Agglutinin Binding Sites Research Articles

Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose.

Several clones of Chinese hamster ovary cells have been selected for their resistance to the toxic effects of wheat germ agglutinin. The clones do not bind wheat germ agglutinin as well as parent cells and are 5- to 250-fold more resistant to the toxic effects of the lectin. Of three clones studied in detail, all exhibit a decrease in wheat germ agglutinin binding affinity. Two have normal numbers of wheat germ agglutinin binding sites, while one (Clone 13) has a 65% decrease in binding sites. Crude membrane preparations of the clones have a decrease in sialic acid content relative to parent cells, and Clone 13 membranes are also deficient in galactose, while the mannose and hexosamine contents of all three clones are normal. The membrane sugar deficiencies affect both glycoproteins and glycolipids. Sialyl-lactosylceramide is the major glycolipid in parent cells, while Clones 1 and 1021 have lactosylceramide and Clone 13 has glucosylceramide as the predominant glycolipid. Labeling experiments with N-[G-3H]acetylmannosamine suggest that Clone 1021 cells have a block in the transfer of sialic acid from CMP-sialic acid to glycoprotein and glycolipid acceptors. Yet CMP-sialic acid:glycoprotein sialyl-transferase activity in cell lysates of Clone 1021 cells is 80% of normal. While CMP-sialic acid:lactosylceramide sialyl-transferase activity is only 25% of normal, it can be restored to normal or elevated levels by sodium butyrate induction without an associated increase in cellular sialyl-lactosylceramide content. Similarly, the galactose-deficient Clone 13 can synthesize UDP-galactose and has normal levels of UDP-galactose:glycoprotein galactosyltransferase and UDP-galactose:glucosylceramide galactosyltransferase when assayed in vitro. The glycosyltransferases of both these clones can utilize their own glycoproteins as sugar acceptors in in vitro assays. These data suggest that the variant cells fail to carry out specific glycosyltransferase reactions in vivo despite the fact that they possess the appropriate nucleotide sugars, glycoprotein and glycolipid acceptors, and glycosyltransferases.

Precipitation and carbohydrate-binding specificity studies on wheat germ agglutinin

The ability of wheat germ agglutinin to form precipitates with a series of synthetic carbohydrate-protein conjugates and with carcinoembryonic antigen and its Smith degradation products was investigated. The precipitation reaction between wheat germ agglutinin and p-azophenyl 2-acetamido-2-deoxy-β- d-glucopyranoside-bovine serum albumin was selected to examine the capacity of a large number of sugar haptens to inhibit this system. Our results indicate that the wheat germ agglutinin binding site is complementary to a sequence of three β-(1→4)-linked N- acetyl- d-glucosamine units ( N, N′, N″-triacetyl chitotriose). The internal carbohydrate portion of carcinoembryonic antigen probably contains two such units and wheat germ agglutinin precipitates with untreated as well as sequentially Smith degraded carcinoembryonic antigen. Compared with other reports certain discrepancies in the relative binding affinities of per N-acetylated chitodextrins and N- acetyl- d-glucosamine were found. These differences are discussed in terms of the methods used and the proposed subsite hypothesis of Allen, A. K., Neuberger, A. and Sharon, N. (1973) Biochem. J. 131, 155–162.

Qualitative and quantitative interactions of lectins with untreated and neuraminidase-treated normal, wild-type, and temperature-sensitive polyoma-transformed fibroblasts

The lectin receptors of confluently grown hamster BHK, wild type polyoma virus transformed PyBHK, and temperature-sensitive polyoma transformed ts3-PyBHK fibroblasts were investigated using cell agglutination, quantitative (125I)lectin binding, and ferritin-lectin labeling. PyBHK and permissively grown ts3-PyBHK cells agglutinated more strongly with Ricinus communis I agglutinin (RCA-I)compared to BHK and nonpermissively grown ts3-PyBHK, although saturation binding of (125I)RCA-I to these cells at 4 degrees resulted in a twofold difference in lectin-binding sites on BHK and nonpermissively grown ts3-PyBHK cells (1.0-1.3 x 10 7 sites/cell) compared to PyBHK and permissively grown ts3-PyBHK (0.4-0.6 x 10 7 sites/cell). These cells bound equivalent amounts of (125I)concanavalin A (0.8-1 x 10 7 sites/cell) and (125I)wheat germ agglutinin (1-2.2 x 10 7 sites/cell). Under these binding conditions little endocytosis occurred, as judged by the subsequent release of greater than 90% cell-bound (125I)RCA-I by the RCA-I inhibitor lactose and localization of ferritin-RCA-I exclusively to the extracellular plasma membrane surface. However, if the binding is performed at 22 degrees, only 50% of the bound lectin can be removed by lactose, and ferritin-RCA-I is localized inside the cell within endocytotic vesicles. The relative mobility of RCA-I receptors was examined on ts3-PyBHK cells by the ability of ferritin-RCA-I to induce clustering of its receptors at 22 degrees. RCA-I receptors on permissively grown ts3-PyBHK cells appeared to be more mobile than on nonpermissively grown cells. BHK and PyBHK cells were treated with neuraminidase, and the resulting enzyme-treated cells were assayed for lectin agglutinability and quantitative binding of RCA-I, concanavalin A, and wheat germ agglutinin. Neuraminidase treatment resulted in decreased concanavalin A and wheat germ agglutinability and a slight increase in RCA-I agglutinability. The enzyme-treated BHK and PyBHK cells bound less (125I)wheat germ agglutinin (2.8 x 10 6 and 2.2 x 10 6 sites/cell, respectively) and 2.5 and 6.2 times more (125I)RCA-I (2.5-3 x 10 7) and 3.5-4 x 10 7 sites/per cell, respectively). There was no change in the number of concanavalin A binding sites after neuraminidase treatment. The increase in RCA-I binding sites approximated the decrease in wheat germ agglutinin binding sites indicating that the predominant penultimate oligosaccharide residue to sialic acid on these cells is D-Gal.

On the mechanism of the increased aggregation by neuraminidase of 16C malignant rat dermal fibroblasts in vitro.

ABSTRACT The aggregation of briefly trypsinized 16 C malignant rat dermal fibroblasts as measured by gyratory shaker/electronic particle counting technique is increased by neuraminidase. Enzyme treatment also exposes soybean and Ricinus communis agglutinin-binding sites as judged by cell agglutination. Desialysed bovine submaxillary mucin and desialysed, agalacto fetuin reversed the neuraminidase-stimulated increase in aggregation, whereas desialysed fetuin and ovalbumin did not. The effective glycoproteins also acted as acceptors in cellular sialyl- and galactosyl-transferase reactions, and these activities are detected in plasma membrane fractions. The possibility that neuraminidase increases cellular aggregation by generating acceptor sites for interaction with cell surface glycosyltransferases, is discussed.

The fluorescence of wheat germ agglutinin and of its complexes with saccharides

Wheat germ agglutinin, upon excitation at 280 nm, presents a typical tryptophan fluorescence spectrum centered at 348 nm with a quantum yield of 0.095. Upon addition of N-acetyl-D-glucosamine or of its β(1→4) linked oligomers (GlcNAc)2,3,4, a blue shift of the fluorescence maximum and an increase in the fluorescence quantum yield of wheat germ agglutinin are observed. These are indications of the involvement of tryptophan residue(s) in the binding of saccharides. The changes with pH of the fluorescence intensity of wheat germ agglutinin and of its complexes with saccharides suggest an interaction between carboxyl and tryptophan residue(s) in the binding site which results in fluorescence quenching. Association constants for the binding to wheat germ agglutinin of (GlcNAc)2, (GlcNAc)3 and (GlcNAc)4, were determined spectrofluorimetrically, and found to be 1.3 × 104 M−1, 2.2 × 104M−1 and 3.6 × 104M−1, respectively. Addition of the oligosaccharides (GlcNAc)2,3,4 to wheat germ agglutinin produced greater enhancement of the fluorescence intensity than did addition of the corresponding monosaccharide. Our findings corroborate an earlier suggestion that the binding site of wheat germ agglutinin consists of several adjacent subsites.

Binding of soybean agglutinin by normal and trypsin-treated red blood cells

Trypsinized human erythrocytes were found to bind 3 times as much soybean agglutinin as the untreated cells. With rabbit erythrocytes, no effect of trypsinization on the binding of the agglutinin was observed. However, the susceptibility to agglutination increased 100- to 200 fold for all erythrocytes. The increased erythrocyte agglutinability can therefore not be explained simply by an increase in the number of agglutinin binding sites.

Difference in Topology of Normal and Tumour Cell Membranes shown by Different Surface Distributions of Ferritin-conjugated Concanavalin A

THE observations of Burger and Goldberg1 on the differential agglutinability of malignant cells by the saccharide-binding protein, wheat germ agglutinin (WGA), have prompted other investigators to study the nature of tumour cell surfaces using similar reagents. A variety of other plant agglutinins specifically agglutinate certain oncogenic virus-transformed cells at much lower concentrations than those required to agglutinate their normal parental lines. In addition to WGA1,2 (specific for binding N-acetyl-D-glucosamine-like residues1), these include concanavalin A (Con A)3,4 (specific for binding α-D-glucose or α-D-mannose-like residues5), soy bean agglutinin (SBA)6,7 (specific for binding N-acetyl-D-galactosamine and D-galactose-like residues6) and Ricinus communis agglutinin (specific for binding D-galactose and L-arabinose-like residues) (G. L. N. and J. Blaustein, in preparation). To explain this differential agglutinability phenomenon, Burger2 proposed that the agglutinin-binding sites on normal cells could have undergone three possible types of change after transformation: (a) the normal parent cell may have no agglutinin sites and after transformation a complete de novo synthesis of agglutinin sites occurs; (b) the normal parent cell may have agglutinin sites, but not enough for agglutination and transformation results in an increase in the number of agglutinin sites; or (c) the normal parent cell agglutinin sites remain constant in number after transformation; but this process results in an exposure of “cryptic” agglutinin sites and the transformed cell is rendered agglutinable. Several workers8,10,19 postulate a fourth type of change: (d) the normal parent cell agglutinin sites remain constant in number after transformation but the topological distribution of agglutinin sites changes to a distribution more favourable for agglutination. The first and second mechanisms have proved to be unlikely as originally suggested2, because agglutinin surface receptors are present on normal cells2 and they are present in the same amounts on normal or transformed cells. This latter finding has been demonstrated in saturation binding experiments using purified 125I-labelled WGA8, Con A8,9 and SBA10, with results contrary to earlier reports using 63Ni-labelled Con A3.