Inhibition of lysosomal alpha-mannosidase by swainsonine, an indolizidine alkaloid isolated from Swainsona canescens.

An indolizidine alkaloid (swainsonine) was isolated from the plant Swainsona canescens. Swainsonine is a specific and potent inhibitor of alpha-mannosidase (EC 3.2.1.24) and when administered to animals produces a phenocopy of the genetically based lysosomal storage disease, mannosidosis. Evidence is presented to suggest that swainsonine is a reversible active site-directed inhibitor of lysosomal alpha-mannosidase.     

Effect of swainsonine on the processing and turnover of lysosomal beta-galactosidase and beta-glucuronidase from mouse peritoneal macrophages

The effect of swainsonine, an inhibitor of Golgi alpha-mannosidase II and lysosomal alpha-mannosidase, on the synthesis, processing, and turnover of two glycoproteins, lysosomal beta-galactosidase and lysosomal beta-glucuronidase, has been studied in cultured mouse peritoneal macrophages. No effect of the inhibitor on the relative rates of synthesis of the precursor form of either enzyme was observed. On the other hand, carbohydrate processing of beta-galactosidase and beta-glucuronidase was markedly altered by swainsonine, consistent with a blockage by the inhibitor of the removal of the alpha-1,3- and alpha-1,6-linked mannose residues which occurs in normal processing. In homogenates of both normal and swainsonine-treated cells, the precursor forms of the enzymes were found exclusively in the light membrane fraction on Percoll gradients and the mature forms exclusively in the lysosomal fractions indicating that translocation from Golgi to lysosomes and proteolytic processing in the lysosome were not impaired by the presence of abnormal oligosaccharide side chains. There was no detectable effect of swainsonine during a 4-day chase period on the total cellular turnover of these enzymes which involves two processes, secretion and degradation. In the absence of swainsonine, secretion represented about 40% of the total turnover of beta-galactosidase and about 50% with beta-glucuronidase. The presence of swainsonine increased these proportions to about 60 and 70%, respectively.     

Swainsonine inhibits macrophage receptor-mediated uptake and degradation of a mannosyl-oligosaccharide

Rat pulmonary macrophages were incubated in the presence of a radiolabeled mannosyl-oligosaccharide obtained from ovalbumin. Receptor-mediated endocytosis and degradation of this ligand by the cells was followed in the presence or absence of swainsonine, an inhibitor of alpha-mannosidases. The results indicated that at higher concentrations (greater than 1 microgram/ml) of swainsonine, both the internalization and degradation of the radiolabeled ligand were inhibited. At a concentration of 0.1 microgram/ml of swainsonine, only the degradation was inhibited while the uptake was unaltered. The degradation of the oligosaccharide was blocked due to the inhibition of lysosomal alpha-mannosidase. However, the inhibition of lysosomal alpha-mannosidase was reversible upon withdrawal of swainsonine.     

Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver

An alpha-mannosidase activity has been identified in a preparation of rat liver endoplasmic reticulum and shown to be distinct from the previously described Golgi alpha-mannosidases I and II and the lysosomal alpha-mannosidase. The enzyme was solubilized with deoxycholate and separated from other alpha-mannosidases by passage over concanavalin A-Sepharose to which it does not bind. The endoplasmic reticulum alpha-mannosidase cleaves alpha-1,2-linked mannoses from high mannose oligosaccharides and, unlike Golgi alpha-mannosidase I, is active against p-nitrophenyl-alpha-D-mannoside (Km = 0.17 mM). It has no activity toward GlcNAc-Man5GlcNAc2 peptide, the specific substrate of the Golgi alpha-mannosidase II. The endoplasmic reticulum alpha-mannosidase activity toward p-nitrophenyl-alpha-D-mannoside is relatively insensitive to swainsonine, an inhibitor of both the lysosomal alpha-mannosidase and Golgi alpha-mannosidase II. We propose that the endoplasmic reticulum alpha-mannosidase is responsible for the removal of mannose residues from asparagine-linked high mannose type oligosaccharides prior to their entry into the Golgi.     

Swainsonine is a useful tool to monitor the intracellular traffic of N-linked glycoproteins as a function of the state of enterocytic differentiation of HT-29 cells.

After treatment with swainsonine, an inhibitor of both lysosomal alpha-mannosidase and Golgi alpha-mannosidase-II activities, analysis of [3H]mannose-labeled glycans showed that HT-29 cells, derived from a human colonic adenocarcinoma, displayed distinct patterns of N-glycan expression, depending upon their state of enterocytic differentiation. In differentiated HT-29 cells hybrid-type chains were detected, whereas undifferentiated HT-29 cells accumulated high-mannose-type oligosaccharide, despite our demonstration of Golgi alpha-mannosidase-II activity in both cell populations. Pulse/chase experiments carried out in the presence of swainsonine revealed that the persistence of high-mannose-type chains in undifferentiated HT-29 cells was the result of the stabilization of glycoproteins substituted with these glycans. These data suggest that in undifferentiated HT-29 cells, glycoproteins with high-mannose-type oligosaccharides are delivered to a degradative compartment containing swainsonine-sensitive alpha-mannosidase(s), whereas in differentiated HT-29 cells glycoproteins enter a compartment in which alpha-mannosidase II (Golgi apparatus) is present. Thus, this apparent dual effect of swainsonine on N-glycan trimming may reflect differences in the intracellular traffic of glycoproteins as a function of the state of enterocytic differentiation of HT-29 cells.     

Marked differences in the swainsonine inhibition of rat liver lysosomal alpha-D-mannosidase, rat liver Golgi mannosidase II, and jack bean alpha-D-mannosidase

Swainsonine, a plant toxin, strongly inhibits certain alpha-D-mannosidases but has no effect on others [D. R. P. Tulsiani, T. M. Harris, and O. Touster (1982) J. Biol. Chem. 257, 7936-7939]. The reversible inhibition of jack bean and lysosomal alpha-D-mannosidases has previously been suggested to be similar in nature but quite complex. Specific differences in the action of swainsonine on these two enzymes and on Golgi mannosidase II are reported. (a) The inhibition of the jack bean mannosidase, but not rat liver lysosomal alpha-D-mannosidase or Golgi mannosidase II, is increased by preincubation with the alkaloid. (b) The inhibition of the jack bean and lysosomal enzymes, but not mannosidase II, is competitive at inhibitor concentrations of less than or equal to 0.5 microM. (c) The inhibition of jack bean alpha-mannosidase is largely irreversible, its very limited reversibility being partially dependent upon the swainsonine concentration used and on the time of preincubation with the inhibitor. On the other hand, the inhibition of lysosomal alpha-mannosidase is largely reversible, as shown by dilution experiments and by the use of [3H]swainsonine. Golgi mannosidase II shows intermediate reversibility, the results indicating two modes of binding; one rapid and irreversible, the other much slower and reversible.     

A human lysosomal alpha-mannosidase specific for the core of complex glycans.

A novel lysosomal alpha-mannosidase, with unique substrate specificity, has been partially purified from human spleen by chromatography through concanavalin A-Sepharose, DEAE-Sephadex, and Sephacryl S-300. This enzyme can catalyze the hydrolysis of only 1 mannose residue, that which is alpha(1----6)-linked to the beta-linked mannose in the core of N-linked glycans, as found in the oligosaccharides Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc and Man alpha(1----6)Man beta(1----4) GlcNAc. The newly described alpha-mannosidase does not catalyze the hydrolysis of mannose residues outside of the core, even if they are alpha(1----6)-linked, and is not active on the other alpha-linked mannose in the core, which is (1----3)-linked. The narrow specificity of the novel mannosidase contrasts sharply with that of the major lysosomal alpha-mannosidase, which is able to catalyze the degradation of oligosaccharides containing diverse linkage and branching patterns of the mannose residues. Importantly, although the major mannosidase readily catalyzes the hydrolysis of the core alpha(1----3)-linked mannose, it is poorly active towards the alpha(1----6)-linked mannose, i.e. the very same mannose residue for which the newly characterized mannosidase is specific. The novel enzyme is further differentiated from the major lysosomal alpha-mannosidase by its inability to catalyze the efficient hydrolysis of the synthetic substrate p-nitrophenyl alpha-mannoside, and by the strong stimulation of its activity by Co2+ and Zn2+. Similarly to the major mannosidase, it is strongly inhibited by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, but not by deoxymannojirimycin. The presence of this novel alpha-mannosidase activity in human tissues provides the best explanation, to date, for the structures of the oligosaccharides stored in human alpha-mannosidosis. In this condition the major lysosomal alpha-mannosidase activity is severely deficient, but apparently the alpha(1----6)-mannosidase is unaffected, so that the oligosaccharide structures reflect the unique specificity of this enzyme.     

Fucosylated hybrid-type N-glycans on the secreted human epidermal growth factor receptor from swainsonine-treated A431 cells

N-Glycans linked to the human secreted form of epidermal growth factor receptor were isolated from A431 cells after swainsonine treatment. Analysis of the oligosaccharides by (1)H NMR spectroscopy and mass spectrometry shows the presence of oligomannose- and (alpha2-3)-sialylated hybrid-type glycans. The major hybrid-type oligosaccharide chains are fucosylated at the Asn-bound GlcNAc residue. Smaller amounts of the hybrid-type structures are also fucosylated at peripheral GlcNAc residues, constituting the sialyl-Le(x) antigen. No complex-type glycans are found, suggesting the absence of alpha-mannosidase III. An assay for alpha-mannosidase III on the A431 cells in the absence and presence of 6 microM swainsonine shows that Man(5)GlcNAc(2) is not converted into Man(3)GlcNAc(2), thereby confirming that these cells do not contain alpha-mannosidase III activity.     

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of cells with 0.2 mg/ml low-density lipoprotein and 50 microM chloroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without chloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with lipoprotein-deficient serum revealed that polyunsaturated cholesteryl esters (linoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA:cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We conclude that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydrolyzes polyunsaturated cholesteryl esters.     

Disruption of alpha-mannosidase processing induces non-canonical hybrid-type glycosylation

Golgi alpha-mannosidase II is essential for the efficient formation of complex-type glycosylation. Here, we demonstrate that the disruption of Golgi alpha-mannosidase II activity by swainsonine in human embryonic kidney cells is capable of inducing a novel class of hybrid-type glycosylation containing a partially processed mannose moiety. The discovery of 'Man(6)-based' hybrid-type glycans reveals a broader in vivo specificity of N-acetylglucosaminyltransferase I, further defines the arm-specific tolerance of core alpha1-6 fucosyltransferase to terminal alpha1-2 mannose residues, and suggests that disruption of Golgi alpha-mannosidase II activity is capable of inducing potentially 'non-self' structures.     

Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products

We have previously shown that oxidized low-density lipoprotein (LDL) induces damage to the macrophage lysosomal membranes, with ensuing leakage of lysosomal contents and macrophage cell death. Cholesterol oxidation products (ChOx) have been reported to be the major cytotoxic components of oxidized LDL/LDL- and also to stimulate cholesterol accumulation in vascular cells. In the present study, we characterized the initial events during macrophage damage induced by cholesterol oxidation products (ChOx). Within 24 h of exposure, ChOx caused lysosomal destabilization, release to the cytosol of the lysosomal marker-enzyme cathepsin D, apoptosis, and postapoptotic necrosis. Enhanced autophagocytosis and chromatin margination was found 12 h after the exposure to ChOx, whereas apoptosis and postapoptotic necrosis was pronounced 24 and 48 h after the exposure. Some lysosomal vacuoles were then filled with degraded cellular organelles, indicating phagocytosis of apoptotic bodies by surviving cells. Because caspase-3 activation was detected in the ChOx-exposed cells, lysosomal destabilization may associate with the leakage of lysosomal enzymes, and activation of the caspase cascade. MnSOD mRNA levels were markedly increased after 24 h of exposure to ChOx, suggesting associated induction of mitochondrial protection repair or turnover. We conclude that ChOx-induced damage to lysosomes and mitochondria are sequelae to the cascade of oxysterol cytotoxic events. The early disruption of lysosomes induced by ChOx, with resultant autophagocytosis may be a critical event in apoptosis and/or necrosis of macrophages/foam cells during the development of atherosclerotic lesions.     

Insect cells encode a class II alpha-mannosidase with unique properties

Previously, we cloned and characterized an insect (Sf9) cell cDNA encoding a class II alpha-mannosidase with amino acid sequence and biochemical similarities to mammalian Golgi alpha-mannosidase II. Since then, it has been demonstrated that other mammalian class II alpha-mannosidases can participate in N-glycan processing. Thus, the present study was performed to evaluate the catalytic properties of the Sf9 class II alpha-mannosidase and to more clearly determine its relationship to mammalian Golgi alpha-mannosidase II. The results showed that the Sf9 enzyme is cobalt-dependent and can hydrolyze Man(5)GlcNAc(2) to Man(3)GlcNAc(2), but it cannot hydrolyze GlcNAcMan(5)GlcNAc(2). These data establish that the Sf9 enzyme is distinct from Golgi alpha-mannosidase II. This enzyme is not a lysosomal alpha-mannosidase because it is not active at acidic pH and it is localized in the Golgi apparatus. In fact, its sensitivity to swainsonine distinguishes the Sf9 enzyme from all other known mammalian class II alpha-mannosidases that can hydrolyze Man(5)GlcNAc(2). Based on these properties, we designated this enzyme Sf9 alpha-mannosidase III and concluded that it probably provides an alternate N-glycan processing pathway in Sf9 cells.     

Synthesis of lysosomal alpha-mannosidase in normal and mannosidosis fibroblasts

The biosynthesis and secretion of lysosomal alpha-mannosidase was studied in metabolically labelled fibroblasts from controls and two patients with mannosidosis. Normal fibroblasts secrete alpha-mannosidase as a 110kDa polypeptide. Intracellularly alpha-mannosidase is represented by several polypeptides with apparent Mrs ranging from 40 to 67kDa. In two mannosidosis cell lines none of intra- and extracellular polypeptides of alpha-mannosidase were detectable. The mannosidosis fibroblasts secreted acid alpha-mannosidase activity at one third of the normal rate. In contrast to normal cells the secretion was not enhanced by NH4C1 and the secreted activity was not immunoprecipitable, indicating that the acid alpha-mannosidase activity secreted by mannosidosis fibroblasts is not related to the lysosomal alpha-mannosidase.     

A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis

A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha- mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild- type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the alpha-mannosidase precursor by HMW570 was not accompanied by major alterations in N- linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominantly located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.     

Structure-activity relationship of swainsonine. Inhibition of human alpha-mannosidases by swainsonine analogues.

The inhibitory properties of a series of synthetic epimers and analogues of swainsonine towards the multiple forms of human alpha-mannosidases were studied in vitro and in cells in culture. Of the five epimers tested, only the 8a-epimer and 8,8a-diepimer of swainsonine were specific and competitive inhibitors (Ki values of 7.5 x 10(-5) and 2 x 10(-6) M respectively) of lysosomal alpha-mannosidases in vitro and induced storage of mannose-rich oligosaccharides in human fibroblasts in culture. The structures of these storage products indicated that processing alpha-mannosidases had also been inhibited. This was consistent with the observed inhibition in vitro of these enzymes by these compounds. In contrast, the 8-epimer, 1,8-diepimer and 2,8a-diepimer of swainsonine had no appreciable effect on any alpha-mannosidases. The corresponding open-chain analogues of swainsonine, namely 1,4-dideoxy-1,4-imino-D-mannitol, of the 8a-epimer, namely 1,4-dideoxy-1,4-imino-D-talitol, and of the 8,8a-diepimer, namely 1,4-dideoxy-1,4-imino-L-allitol, were weaker competitive inhibitors of lysosomal alpha-mannosidase, with Ki values of 1.3 x 10(-5), 1.2 x 10(-4) and 1.2 x 10(-4) M respectively. These analogues also proved less effective at inducing oligosaccharide accumulation and in disturbing glycoprotein processing. These compounds offer the opportunity to determine which alterations in the chirality of the swainsonine molecule affect its inhibitory specificity. A comparison of their biological activities has identified reagents that will be useful for studying steps in the biosynthesis and catabolism of glycoproteins and that may be of potential value in chemotherapy.     

Glycoprotein metabolism in normal and beta-mannosidase-deficient cultured goat skin fibroblasts.

Cultured skin fibroblasts established from goats affected with beta-mannosidosis, an inherited neurovisceral storage disorder, showed an absence of lysosomal beta-mannosidase activity and the corresponding accumulation of a trisaccharide (TS) with the structure Man beta (1----4)GlcNAc beta (1----4)GlcNAc (0.4 mumol/g) and lesser amounts (0.15 mumol/g) of a Man beta (1----4)GlcNAc disaccharide (DS). By using purified storage TS isolated from fibroblasts metabolically labelled with [3H]GlcN, no conversion of TS into DS could be demonstrated in homogenates of affected cells at either lysosomal pH (4.4) or cytosolic pH (6.1), or in the culture medium (pH 7.0) of affected cells. Both TS and DS were secreted into the culture medium by affected fibroblasts. When affected fibroblasts were treated with tunicamycin before labelling with [3H]GlcN, the accumulation of both labelled TS and DS was completely inhibited. Treatment of both affected and normal goat fibroblasts with swainsonine resulted in the inhibition of lysosomal alpha-mannosidase activity and in the accumulation of the same labelled oligosaccharides in both. The major storage pentasaccharide from both normal and affected swainsonine-treated fibroblasts was sensitive to digestion with alpha-mannosidase and endo-beta-N-acetylhexosaminidase D, suggesting a branched mannose structure and a chitobiose core. In the absence of evidence for the existence of unusual N-linked glycoprotein-associated chitotriose oligosaccharide structures in affected goat fibroblasts, it must be concluded that degradative pathways for N-linked oligosaccharides are similar in both normal and affected goat fibroblasts, and that these pathways differ from catabolic pathways in human fibroblasts.     

Elevated temperature produces cystine depletion in cystinotic fibroblasts

Increasing the incubation temperature of cystinotic fibroblasts to 40 or 43 degrees C produces a 70-80% decrease in lysosomal cystine content within 24-48 h. This effect is probably mediated by an altered substrate affinity for another lysosomal transport protein.     

Different adhesion and migration properties of human HCV29 non-malignant urothelial and T24 bladder cancer cells: role of glycosylation

In tumour cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. This study used cell lines having very different behaviour in vivo: HCV29 non-malignant transitional epithelium and T24 bladder transitional cell carcinoma. These differences in behaviour might be due in part to differences in cellular glycosylation patterns. Glycan chain analysis of their glycoproteins was performed with the use of specific lectins. The functional role of carbohydrates was studied by treating these cells with swainsonine, an inhibitor of Golgi alpha-mannosidase II, and in vitro adhesion and migration assays. The adhesion of swainsonine-treated HCV29 and T24 cells was increased on fibronectin and type IV collagen by 1.5- and 2-fold, respectively, whereas adhesion on laminin was virtually unchanged after swainsonine-treatment in HCV29 cells and was increased in T24 cells. Swainsonine treatment reduced the rate of T24 cell migration by 20%. We concluded that beta1-6 branched tri- and tetraantennary complex-type glycans have an important function in adhesion and migration in the studied cell lines. These data support the view that oligosaccharides are involved in several steps of the metastatic process.