}Iistoehemistry 41,365--368 (1975) �9 by Springer-Verlag 1975

Short Communication

Uhrastructural Detection of Lectin Receptors by Cytochemical Affinity Reaction Using

Mannan-Iron Complex Jiirgen Roth

I~stitute of Pathology, Friedrieh-Sehiller-University Jena, DDP~-69 Jena, German Democratic Republic

Ha r t m u t Franz State Institute for Immune Preparates and Nutrition Media,

DI)R-II2 Berlin, German Democratic l~epublic

Received November 15, 1974

Summary. A two-step affinity reaction is described for electron microscopic demonstration of the Concanavalin A as well as the Lens culinaris lectin receptors by means of the yeast mannan-iron complex. First the tissue was incubated in the leetin. Afterwards the incubation in the yeast mannan-iron complex was performed and reaction takes place between the still free second sugar binding site of membrane bound lectin molecules and the polysaccharides. This membrane receptor-lectin-polysaceharide complex is revealed by the electron dense iron core of the yeast mannan-iron complex. The specificity of the reactions could be demonstrated by addition of the hal)ten or by incubation in the yeast mannan-iron complex only.

The proposed technique has proved useful for demonstration of lectin receptors in the small intestine.

Lectins react in a specific way comparable to an antigen-antibody-reaction with certain terminal or internal sugar residues of membrane bound glycoproteins and glycolipids (for review, see Sharon and Lis, 1972). They have been used for electron microscopic studies concerning distribution and mobility of lectin receptors of normal and transformed cells (for review, see Nicolson, 1974) as well as for ultrahistochemical studies (Roth, 1973; 1974). For these purposes leetin affinity reactions have been developed (Bernhard and Avrameas, 1971; Smith and Revel, 1972; Roth and Thoss, 1974) or ferritiu and peroxidase coupling of lectins has been performed (Nieolson and Singer, 1971; Huet and Garrido, 1972; Stobo and l~osenthal, 1972; Gonatas and Avrameas, 1973). The preparation and purification of biological active lectin conjugates is a relatively laborious pro- cedure.

We report a simple two-step affinity reaction (Fig. 1) with a high degree of specificity for ultrastructural demonstration of Concanavalin A and Lens culinaris lectin receptors. The basis for this affinity reactions consists in the specific reac- tion of Coneanavalin A and Lens culinaris lectin with mannans (So and Goldstein, 1968; Young et al., 1971). In consequence we synthetisized yeast mannan-iron complexes (Franz and Roth, 1974) which were utilized in eytochemical procedure simultaneously as receptor for membrane receptor bound lectin as well as an electron dense marker for ]ocMization of membrane receptor-lectin complexes.

366 J. i%oth and H. Franz

} Fig. 1. Schematic representation of the two step affinity reaction. 1-cell with lectin receptors,

2-1ectin, 3-mannan-iron complex

During preparat ion of the manuscr ipt Martin and Spicer (1974) reported the demonstra t ion of Concanavalin A receptors by means of a commercial available i ron-dextran complex.

Small pieces of tile routine intestine were prefixed in 4% formaldehyde (freshly prepared from paraformaldehyde) --0.1 M phosphate buffer (pH 7.4) for 30 rain at room temperature. After rinsing in the buffer the tissue samples were incubated in Concanavalin A (100 tzg/ml, Pharmaeia Uppsala) or Lens culinaris lectin (1 mg/ml) for 15 rain at room temperature. Following rinsing in buffer (3 times for 5 min) incubation in the yeast mannan-iron complex (ratio mannan'iron = 85%:15 %) takes place for 15 rain at room temperature. After washing in buffer postfixation in 2% osmium tetroxide-0.1 M eacodylate buffer (pH 7.4) was done for 1 hour at 4~ The thin sections were not counterstained.

For control of specificity of the reaction 0.2 M c~-methyl-D-mannopyranoside (Calbioehem, A grade) was added to Concanavalin A or Lens culinaris lectin as well as to the yeast mannan- iron complex 30 rain before tissue incubation. Furthermore tissue samples were incubated in the yeast mannan-iron complex only.

After performance of the two-step affinity reaction a specific labelling at the luminal plasma membrane of the intestinal cells could be observed (Fig. 2) cor- responding to the results obtained by Coneanavalin A-peroxidasc technique (Bernhard and Avrameas, 1971) or incubation in ferritin-labelled Concanavalin A and Lens culinaris lectin (Roth et al., /974). A stronger reaction was visible when Coneanavalin A was used instead of Lens culinaris lectin. Beside labelling of the microvitli and the luminal plasma membrane of goblet cells also intracellular labelling of the mucus of ruptured goblet cells was demonstrable. Generally in intact cells no labelling of intracytoplasmie structures or lateral plasma membranes

occurred. After addit ion of the hap ten to lectins and the polysaceharid-iron complex

(Fig. 2) or after incubat ion in the yeast mannan- i ron complex only (Fig. 3) no labelling at the intestinal cells could be observed. That demonstrates the specific binding of yeast mannan- i ron complexes by membrane receptor bound lectin. I n absence of lectins the yeast mannan- i ron complex does not bind to the membrane

non-specifically. Size and electron densi ty of the iron core of the polysaeeharid-iron complex

allowed a high resolution and exact localization. I n our laboratories fur ther experiments using other specific polysaeeharid-metal complexes for the demon- strat ion of lectins are in progress.

Mannan-Iron Complex for Demonstration of Lectin Receptors 367

Fig. 2. Concanavalin A-yeast mannan-iron complex reaction. Jejunum, mouse. Densely packed iron particles at the outer surface of microvilli plasma membrane. • 120000

Fig. 3. Negative reaction after addition of the mannopyranoside to the lectin and yeast mannan-iron complex. Jejunum, mouse. • 100000

Fig. 4. No iron particles are visible at the microvilli membrane after incubation in the yeast mannan-iron complex only. Duodenum, mouse. • 100000

References Bernhard, W., Avrameas, S.: Ultrastructurat visualization of cellular carbohydrate com-

ponents by Coneanavalin A. Exp. Cell Res. 64, 232-236 (1971) Yranz, H., Roth, J. : Preparation of carbohydrate-iron complexes as electron dense receptors

for membrane bound lectins. Histochemistry 41, 361-363 (1975) Gonatas, N. K., Avrameas, S.: Detection of plasma membrane carbohydrates with lectin

peroxidase conjugates. J. Cell Biol. 59, 436-443 (1973) Huet, Ch., Garrido, J.: Ultrastructural visualization of cell coat components by means of

wheat germ agglutinin. Exp. Cell Res. 7g, 523-527 (1972) Martin, B. J., Spieer, S. S. : Coneanavalin A-iron dextran technique for staining cell surface

mucosubstanees. J. Histochem. Cytochem. 22, 206-207 (1974) Nicolson, G. L.: The interaction of leetins with animal cell sm'faces, int. Rev. Cytol. 39,

89-190 (1974)

368 J. Roth and It. Franz

Nicolson, G. L., Singer, S. J.: Ferritin-conjugated plant agglutinins as specific saccharide stains for electron microscopy: application to saccharides bound to cell membranes. Proc. nat. Acad. Sci. (Wash.) 68, 942-945 (1971)

Roth, J. : Ultrahistochemical demonstration of saceharide components of complex carbo- hydrates at the alveolar cell surface and at the mesothclial cell surface of the pleura visceralis of mice by means of Concanavalin A. Exp. Path. 8, 157-167 (1973)

Roth, J. : Electron microscopic demonstration of saeeharide moieties in the hypophase of the alveolar surfactant system by lectins. Respir. Physiol., in press (1974)

l%oth, J.,Thoss, K.: Light and electron microscopic demonstration of D-mannose and D- glucose like sites at the cell surface by means of the lectin from the Lens culinaris. Ex- perientia (Basel) 30, 414 (1974)

Roth, J., Wagner, M., Thoss, K.: Verwendung yon Lektinen in der Immunelektronen- mikroskopie. 3. Arbeitstagung ,,Markierte AntikSrper", Berlin, 23.-24. 5. 1974.

Sharon, N , Lis, H.: Lectins:eell-agglutinating and sugar specific proteins. Science 177, 949-959 (1972)

Smith, S. B., Revel, J. P. : Mapping of Concanavalin A binding sites on the surface of several cell types. Deve]op. Biol. 27, 434-441 (1972)

So, L.L., Goldstein, I . J . : Protein-carbohydrate interactions XIII. The interaction of Concanavalin A with ~-mannans from a variety of microorganism. J. biol. Chem. 243, 2003-2007 (1968)

Stobo, J. D., Rosenthal, A. S. : Biologically active Concanavalin A complexes suitable for light and electron microscopy. Exp. Cell Res. 79, 443=447 (1972)

Young, bT. M., Leon, M. A., Takahashi, T. : Studies on a phytohemagglutinin from the lentil. III. Reaction of Lens culinaris hemagglutinin with polysaccharides, glycoproteins, and lymphocytes. J. biol. Chem. 25, 1596-1601 (1971)

Dr. reed. J. Roth Pathologisehes Institut der Universiti~t DDR-69 Jena Ziegelmfihlenweg 1 German Democratic Republic