Swainsonine, a potent mannosidase inhibitor, elevates rat liver and brain lysosomal alpha-D-mannosidase, decreases Golgi alpha-D-mannosidase II, and increases the plasma levels of several acid hydrolases

Swainsonine, a toxic plant alkaloid reported to be the agent that induces in animals a neurological condition very similar to the hereditary lysosomal storage disease mannosidosis, and to inhibit the formation of complex glycoproteins of the asparagine-linked class, was recently shown [D.R.P. Tulsiani, T.M. Harris, and O. Touster, (1982) J. Biol. Chem. 257, 7936-7939] to be a highly potent and specific inhibitor of Golgi mannosidase II in addition to being a strong inhibitor of lysosomal mannosidase. In the present study the effect of administered swainsonine on tissue enzyme levels was investigated. The activity of Golgi mannosidase II was markedly decreased (22% of control) without changes occurring in the activities of several other Golgi enzymes. However, the effects of swainsonine on lysosomal enzymes was unexpected. In liver, acid mannosidase increased markedly, instead of decreasing as would be expected from a compound reported to induce a mannosidosis-like condition. Similarly, the principal change in brain was a substantial increase in lysosomal mannosidase levels. In plasma, most lysosomal enzymes increased. These results indicate that the pathological effects of swainsonine are not solely attributable to its being an inhibitor of lysosomal alpha-D-mannosidase and are probably a consequence of abnormal processing of glycoproteins.     

Topology of mannosidase II in rat liver Golgi membranes and release of the catalytic domain by selective proteolysis

The orientation of mannosidase II, an integral Golgi membrane protein involved in asparagine-linked oligosaccharide processing, has been examined in rat liver Golgi membranes. Previous studies on mannosidase II purified from Golgi membranes revealed an intact subunit of 124,000 daltons, as well as a catalytically active 110,000-dalton degradation product generated during purification (Moremen, K. W., and Touster, O. (1985) J. Biol. Chem. 260, 6654-6662). In Triton X-100 extracts of Golgi membranes, the intact enzyme was cleaved by a variety of proteases to generate degradation products similar to those observed previously. At appropriate concentrations, chymotrypsin, pronase, and proteinase K generated 110,000-dalton species, while trypsin and Staphylococcus aureus V8 protease generated 115,000-dalton forms. Cleavage by chymotrypsin under mild conditions (10 micrograms/ml, 10 min, 20 degrees C) resulted in a complete conversion to a catalytically active 110,000-dalton form of the enzyme which was extremely resistant to further degradation. Attempts to demonstrate these protease digestions in nonpermeabilized Golgi membranes were unsuccessful, a result suggesting that the protease-sensitive regions are not accessible on the external surface of the membrane. In Golgi membranes permeabilized by treatment with 0.5% saponin, mannosidase II could readily be cleaved to the 110,000-dalton form by digestion with chymotrypsin under conditions similar to those which result in a proteolytic inactivation of galactosyltransferase, a lumenal Golgi membrane marker. Although mannosidase II catalytic activity was not diminished by this chymotrypsin digestion, as much as 90% of the enzyme activity was converted to a nonsedimentable form. To examine the effect of the proteolytic cleavage on the partition behavior of the enzyme, control and chymotrypsin-treated Triton X-114 extracts of Golgi membranes were examined by phase separation at 35 degrees C. The undigested enzyme partitioned into the detergent phase consistent with its location as an integral Golgi membrane protein, while the 110,000-dalton chymotrypsin-digested enzyme partitioned almost exclusively into the aqueous phase in a manner characteristic of a soluble protein. These results suggest that mannosidase II catalytic activity resides in a proteolytically resistant, hydrophilic 110,000-dalton domain. Attachment of this catalytic domain to the lumenal face of Golgi membranes is achieved by a proteolytically sensitive linkage to a 14,000-dalton hydrophobic membrane anchoring domain.     

The purification and characterization of mannosidase IA from rat liver Golgi membranes

Rat liver Golgi membranes contain two alpha 1,2-specific mannosidases (IA and IB) (Tulsiani, D. R. P., Hubbard, S. C., Robbins, P. W., and Touster, O. (1982) J. Biol. Chem. 257, 3660-3668). Mannosidase IA has now been purified to apparent homogeneity by detergent extraction and (NH4)2SO4 precipitation, followed by Sephacryl S-300, ion-exchange, and hydroxylapatite chromatography. The enzyme was homogeneous by nondenaturing polyacrylamide gel electrophoresis with different gel concentrations, and Ferguson plot analysis indicated an Mr of 230,000 for the native enzyme. Although electrophoresis under denaturing conditions generally gave a subunit Mr of 57,000, electrophoresis of less than 1 microgram of protein yielded a faint doublet of Mr 57,000 and 58,000. Thus, the enzyme appears to be a tetramer with four very similar subunits. The enzyme bound to concanavalin A-Sepharose 4B only when it was kept in contact with the lectin for 16 h. Endoglycosidase H treatment resulted in loss of its binding to the lectin, without leading to a detectable change in the size of the enzyme subunit. On electrophoretic gels, the enzyme gave a faint positive stain with periodic acid-Schiff's base. The enzyme contained about 0.9% hexose by direct analysis. It did not bind to affinity resins specific for neuraminic acid, galactose, or N-acetylglucosamine. All these studies suggest that the enzyme is a glycoprotein containing only one or two clusters of high mannose oligosaccharide. Mannosidase IA is active toward oligosaccharides containing alpha 1,2-linked mannosyl residues. [3H]Man9GlcNAc, [3H] Man8GlcNAc, [3H]Man7GlcNAc, and [3H]Man6GlcNAc are good substrates. Man9GlcNAc, the best substrate, yields Man8, Man7, and Man6 derivatives with structures suggesting that the sequence of release of mannose residues is rather specific. Immunoprecipitation studies using polyclonal antibody (IgG) prepared against homogeneous mannosidase IA cross-reacted with mannosidase IB, a result suggesting that these two enzymes share antigenic determinants. However, no cross-reactivity was observed with rat liver cytosolic and lysosomal alpha-D-mannosidases or with Golgi mannosidase II.     

A novel Golgi mannosidase inhibitor: Molecular design,synthesis, enzyme inhibition,and inhibition of spheroid formation

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell–cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.     

Biosynthesis and modification of Golgi mannosidase II in HeLa and 3T3 cells

The biosynthesis and post-translational modification of mannosidase II, an enzyme required in the maturation of asparagine-linked oligosaccharides in the Golgi complex, has been investigated. Antibody raised against this enzyme purified from rat liver Golgi membranes was used to immunoprecipitate mannosidase II from rat liver, 3T3 cells, or HeLa cells. Mannosidase II immunoprecipitated from rat liver Golgi membranes, when analyzed by polyacrylamide gel electrophoresis, migrated with an apparent molecular weight of approximately 124,000. In contrast, the enzyme purified from rat liver Golgi membranes was shown to contain both the 124,000-dalton component and a 110,000-dalton polypeptide believed to result from degradation of intact mannosidase II during purification. Mannosidase II from 3T3 and HeLa cells migrated on polyacrylamide gels with apparent molecular weights of approximately 124,000 and 134,000-136,000, respectively. When immunoprecipitated from radiolabeled cultures, mannosidase II from both cell types was similar in the following respects: (a) the initial synthesis product had an apparent molecular weight of approximately 124,000; (b) in cultures treated with tunicamycin the initial synthesis product had an apparent molecular weight of approximately 117,000; (c) endoglycosidase H digestion of the initial synthesis product gave an apparent molecular weight similar to the tunicamycin-induced polypeptide; (d) the mature enzyme was mostly (HeLa) or entirely (3T3) resistant to digestion by endoglycosidase H. Loss of [35S]methionine from intracellular mannosidase II occurred with a half-life of approximately 20 h; there was no appreciable accumulation of labeled immuno-reactive material in the medium. HeLa mannosidase II, but not the 3T3 enzyme, was additionally modified 1-3 h after synthesis, the initial synthesis product being converted to a doublet with an apparent molecular weight of approximately 134,000-136,000. Evidence is presented that this mobility shift may result from O-glycosylation. Mannosidase II from both cell types could be labeled with [32P]phosphate or [35S]sulfate. The latter is apparently attached to oligosaccharide as indicated by inhibition of labeling by tunicamycin; the former was shown with the HeLa enzyme to be present as serine phosphate moieties. In addition, [3H]palmitate could be incorporated into the enzyme in 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification to homogeneity and properties of mannosidase II from mung bean seedlings

Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.     

Purification and characterization of the alpha-D-mannosidase of rat liver cytosol.

Three forms of alpha-D-mannosidase have previously been identified in rat liver, and each is localized in a different subcellular fraction: lysosomes, Golgi membranes, and cytosol. This communication reports the purification and characterization the cytosolic form. The enzyme was purified 12,000-fold in good yield to approximately 90% purity with the aid of the competitive inhibitor mannosylamine and dithioerythritol as stabilizers. The molecular weight of the enzyme is in the range of 372,000 to 490,000 depending on the method used. Since the subunit molecular weight is 110,000 by sodium dodecyl sulfate polyacrylamide electrophoresis, the enzyme is probably a tetramer. The pH optimum was shown to be between 5.5 and 5.9 (in the presence of 1 mM CoCl2) with the substrate p-nitrophenyl-alpha-D-mannoside. Normal Michaelis-Menten kinetics were observed with a Km of 0.14 mM. Mannosylamine was a competitive inhibitor with a Ki of 0.007 mM. The purified enzyme, stabilized by Co2+, Mn2+, and Fe2+ under some conditions, was unstable at low protein concentrations. Since an electrophoresed sample showed a positive periodic acid-Schiff stain, the enzyme may contain carbohydrate. The availability of purified cytosolic alpha-D-mannosidase should now make it possible to carry out substrate specificity, immunological, and structural studies which may shed light on the biological role of this enzyme.     

The uptake of swainsonine, a specific inhibitor of alpha-D-mannosidase, into normal human fibroblasts in culture

Swainsonine, an indolizidine alkaloid, found in plants of the genus Swainsona, has been shown to be a strong inhibitor in vitro of the alpha-D-mannosidase activity in normal human fibroblasts. Therefore, inhibition of alpha-D-mannosidase activity in extracts of harvested cells grown with swainsonine in the medium has been used to follow the association of the alkaloid with normal human fibroblasts in culture. Swainsonine that could not be removed by extensive washing became associated with the cells within 1 min, and it is concluded that the alkaloid is internalized rapidly by the cells. The amount of swainsonine taken up into the cells depended on the length of time in contact and the concentration of swainsonine in the medium, but at 37 degrees C a plateau of internalized swainsonine occurred after 2 hr with extracellular concentrations of swainsonine of 100 microM or greater. At lower concentrations of swainsonine the rate of uptake was found to be temperature-dependent, increasing greatly at 20 degrees C. The rapidity and temperature sensitivity of the uptake, together with the observation that mannose or mannose-6-phosphate did not prevent the association, suggest that swainsonine enters the cells by permeation rather than by endocytosis. When swainsonine is withdrawn from the culture medium, there is a decrease with time of cell-associated swainsonine. The kinetics of uptake and release of swainsonine and its slightly basic nature make it likely that swainsonine is concentrated initially in the lysosomes. This rapid, but reversible, concentration of swainsonine in lysosomes would be consistent with the observed effects of the toxin in vivo.     

Protein matrix effects on glycan processing by mannosidase II and sialyl transferase from rat liver

The effect of the protein environment on the reaction sequence and the relative rates of two two-step reactions involved in the biosynthesis of complex glycans in glycoproteins has been explored by comparing the processing of biotinylated substrates either free or bound to avidin. By use of biotinyl and biotinamidohexanoyl derivatives, the display of the glycan in a proximal and distal association with the avidin surface could also be assessed. Mannosidase II removes two Man residues from the substrate GlcNAcMan5GlcNAc2-R to yield GlcNAcMAn3GlcNAc2-R. The NMR spectra of the substrate, intermediate, and product showed that the first Man is removed from the 6-arm of the substrate. The rate constants for the first and second step (estimated by direct analysis of the reactants by anion-exchange chromatography with a pulsed amperometric detector) were determined to be about 0.05 and 0.08 min-1, respectively, for the free substrates. In the proximal complex k1 was reduced 80-fold, and the k2 step could not be observed under the same conditions. In the distal complex both k1 and k2 were reduced about 8-fold. Sialyl transferases transfer Sia from CMP-Sia to the biantennary substrate Gal2GlcNAc2-Man3GlcNA2-R to yield the product Sia2Gal2-GlcNAc2Man3GlcNAc2-R with the Sia linked either 2-3 or 2-6 to the Gal residues. The NMR spectra showed that the first step involved the Gal on the 3-arm of the substrate and that both Sia residues were added 2-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical studies on rat liver Golgi apparatus. II. Further characterization of isolated Golgi fraction.

Although the preparation of rat liver Golgi apparatus isolated by our method contains appreciable activities of NADH- and NADPH-cytochrome c reductases and glucose-6-phosphatase, these enzymes as well as thiamine pyrophosphatase of the extensively fragmented Golgi fraction are partitioned in aqueous polymer two-phase systems quite differently from those associated with microsomes. Similarly, the partition patterns of acid phosphatase and 5'-nucleotidase of the Golgi fragments differ from those of homogenized lysosomes and plasma membrane, respectively. It is concluded that most, if not all, of these marker enzymes in the Golgi fraction cannot be ascribed to contamination by the non-Golgi organelles. In sucrose density gradient centrifugation the NADH- and NADPH-cytochrome c reductase activities of the Golgi fraction behave identically with galactosyltransferase but differently from the reductase activities of microsomes, again indicating that the reductases are inherently associated with the Golgi apparatus. NADPH-cytochrome c reductase of the Golgi preparation is immunologically identical with that of microsomes. The marker enzymes mentioned above and galactosyltransferase behave differently from one another when the Golgi fragments are subjected to partitioning in aqueous polymer two-phase systems, suggesting that these enzymes are not uniformly distributed in the Golgi apparatus structure.     

Lysosomal alpha-D-mannosidase of rat liver. Purification and comparison with the golgi and cytosolic alpha-D-mannosidases

Rat liver contains alpha-D-mannosidases in lysosomes, Golgi membranes, and cytosol. The lysosomal enzyme has now been purified approximately 30,000-fold over the crude extract and is free of at least 13 other lysosomal hydrolases. The enzyme has an apparent molecular weight of 335,000 by molecular sieve chromatography and 200,000 by sucrose density centrifugation. It is a glycoprotein, as evidenced by its binding to a concanavalin A affinity column and by a positive periodic acid-Schiff stain. The enzyme has a pH optimum near 4.6. Although it is generally insensitive to a large variety of inorganic salts, chelating agents, and sulfhydryl reagents, prolonged exposure to ethylenediaminetetraacetic acid caused loss of activity, which could be restored by the addition of ZnSO4. Substrate specificity studies were performed on the purified lysosomal alpha-D-mannosidase, as well as on the purified Golgi and cytosolic alpha-D-mannosidases. The three enzymes exhibited only very limited activity on native glycoproteins, but were found to be active on glycopeptides and oligosaccharides, hydrolyzing 1 yields 2 and 1 yields 3 linkages, except that the Golgi enzyme had negligible activity towards the latter linkage. Immunological comparisons by antibody precipitation tests and double-diffusion plates indicated that the three enzymes are not immunologically related. The alpha-D-mannosidase isolated from rat epididymis was found to be immunologically very similar, if not identical, to the lysosomal enzyme isolated from rat liver.     

Ganglioside biosynthesis in Golgi apparatus of rat liver. Stimulation by phosphatidylglycerol and inhibition by tunicamycin

Golgi vesicles were isolated and purified from rat liver, in which the specific activities of glycosyltransferases (e.g. GM3:CMP-NeuAc sialyltransferase, GD3 synthase; GM3:UDP-GalNAc galactosaminyltransferase, GM2 synthase) were 50-60-times enriched relative to microsomes or total homogenate. Synthesis of gangliosides GM2 and GM1 in such Golgi vesicles is, in the absence of any detergents, stimulated 6-fold and 20-fold respectively by phosphatidylglycerol. Other phospholipids like phosphatidylethanolamine and phosphatidylserine are also significantly stimulatory. With 50 micrograms Golgi protein and 1 nmol UDP-GalNAc, optimal stimulation of GM2 synthase was obtained with 20 micrograms of phosphatidylglycerol and 7.5 nmol of the lipid acceptor GM3. Under the same experimental conditions this stimulation exceeds (by about 40%) that obtained with optimal amount (200 micrograms) of the detergent octylglucoside. Phosphatidylglycerol, on the other hand, has virtually no stimulatory activity on the synthesis of ganglioside GD3 either in the presence of Mg2+ or Mn2+, indicating that facilitation by phospholipid of GM3 transport into Golgi vesicles was not the basis of stimulation of GM2 synthesis. Tunicamycin inhibits the synthesis of gangliosides GM2 and GM1 in isolated Golgi vesicles, but only in the absence of detergents. In the presence of phosphatidylglycerol, GM2 synthesis, for example, was inhibited by 60% by 2 micrograms tunicamycin and more than 85% by 10 micrograms tunicamycin, per 50 micrograms Golgi membrane protein. The inhibition was stronger on GM1 synthesis: 85% with 2.5 micrograms of the antibiotic. The dependence on phosphatidylglycerol and the degree of inhibition by tunicamycin of the synthetic activities are strictly dependent on the intactness of the Golgi vesicles: both phenomena become increasingly less evident when the vesicles are pelleted, and frozen and thawed several times, and completely disappear when the vesicles are solubilized by detergents or disrupted by ultrasonication. Furthermore, tunicamycin inhibition is reversible by increased concentration of phosphatidylglycerol. All these results indicate that phosphatidylglycerol does not stimulate, and tunicamycin does not inhibit, the transferases themselves; rather, the two opposing effects might relate to carrier-mediated transport, e.g. of nucleotide sugars, across Golgi vesicles.     

Effects of swainsonine on rat liver and kidney: biochemical and morphological studies

Among the reported effects of the plant toxin swainsonine in animals are a decreased level of Golgi mannosidase II activity, an increase in lysosomal alpha-D-mannosidase activity, oligosaccharide accumulation, vacuolization of cells, and neurological changes. We now find that, in the rat, the alkaloid rapidly induces vacuolization of both liver and kidney cells, but oligosaccharides accumulate only in the latter. We demonstrate by enzyme- and immunocytochemistry that the induced pleomorphic vacuoles are lysosomal in nature. The vacuoles do not appear to be derived from the Golgi apparatus, which retains its typical ultrastructural appearance, but are formed by autophagy. In swainsonine-fed rats, the lysosomal system is highly developed in hepatocytes, Kupffer cells, and cells of the proximal convoluted tubules. The relation of this hypertrophy of the lysosomal system to the known effects of swainsonine on glycoprotein biosynthesis and on Golgi and lysosomal alpha-mannosidases is not clear. In addition, in liver there occurs a marked increase in mitotic figures in the hepatocytes. This occurred in the absence of both cell death and increased liver size as estimated by gross morphology.     

Immunocytochemical localization of lysosomal beta-galactosidase in rat liver

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta- galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta- galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.     

Localization of dolichol in the lysosomal fraction of rat liver

The distribution of dolichol and/or dolichol esters in subcellular fractions prepared from a rat liver homogenate has been investigated. After saponification of the various fractions dolichol was isolated and quantitated by high performance liquid chromatography in three systems. The degree of purity of the subcellular preparations was examined by marker enzymes and by electron microscopy. Using differential centrifugation it was found that the level of dolichol was highest in the mitochondria-lysosome fraction. Upon further resolution of this fraction by sucrose density centrifugation it was found that the majority of the dolichol was associated with the lysosome-rich fraction. In contrast, the mitochondrial fraction had only a low level of dolichol. This novel observation was confirmed by the finding that dolichol was greatly enriched in a highly purified lysosome fraction preparations isolated by Metrizamide density centrifugation. The enrichment of dolichol in this purified preparation paralleled the observed enrichment of the lysosomal enzyme activity in this fraction. All of these data suggest that the majority of cellular dolichol and/or dolichol esters is localized in the lysosome fraction. The significance of this finding in relation to the metabolism of dolichol is discussed.