Galactosyltransferase and Concanavalin A Agglutination of Cells

Abstract

A correlation has been observed between concanavalin A agglutination of various cell types and the presence of surface membrane galactosyltransferase (1-0-a-D-Galactosyl-myo-inositol: raffinose galactosyltransferase, EC 2.4.1.67) activity. Moreover, a reduction to less than 50% of cell surface galactosyltransferase activity occurred after treatment with concanavalin A; other cell surface glycosyltransferase enzyme activities examined were unaffected by concanavalin A treatment. To confirm the participation of cell surface galactosyltransferase in concanavalin A-induced cell agglutination, the enzyme from rabbit erythrocytes was solubilized by sonication and purified by preparative polyacrylamide gel electrophoresis. It was possible to achieve a purified preparation of rabbit erythrocyte galactosyltransferase by separation on concanavalin A-Sepharose. The purified enzyme showed visible immunoprecipitation (Ouchterlony) with concanavalin A. Furthermore, human erythrocytes, which are not normally agglutinated by concanavalin A, became agglutinable by the lectin when the erythrocytes were preincubated with purified galactosyltransferase. These experiments suggest a direct and possible specific role of cell surface galactosyltransferase enzyme in the mechanism of concanavalin A agglutination of cells.Concanavalin A (Con A), a lectin isolated from Jack beans, is known to preferentially agglutinate certain cell types. Agglutination by Con A has been demonstrated for virally transformed cells (1), chick embryonic retinal cells (2), human fetal intestinal cells (3), mitotically active intestinal crypt cells (4), and various erythrocytes (5). Although the mechanism of agglutination remains unclear, it is known that Con A binding involves interaction with the carbohydrate portion of cell membrane glycoproteins (6). However, the relationship of binding to agglutination is unresolved. Some investigators have reported a differential quantitative binding between agglutinable and nonagglutinable cells (7-9), while others reported that many cells agglutinated by Con A bind as much Con A as those not agglutinated by Con A (10, 11).The latter data have led to suggestions of a topographic explanation for Con A agglutinability (12,13

Cell Surface Galactosyltransferase and Lectin Agglutination of Thymus and Spleen Lymphocytes

“…S cells showed only minimal changes in galactosyltransferase activity over 72 hr, either with or without Con A. Con A appeared to cause a slight depression of surface galactosyltransferase activity in S cells. We have noted a depression of transferase activity by Con A with a variety of other cells, and have interpreted this as supporting the hypothesis that Con A binds to the enzyme, the acceptor, or to both (7).…”

“…Our previous experiments with rabbit erythrocyte plasma membranes suggested that galactosyltransferase enzyme was directly involved in Con A agglutination of these cells (7). It …”

“…Incubation mixtures contained 0.05 ml of a cell suspension of T or S cells and 0.05 ml of Con A or WGA at various concentrations. Degree of agglutination (0 to ++++) was determined after a 30-min incubation in a 370 water bath, as described (7). Control diameter glass fiber filters (Grade 934AH, Reeve Angel), and the filters were washed twice more with 5% phosphotungstic acid-2 M HCl and then three times with cold absolute ethanol.…”

“…A recent study from this laboratory showed a correlation between Con A agglutination of various cells and the activity of cell surface galactosyltransferase (EC 2.4.1.67; 1-O-a-Dgalactosyl-myo-inositol: raffinose galactosyltransferase) (7). Agglutinable In this study we demonstrate that T lymphocytes of neonatal rates were preferentially agglutinated and blast transformed by Con A, while spleen lymphocytes (S cells) were agglutinated by wheat germ agglutinin (WGA) and not blast transformed by Con A.…”

Biosynthesis of glycoprotein-glycosyl transferases during the cell cycle.

“…I was particularly interested in glycoprotein-glycosyl transferases as membrane components for the following reasons. First , this enzyme system is involved in the biosynthesis of glycoproteins that are major constitutents of plasma membrane and one of them has been reported to be a glycoprotein itself (Podolsky et al 1974). Secondly, the glycosyl transferases are localized both in the plasma membrane and in the Golgi membrane.…”