Halina Lis

Different locations of carbohydrate-containing sites in the surface membrane of normal and transformed mammalian cells

SummaryA soybean agglutinin was found to agglutinate mouse, rat and human cell lines transformed by viral carcinogens, but not hamster cells transformed by viral or non-viral carcinogens. Normal cells from which the transformed cells were derived were not agglutinated by this agglutinin, but they were rendered agglutinable after short incubation with trypsin or pronase. The transformed hamster cells, on the other hand, became agglutinable only after prolonged treatment with pronase. The agglutination was specifically inhibited by N-acetyl-d-galactosamine, indicating that N-acetyl-d-galactosamine-like saccharides are part of the receptor sites for soybean agglutinin on the surface membrane. Such sites exist in a cryptic form in normal cells; they are exposed in transformed mouse, rat and human cells, but become less accessible in transformed hamster cells. The receptor sites for soybean agglutinin differ from the receptors for two other plant agglutinins (wheat germ agglutinin that interacts with N-acetyl-d-glucosamine-like sites and Concanavalin A that interacts with α-d-glucopyranoside-like sites) which become exposed upon transformation of all lines tested. In normal hamster cells, the receptors for all three agglutinins become exposed after incubation with trypsin, but the exposure of N-acetyl-d-galactosamine-like sites requires the longest enzyme treatment. The results indicate a difference in the location of different carbohydrate-containing sites in the surface membrane. The differences in the exposure of carbohydrate-containing sites in the membrane could not be correlated with the levels of carbohydrate-splitting glycosidases in normal and transformed cells.

Lectins: Their Chemistry and Application to Immunology

Publisher Summary Living organisms abound in a large variety of proteins that possess the ability to specifically bind different compounds. Most important among these are the enzymes that combine specifically with the substrates and inhibitors, and antibodies that bind antigens. A third class of proteins with specific combining sites, found mainly in plants, has recently moved to the forefront of biologic research; these are lectins. The main characteristics of lectins are their ability to bind sugars, to agglutinate cells, and to stimulate lymphocytes. In all their activities, lectins exhibit varying degrees of specificity. Most lectins interact preferentially with a single sugar structure. For some lectins, the specificity is broader and includes a number of closely related sugars; whereas certain lectins interact only with complex carbohydrate structures. Through their sugar-combining sites, lectins interact specifically with polysaccharides and glycoproteins to form precipitates. This reaction is similar to the precipitin reaction between antibody and antigen, in that it is specific, exhibits concentration dependence on lectin and polysaccharide, and might be inhibited specifically by low molecular weight “haptens”—compounds identical with or derived from the sugar(s) for which, the lectin is specific. Therefore, lectins are similar to antibodies in many respects.

The β1 → 2‐d‐xylose and α1 → 3‐l‐fucose substituted N‐linked oligosaccharides from Erythrina cristagalli lectin

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 1H-NMR showed that the major component was the fucose-containing heptasaccharide Man alpha 3(Man alpha 6)(Xyl beta 2)Man beta 4GlcNAc beta 4(Fuc alpha 3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components. The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Man alpha 3(Man alpha 6)Man beta 4GlcNAc beta 4GlcNAc] by a D-xylose residue linked beta 1----2 to the beta-mannosyl residue and an L-fucose residue linked alpha 1----3 to the reducing terminal N-acetylglucosamine residue. The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.

Purification of soybean agglutinin by affinity chromatography On sepharose-N-ϵ-aminocaproyl-β-D-galactopyranosylamine

Affinity chromatography has been successfully used for the purification of proteins with specific binding sites, such as enzymes, antibodies and lectins [l-3]. Soybean agglutinin (SBA), a lectin isolated from soybean oil meal [4,5] has been shown to bind specifically N-acetyl-D-galactosamine and D-galactose [6]. This property has now been utlized by us for the purification of SBA on a column made of a conjugate of Sepharose and N-e-aminocaproyl-/3-D-galactopy ranosylamine (SAG). The method used for the preparation of SAG was essentially the same as that developed very recently by Blumberg et al. [7] for the binding of p-L-fucopyranosylamine to Sepharose. N-e-aminocaproylQ-Dgalactopyranosylamine was prepared by a reaction of /3-D-galactopyranosylamine (1 Q-amino-l -deoxy-Dgalactopyranoside) and N-benzyloxycarbonyl-eaminocaproic acid [8], and removal of the benzyloxycarbonyl group by hydrogenolysis. The product was coupled to Sepharose by the cyanogen bromide method of Ax&r et al. [9] as described by Blumberg et al. [lo]. SBA was adsorbed from a partially purified extract of soybean oil meal to a column made of SAG, and the active material was eluted from the column by a solution of D-galactose. Separation of SBA from minor agglutinating components present in the soybean oil meal [ 111 was achieved by chromatography on DEAE cellulose. From 50 g of soybean oil meal, 80 mg of purified SBA, with a specific ac-

Use of Lectins for the Study of Membranes

For many years it has been known that plant extracts possess the ability to agglutinate erythrocytes (Bird, 1959, Boyd, 1963; Sharon and Lis, 1972). It was recognized quite early that this agglutination is the result of the specific interaction of certain proteins found in the extracts with sugars on the surface of erythrocytes (Sumner and Howell, 1936). However, the vast possibilities which these proteins, presently known as lectins, open for the study of cell surfaces and membranes in general are only now being appreciated (Sharon and Lis, 1972; Burger, 1973; Lis and Sharon, 1973). This has occurred simultaneously with the growing recognition of the important role that sugars located on the cell surface play in the life of cells (Roseman, 1970; Winzler, 1970; Ginsburg and Kobata, 1971; Ashwell and Morell, 1974). Although sugars comprise only a small proportion (2–10%) of the weight of the cellular membrane, they are believed to provide cells with recognition patterns, give them individuality, and play a decisive role in the “social life” of the cell. Any reagent which is specific for sugars on surfaces is therefore an important aid for cell biologists.

Lectins in higher plants

Publisher Summary This chapter discusses physicochemical properties of lectins in higher plants. Lectins are best defined as sugar-binding and cell-agglutinating proteins of nonimmune origin. In contrast to antibodies, which are structurally similar, lectins vary in composition, MW, subunit structure, and number of sugar-binding sites. The presence of a lectin in a plant is readily detected by testing whether an extract of the plant agglutinates erythrocytes and by demonstrating that the agglutination is sugar-specific. Blood group-specific lectins are identified with the aid of a panel of typed human erythrocytes. The most common cell modification is mild digestion with trypsin or other proteolytic enzymes or with neuraminidase, an enzyme that removes sialic acid from complex carbohydrates. Lectins may also be detected by their ability to form precipitates with polysaccharides or glycoproteins, either in liquid or semisolid media. Such interactions also provide information regarding lectin specificity, and on the constituent sugars and glycosidic linkages of the polysaccharide or glycoprotein precipitated.

Isolation and properties of N-acetyllactosamine-specific lectins from nine erythrina species

Abstract Lectins from seeds of nine species of Erythrina have been purified by affinity chromatography on columns of lactose coupled to Sepharose and their properties compared with those of the lectin from Erythrina cristagalli . All lectins are glycoproteins of M , ca 60 000 composed of two identical or nearly identical subunits. They contain between 3–10% carbohydrates comprised of N -acetylglucosamine, mannose, fucose and xylose. The amino acid composition of all Erythrina lectins is very similar. The N -terminal amino acid is valine, with the exception of the lectin from E. flabelliformis in which it is alanine. To the extent tested, identities or near identities have been found in the N -terminal sequences (up to 15 residues in some cases) of the lectins. Hapten inhibition experiments of agglutination have shown that the lectins are specific for N -acetyllactosamine, this disaccharide being 10–30 times more inhibitory than D -galactose and 10–20 times more than N -acetyl- D -galactosamine. All lectins agglutinate human erythrocytes equally well, irrespective of blood type, at minimal concentrations of 5–20 μg/ml. Six of the lectins are also very effective in agglutinating rabbit erythrocytes and are mitogenic for human peripheral blood lymphocytes, whereas three of them are considerably weaker hemagglutinins for rabbit erythrocytes, and two of these are also very weak mitogens. Our results, while demonstrating striking similarities in the molecular properties and sugar specificity of all Erythrina lectins studied, suggest the existence of differences at or close to the carbohydrate-binding site.

Purification of wheat germ agglutinin by affinity chromatography on a sepharose-bound N-acetylglucosamine derivative.

Abstract Sepharose-2-acetamido-N-(ϵ-aminocaproyl)-2-deoxy-β-D-glucopyranosylamine was prepared by a reaction of 2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-β-D-glucopyranosylamine and N-(benzyloxycarboxyl)-ϵ-aminocaproic acid, removal of the 0-acetyl and the benzyloxycarboxyl groups and coupling to Sepharose. The product was used for the purification of wheat germ agglutinin, by adsorption from a crude wheat germ extract and elution with 0.1M acetic acid. The purified agglutinin was homogeneous on SDS-polyacrylamide gel electrophoresis and had a specific hemagglutinating activity of 3000 u/mg when tested on trypsinized rabbit erythrocytes. It was rich in cysteine, cystine and glycine, and contained no sugar.

Quantitation of N-acetyl-d-galactosamine-like sites on the surface membrane of normal and transformed mammalian cells

Measurements of the binding of 125I-labeled soybean agglutinin to cells cultured with fetal calf serum have shown, that there can be a similar number of d-GalNAc-like sites exposed on normal and transformed mouse and rat cells; that there were only 10 % of such sites on transformed hamster cells; and that treatment with pronase can render normal cells agglutinable by soybean agglutinin without increasing the total number of exposed d-GalNAc-like sites.

The Structural Basis for Carbohydrate Recognition By Lectins

1. Different carbohydrate-specific proteins, such as lectins, may combine with the same monosaccharide or oligosaccharide by different H-bonding and hydrophobic side chains. 2. Homologous proteins with distinct specificities may bind different monosaccharides (e.g., for glucose and galactose that differ in the configuration of a single hydroxyl) by the same set of invariant residues that are identically positioned in their tertiary structures. 3. The energetics of protein-carbohydrate interactions cannot be derived from structural information. 4. Nature solves in a variety of different ways the problem of constructing combining sites for carbohydrates, just as it provides diverse solutions for other functions of proteins.