Effect of swainsonine, an inhibitor of glycoprotein processing, on cultured mammalian cells

Abstract

Swainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase 11. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 pg/ml, swainsonine did not affect the growth rate of Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus-181 (SV-IOI), 8-16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5-day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine-grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B-16 melanoma cells were grown for several days in the presence of swainsonine (100-500 ng/ml), these cells showed a 50-100% increase in their ability to bind [3H]concanavalin A, and a substantial decrease in the binding of [3H]wheat germ agglutinin. These alterations suggested an increase in high-mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B-886, a bacterium that binds to high-mannose glycoproteins, was also increased 1.5-to twofold, in cells grown in swainsonine. However, the binding of E. coli SS-142, another bacterial strain that does not bind to high-mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 pglml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [3H]leucine or [3H]proline into protein.There was, however, a significant inhibition in the incorporation of [3H]fucose, [3H]glucosamine, and [3H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, gl...

Oligosaccharide modification by swainsonine treatment inhibits pulmonary colonization by B16-F10 murine melanoma cells.

“…The Fig. 4, SW at concentrations up to 10 ,ug/ml did not impair either leucine or mannose incorporation into trichloroacetic acid-insoluble material, in agreement with previous findings (23). Indeed, at no time has SW ever exerted a toxic effect at these concentrations, even for incubation periods ofup to 3 days (data not shown).…”

Bacterial lectins, cell‐cell recognition and infectious disease

“…Animal cells treated with swainsonine, an inhibitor of the processing of asparagine-linked oligosaccharide units of glycoproteins, express increased levels of oligomannose or hybrid type oligosaccharides on their surfaces, and decreased levels of complex oligosaccharides. Such cells bound increased numbers (1 S-2-fold) of mannose specific E. coli, but binding to the cells of mannose resistant E. coli was unaffected [26].…”

Intestinal Dipeptidyl Peptidase IV Is Efficiently Sorted to the Apical Membrane through the Concerted Action of N- andO-Glycans as Well as Association with Lipid Microdomains

“…When used, benzylGalNAc, an inhibitor of O-glycosylation (39), swainsonine, an inhibitor of mannosidase II (40), fumonisin, an inhibitor of sphingolipid biosynthesis (41), or ␤-cyclodextrin for cholesterol depletion (42) were present in the culture medium during preincubation of the cells in methioninedeficient medium and during the labeling intervals essentially as described before (36). The final concentration of benzyl-GalNAc was 4 mM, that of swainsonine was 4 g/l, that of fumonisin was 10 M, and that of ␤-cyclodextrin was 50 mM (all reagents were obtained from Sigma).…”