Swainsonine: an inhibitor of glycoprotein processing.

Abstract

Swainsonine, an indolizidine alkaloid, inhibits the processing ofasparagine-linked glycoproteins in both cell-free extracts and animal cells in culture. Thus, in a liver particulate enzyme preparation, swainsonine at 0.1-1.0 FM inhibited the mannosidase that releases [3H]mannose from a high mannose glycopeptide but only slightly inhibited the release of glucose from a glucose-labeled glycopeptide. MDCK and Chinese hamster ovary cells in culture incorporate [2-3H]mannose and [6-3H]-glucosamine into both high mannose and complex types ofoligosaccharides. When these cells were incubated with swainsonine and then labeled with mannose or glucosamine, there was a dramatic decrease in the amount oflabel in the complex type ofglycopeptide and a substantial increase in the radioactivity in the high mannose type. This change was monitored by the increase in radioactivity that became susceptible to digestion by endoglucosaminidase H with increasing concentrations of swainosine. The endoglucosaminidase H-released oligosaccharide(s) from swainsonine-treated cells was larger and more homogenous than that from controls and eluted from Bio-Gel P4 at the position of Man9GlcNAc. Several tissue culture cell lines were grown in the presence of swainsonine to determine its effect on cell surface glycoproteins. Cells grown in the alkaloid showed an increased capacity to bind Escherichia coli B886, a bacterium that binds to high mannose glycoproteins. These cells also showed an increasing binding of [3H]concanavalinA.During the synthesis of the oligosaccharide portion of asparagine-linked glycoproteins, a Glc3Man9GlcNAc2 oligosaccharide is transferred from its dolichol derivative to the protein (1). This newly formed glycoprotein is apparently the precursor of both the high mannose and the complex types ofoligosaccharide and undergoes a number ofprocessing or trimming reactions to form these oligosaccharides. These reactions result in the removal of all three glucose residues and a number of mannose units (3). Several enzymes having glucosidase and mannosidase activity have been described in various membrane fractions (4-9). While the mannose residues are being removed, the sugars that characterize the complex oligosaccharides are added from the appropriate sugar nucleotide donors to form the trisaccharide structures, NeuNAc -* galactose --GlcNAc, linked to the mannose core (10).In this paper, we describe a new inhibitor, swainsonine, that blocks the processing of asparagine-linked glycoproteins. Swainsonine is an indolizidine alkaloid isolated from the plant Swainsona canescens (11). The compound has been shown to inhibit lysosomal a-mannosidase and to produce symptoms of a-mannosidosis in cattle that eat the plant (12). We also show that this alkaloid inhibits the a-mannosidase involved in glycoprotein processing and therefore leads to an increase in high mannose oligosaccharides and a decrease in complex types at the cell surface.EXPERIMENTAL PROCEDURES Studies in Cell-Free Systems. The effect of swainsonine on neutral a-m...

Plant lectins are novel Toll-like receptor agonists

“…Moreover fluorescently labeled lectins have been used effectively to detect and sort cell types as well as define kinetics [22], so by designing a labeled lectin TLR binding assay a new avenue into screening for small molecules that interact with TLRs can be pursued. Other standard lectin biology-based tools could also be applied to gain greater understanding of this lectin-TLR activation, for example, demonstrate blockade of lectin activation using known cognate oligosaccharides and/or modulation of protein glycosylation using swainsonine [23].…”

Studies on synthetic pathway of xylose‐containing N‐linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.)

“…When 10 mM swainsonine, an inhibitor of Golgi amannosidase I1 [39], was added to the assay system including M5-PA, a different pyridylaminated derivative was obtained. After a 4-h incubation, the elution times of the pyridylaminated derivative were 10.2 min and 13.7 rnin on ODssilica and amide-silica columns, respectively (Fig.…”

Spontaneous Lysosomal Storage Disease Caused by Sida carpinifolia (Malvaceae) Poisoning in Cattle

“…Similar lesions were also reported in cases of poisoning by plants that contain the alkaloid swainsonine, 1,3,4,9,10,12,20,22 as well as in cases of inherited alpha-mannosidosis in cattle, 21,39 humans, 29 domestic cats, 5,27 and the guinea pig. 28 As in the inherited disease, in the acquired form of alpha-mannosidosis, the alkaloid swainsonine inhibits lysosomal enzyme alpha-mannosidase, 6,8,11,14 resulting in the storage and excretion of partially impaired oligosaccharides. In addition, swainsonine inhibits alpha-mannosidase II from the Golgi complex, modifying the synthesis and excretion of glycoproteins.…”