819–22.
Hedman, K., Pastan, I. &Willingham, M. C. (1986) The organelles of thetrans domain of the cell. Ultrastructural localization of sialoglycoconjugates usingLimax flavus agglutinin.J. Histochem. Cytochem. 34, 1069–77.
Hill, H. D. Jr, Reynolds, J. A. &Hill, R. L. (1977) Purification, composition, molecular weight, and subunit structure of ovine submaxillary mucin.J. Biol. Chem. 252, 3791–8.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.
Mandal, C. &Basu, S., (1987) An unique specificity of a sialic acid binding lectin AchitininH from the hemolymph ofAchatina fulica snail.Biochem. Biophys. Res. Commun. 148, 795–801.
Marchalonis, J. J. &Edelman, G. M. (1968) Isolation and characterization of a hemagglutinin fromLimulus polyphemus.J. Mol. Biol. 32, 453–65.
Mazzuca, M., Roche, A.-C., Lhermitte, M. &Rossel, P. (1977)Limulus polyphemus lectin sites in human bronchial mucosa.J. Histochem. Cytochem. 25, 470–2.
Miller, R. L., Collawan, J. F. &Fish, W. W. (1982) Purification and macromolecular properties of a sialic acid-specific lectin from the slugLimax flavus.J. Biol. Chem. 257, 7574–80.
Mohan, S., Dorai, D. T., Srimal, S., Bachhawat, B. K. &Das, M. K. (1983) Fluorescence studies on the interaction of some ligands with carcinoscorpin, the sialic acid specific lectin, from the horseshoe crab,Carcinoscorpius rotundacauda.J. Biosci. 5, 155–62.
Muresan, V., Iwanij, V., Smith, Z. D. J. &Jamieson, J. D. (1982a) Purification and use of limulin: a sialic acid-specific lectin.J. Histochem. Cytochem. 30, 938–46.
Muresan, V., Sarras, M. P. Jr. &Jamieson, J. D. (1982b) Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas.J. Histochem. Cytochem. 30, 947–55.
Peters, B. P., Ebisu, S., Goldstein, I. J. &Flashner, M. (1979) Interaction of wheat germ agglutinin with sialic acid.Biochemistry 18, 5505–11.
Ravindranath, M. H., Higa, H. H., Cooper, E. L. &Paulson, J. C. (1985) Purification and characterization of anO-acetylsialic acid-specific lectin from a marine crabCancer antennarius.J. Biol. Chem. 260, 8850–6.
Rogers, G. N., Daniels, R. S., Skehel, J. J., Wiley, D. C., Wang, X. F., Higa, H. H. &Paulson, J. C. (1985) Host-mediated selection of influenza virus receptor variants. Sialic acid-α2,6 Gal-specific clones of A/Duck/Ukraine/1/63 revert to sialic acid-α2,3 Gal-specific wild typein ovo.J. Biol. Chem. 260, 7362–7.
Rogers, G. N., Herrler, G., Paulson, J. C. &Klenk, H.-D. (1986) Influenza C virus uses 9-O-acetyl-N-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells.J. Biol. Chem. 261, 5947–51.
Roth, J. (1983) Application of immunocolloids in light microscopy. II. Demonstration of lectin binding sites in paraffin sections by the use of lectin-gold complexes or glycoprotein-gold complexes.J. Histochem. Cytochem. 31, 547–52.
Roth, J. (1987) Light and electron microscopic localization of glycoconjugates with gold-labelled reagents.Scanning Microscopy 1, 695–704.
Roth, J., Bendayan, M., Carlemalm, E., Villiger, W. &Garavito, M. (1981) Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreastic tissue.J. Histochem. Cytochem. 29, 663–71.
Roth, J. &Binder, M. (1978) Colloidal gold, ferritin, and peroxidase as markers for electron microscopic double labelling lectin techniques.J. Histochem. Cytochem. 26, 163–9.
Roth, J., Brown, D. &Orci, L. (1983) Regional distribution ofN-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes.J. Cell Biol. 96, 1189–96.
Roth, J., Lucocq, J. M. &Charest, P. M. (1984) Light and electron microscopic demonstration of sialic acid residues with the lectin fromLimax flavus. A cytochemical affinity technique with the use of fetuin-gold complexes.J. Histochem. Cytochem. 32, 1167–76.
Roth, J., Neupert, G. &Bolck, F. (1975) Concanavalin A receptors in the plasma membrane of rat liver cells: comparative electron microscopic studies on normal cells and on cellsin vivo transformed by diethylnitrosamine.Exp. Pathol. 10, 143–55.
Roth, J. &Taatjes, D. J. (1985) Glycocalyx heterogeneity of rat kidney urinary tubule: demonstration with a lectin-gold technique specific for sialic acid.Eur. J. Cell Biol. 39, 449–57.
Roth, J., Taatjes, D. J., Bitter-Suermann, D. &Fine, J. (1987) Polysialic acid units are spatially and temporally expressed in developing postnatal rat kidney.Proc. Natl Acad. Sci. USA 84, 1969–73.
Roth, J., Taatjes, D. J., Lucocq, J. M., Weinstejn, J. &Paulson, J. C. (1985) Demonstration of an extensivetrans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation.Cell 43, 287–95.
Roth, J., Taatjes, D. J., Weinstein, J., Paulson, J. C., Grebnwell, P. &Watkins, W. M. (1986) Differential subcompartmentation of terminal glycosylation in the Golgi apparatus of intestinal absorptive and goblet cells.J Biol Chem. 261, 14307–12.
Schauer, R. (1982) Chemistry, metabolism, and biological functions of sialic acids.Adv. Carbohyd. Chem. Biochem. 40, 131–234.
Shibuya, N., Goldstein, I. J., Broekaert, W. F., Nsimba-Lubaki, M., Peeters, B. &Peumans, W. J. (1987a) The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(α2,6)Gal/GalNAc sequence.J. Biol. Chem. 262, 1596–1601.
Shibuya, N., Goldstein, I. J., Broekaert, W. F., Nsimba-Lubaki, M., Peeters, B. &Peumans, W. J. (1987b) Fractionation of sialylated oligosaccharides, glycopeptides, and glycoproteins on immobilized elderberry (Sambucus nigra L.) bark lectin.Arch. Biochem. Biophys. 254, 1–8.
Slot, J. &Geuze, H. J. (1981) Sizing of protein A-colloidal gold probes for immunoelectron microscopy.J. Cell Biol. 90, 533–6.
Slot, J. W. &Geuze, H. J. (1985) A new method of preparing gold probes for multiple-labelling cytochemistry.Eur. J. Cell Biol. 38, 87–93.
Stathis, E. C. &Fabrikanos, A. (1958) Preparation of colloidal gold.Chem. Ind. 27, 860–1.
Taatjes, D. J., Schaub, U. &Koth, J. (1987a) Light microscopical detection of antigens and lectin binding sites with gold-labelling reagents on semi-thin Lowicryl K4M sections: usefulness of the photochemical silver reaction for signal amplification.Histochem. J. 19, 235–45.
Taatjes, D. J., Chen, T.-H., Ackerström, B., Björck, L., Carlemalm, E. &Roth, J. (1987b) Streptococcal protein G-gold complex: comparison with staphylococcal protein A-gold complex for spot blotting and immunolabelling.Eur. J. Cell Biol. 45, 151–9.
Tai, T., Sze, L., Kawashima, I., Saxton, R. E. &Irie, R. F. (1987) Monoclonal antibody detects monosialogangliosides having a sialic acid α2,3-galactosyl residue.J. Biol. Chem. 262, 6803–7.
Towbin, H., Staehelin, T. &Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl Acad. Sci. USA 76, 4350–4.
Yamada, K. &Shimizu, S. (1979) The use of peroxidaselabelledLimulus polyphemus agglutinin for the histochemistry of sialic acid-containing glycoproteins in light microscopy.Histochem. J. 11, 457–71.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Taatjes, D.J., Roth, J., Peumans, W. et al. Elderberry bark lectin-gold techniques for the detection of Neu5Ac (α2,6) Gal/GalNAc sequences: applications and limitations. Histochem J 20, 478–490 (1988). https://doi.org/10.1007/BF01002646
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01002646