Inhibitor Of Alpha-mannosidase Research Articles

Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity.

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.

The N-glycan processing in HT-29 cells is a function of their state of enterocytic differentiation. Evidence for an atypical traffic associated with change in polypeptide stability in undifferentiated HT-29 cells.

When the human colon cancer cells HT-29 undergo enterocytic differentiation, they correctly process their N-glycans, whereas their undifferentiated counterpart are unable to process Man9-8-GlcNAc2 species, the natural substrate of alpha-mannosidase I. As this enzyme is fully active in both HT-29 cell populations, we hypothesize that N-glycoproteins are unable to reach the cis Golgi, the site where alpha-mannosidase I has been localized. We have demonstrated this point by using 1-deoxymannojirimycin, leupeptin, and monensin. In the presence of 1-deoxymannojirimycin, a specific inhibitor of alpha-mannosidase I, differentiated HT-29 cells, as expected, accumulate Man9-8-GlcNAc2 species, whereas in undifferentiated HT-29 cells these compounds continue to be rapidly degraded. In contrast, the use of leupeptin, a specific inhibitor of thiol and serine proteases, leads to the accumulation of these oligosaccharides in undifferentiated HT-29 cells. Monensin, a carboxylic ionophore that perturbs distal Golgi functions, is unable to stabilize these compounds. Therefore, we conclude that N-linked glycoproteins in undifferentiated HT-29 cells rapidly egress from the exocytic pathway to a leupeptin-sensitive degradative compartment without entering a monensin-sensitive compartment. These results favor the hypothesis that a direct pathway should exist between the rough endoplasmic reticulum and a leupeptin-sensitive degradative compartment in undifferentiated HT-29 cells. The emergence of this new pathway could explain why protein stability and N-glycan processing may vary as a function of the state of cell differentiation.

Mannostatin A, a new glycoprotein-processing inhibitor

Mannostatin A is a metabolite produced by the microorganism Streptoverticillium verticillus and reported to be a potent competitive inhibitor of rat epididymal alpha-mannosidase. When tested against a number of other arylglycosidases, mannostatin A was inactive toward alpha- and beta-glucosidase and galactosidase as well as beta-mannosidase, but it was a potent inhibitor of jack bean, mung bean, and rat liver lysosomal alpha-mannosidases, with estimated IC50's of 70 nM, 450 nM, and 160 nM, respectively. The type of inhibition was competitive in nature. This compound also proved to be an effective competitive inhibitor of the glycoprotein-processing enzyme mannosidase II (IC50 of about 10-15 nM with p-nitrophenyl alpha-D-mannopyranoside as substrate, and about 90 nM with [3H]mannose-labeled GlcNAc-Man5GlcNAc as substrate). However, it was virtually inactive toward mannosidase I. The N-acetylated derivative of mannostatin A had no inhibitory activity. In cell culture studies, mannostatin A also proved to be a potent inhibitor of glycoprotein processing. Thus, in influenza virus infected Madin Darby canine kidney (MDCK) cells, mannostatin A blocked the normal formation of complex types of oligosaccharides on the viral glycoproteins and caused the accumulation of hybrid types of oligosaccharides. This observation is in keeping with other data which indicate that the site of action of mannostatin A is mannosidase II. Thus, mannostatin A represents the first nonalkaloidal processing inhibitor and adds to the growing list of chemical structures that can have important biological activity.

Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I.

Kifunensine, produced by the actinomycete Kitasatosporia kifunense 9482, is an alkaloid that corresponds to a cyclic oxamide derivative of 1-amino mannojirimycin. This compound was reported to be a weak inhibitor of jack bean alpha-mannosidase (IC50 of 1.2 x 10(-4) M) (Kayakiri, H., Takese, S., Shibata, T., Okamoto, M., Terano, H., Hashimoto, M., Tada, T., and Koda, S. (1989) J. Org. Chem. 54, 4015-4016). We also found that kifunensine was a poor inhibitor of jack bean and mung bean aryl-alpha-mannosidases, but it was a very potent inhibitor of the plant glycoprotein processing enzyme, mannosidase I (IC50 of 2-5 x 10(-8) M), when [3H]mannose-labeled Man9GlcNAc was used as substrate. However, kifunensine was inactive toward the plant mannosidase II. Studies with rat liver microsomes also indicated that kifunensine inhibited the Golgi mannosidase I, but probably does not inhibit the endoplasmic reticulum mannosidase. Kifunensine was tested in cell culture by examining its ability to inhibit processing of the influenza viral glycoproteins in Madin-Darby canine kidney cells. Thus, when kifunensine was placed in the incubation medium at concentrations of 1 microgram/ml or higher, it caused a complete shift in the structure of the N-linked oligosaccharides from complex chains to Man9(GlcNAc)2 structures, in keeping with its inhibition of mannosidase I. On the other hand, even at 50 micrograms/ml, deoxymannojirimyucin did not prevent the formation of all complex chains. Thus kifunensine appears to be one of the most effective glycoprotein processing inhibitors observed thus far, and knowledge of its structure may lead to the development of potent inhibitors for other processing enzymes.

Basement membrane invasion by first trimester human trophoblast: requirement for branched complex-type Asn-linked oligosaccharides

Trophoblast cells of normal first trimester human placenta share with malignant tumor cells the ability for significant cellular proliferation and invasion of basement membranes. Because tumor cell metastasis in vivo and invasion of basement membranes in vitro have recently been shown to require the expression of -GlcNAc beta 1-6 Man alpha 1-6 Man beta 1-branched complex type Asn-linked oligosaccharides in tumor cell surface glycoproteins, we decided to determine if such structures were also necessary for invasion by trophoblast cells. We report here that invasive first trimester trophoblasts express leukoagglutinin-reactive beta 1-6 branched Asn-linked oligosaccharides on their surface. Moreover, basement membrane invasion by trophoblast was significantly inhibited by pretreating the cells with swainsonine, a non-toxic inhibitor of Golgi alpha-mannosidase II which blocks beta 1-6 branching of Asn-linked oligosaccharides. The first trimester trophoblast cells pretreated with swainsonine attached more avidly to the amnion basement membrane and to an extracellular matrix (ECM) preparation compared to control non-treated erophoblast cells. Swainsonine treatment did not inhibit secretion of gelatinase or plasminogen activator activities by trophoblast cells. These results suggest that expression of beta 1-6 branched oligosaccharides in trophoblast cells may be functionally important for the implantation and placentation processes by reducing cell adhesion to ECM and thereby facilitating trophoblast cell invasion.

Asynchronous Transport to the Cell Surface of Intestinal Brush Border Hydrolases Is Not Due to Differential Trimming of N-Linked Oligosaccharides

Intestinal brush border enzyme glycoproteins are transported to the microvillar membrane at different rates in the differentiated intestinal cell line Caco-2. This asynchronism is due to at least two rate-limiting events, a pre- and an intra-Golgi step (Stieger B., Matter, K., Baur, B., Bucher, K., Höchli, M., and Hauri, H.P. (1988) J. Cell Biol. 106, 1853-1861). A possible cause for the asynchronous protein transport might be differential trimming of N-linked oligosaccharide side chains. The effects of two trimming inhibitors on the intracellular transport of sucrase-isomaltase, a slowly migrating hydrolase, and dipeptidylpeptidase IV, a rapidly migrating hydrolase, are described. 1-Deoxymannojirimycin, an inhibitor of Golgi alpha-mannosidase I, had no influence on the rate of appearance of these hydrolases in the brush border membrane as assessed by subcellular fractionation. In the presence of N-methyl-1-deoxynojirimycin, an inhibitor of glucosidase I, 30-40% of the newly synthesized molecules appeared at the cell surface, and half-time for appearance of this pool was identical to that found in control cells. The reduced maximal transport to the cell surface observed with N-methyl-1-deoxynojirimycin may suggest that proper glycosylation is necessary for an efficient transport from the Golgi apparatus to the microvillar membrane. Inhibition of glucosidase I does not prevent the acquisition of endoglycosidase H resistance. Furthermore, evidence is presented that the processing in the presence of N-methyl-1-deoxynojirimycin leads to glycosylated endoglycosidase H-resistant glycoproteins.

Structure-activity relationship of swainsonine. Inhibition of human α-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.

Effects of the ?-mannosidase inhibitors, 1,4-dideoxy-1,4-imino-d-mannitol and swainsonine, on glycoprotein catabolism in cultured macrophages

Thioglycollate-stimulated murine peritoneal macrophages were cultured for eight days in the presence of swainsonine, or 1,4-dideoxy-1,4-imino-D-mannitol (DIM), or both of these competitive alpha-mannosidase inhibitors together. Analysis of accumulated high-mannose oligosaccharides by reversed phase HPLC after perbenzoylation revealed that DIM- and DIM-plus swainsonine-treated macrophages contained larger amounts of Man7GlcNAc, Man8GlcNAc and Man9GlcNAc, while swainsonine-treated macrophages contained relatively more Man3GlcNAc and Man5GlcNAc. These results are consistent with the known inhibitory effects of DIM and swainsonine on Golgi mannosidases I and II, respectively, and on lysosomal alpha-mannosidase. Depletion of stored oligosaccharides to control values was complete within seven days of terminating swainsonine treatment.

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.

Further studies on the role of glycosylation in thyroxine-binding globulin secretion by human hepatoma (HEP G2) cells.

To investigate the role of glycosylation of T4-binding globulin (TBG) in the secretion of the protein, human hepatoma (Hep G2) cells were continuously labeled with [35S] methionine or [3H]mannose or pulse-chase labeled with [35S] methionine in the absence or presence of 1 microgram/ml swainsonine, an inhibitor of Golgi alpha-mannosidase II and lysosomal alpha-mannosidase. In the presence of this alkaloid, TBG was released into the medium at a faster rate than in control cells (50% being secreted after 35 min and 47 min, respectively), owing to accelerated intracellular transport of the newly synthesized protein. TBG secreted from swainsonine-treated cultures moved faster in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, probably because of the reduced sialylation of TBG consequent to the perturbed processing of the oligosaccharide units. Furthermore, secreted TBG was sensitive to endo-beta-N-acetylglucosaminidase H digestion as shown by the shift in the apparent molecular size in sodium dodecyl sulfate-polyacrylamide gel electro-phoresis from 50,000 to 45,000 daltons. Sensitivity to endo H indicated the presence of hybrid-type oligosaccharide chains with high mannose structures. This was also suggested by the higher incorporation of [3H]mannose in swainsonine-treated cultures. In conclusion, the results of the present study demonstrate that swainsonine accelerates the release of TBG from Hep G2 cells and that complete processing of oligosaccharide moieties is not required for TBG secretion.

Effect of isomers of swainsonine on glycosidase activity and glycoprotein processing.

The chemical synthesis of swainsonine [(1S,2R,8R,8 alpha R)-trihydroxyindolizidine] from trans-1,4-dichloro-2-butene was previously described [Adams, C. E., Walker, F. J., & Sharpless, K. B. (1985) J. Org. Chem. 50, 420-424]. A modification of that synthesis provided two other isomers, referred to here as "Glc-swainsonine" [(1S,2S,8R,8 alpha R)-trihydroxyindolizidine] and "Ido-swainsonine" [(1S,2S,8S,8 alpha R)-trihydroxyindolizidine]. To determine whether these new compounds had biological activity, they were compared to swainsonine as inhibitors of a number of commercially available glycosidases. While swainsonine is a potent inhibitor of jack bean alpha-mannosidase but does not inhibit other glycosidases, its two isomers were inactive on alpha-mannosidase but did inhibit other enzymes. Thus, Glc-swainsonine was an inhibitor of the fungal alpha-glucosidase amyloglucosidase, and this inhibition was of a competitive nature (Ki = 5 X 10(-5) M) with respect to the substrate p-nitrophenyl alpha-D-glucopyranoside. This alkaloid also inhibited beta-glucosidase, but much less effectively than alpha-glucosidase. On the other hand, Ido-swainsonine was more effective toward beta-glucosidase than toward alpha-glucosidase, and this inhibition was also of a competitive nature. None of these inhibitors were effective against beta-mannosidase or alpha- or beta-galactosidase. Glc-swainsonine was also tested against the glycoprotein processing glycosidases. Surprisingly, in this respect, the alkaloid was like swainsonine in that it inhibited mannosidase II but had no effect or only slight effect on glucosidase I, glucosidase II, and mannosidase I. Glc-swainsonine also inhibited glycoprotein processing in cell culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor cell surface carbohydrate and the metastatic phenotype.

The synthesis and expression of cell surface carbohydrates is a developmentally regulated process that appears to affect a number of cell-cell interactions. To determine whether specific oligosaccharide structures present on highly malignant cells are required for expression of the metastatic phenotype, we have isolated lectin resistant tumor cell mutants with defects in the biosynthesis of oligosaccharides. The mutants selected from the highly aggressive lymphoreticular-like tumor line MDAY-D2 were grouped into genetic complementation classes, compared for metastatic ability and for changes in cell surface glycoconjugates. The Asn-linked oligosaccharides and glycolipids of class 1 mutants were deficient in both sialic acid and galactose and the cells showed a greatly attenuated metastatic phenotype compared to the parental cells. A revertant of the class 1 mutation selected in vitro regained the wild type glycoconjugate profile and the highly metastatic phenotype indicating a direct association between the mutation and the loss of metastatic potential. Class 2 mutants remained highly metastatic and had Asn-linked oligosaccharide structures very similar to those found in the wild type cells with N-glycolylneuraminic acid rather than the N-acetylneuraminic acid. Swainsonine, an inhibitor of golgi alpha-mannosidase II, blocks the synthesis of complex-type Asn-linked oligosaccharides resulting in the expression of hybrid-type oligosaccharides at the cell surface and the cells display a lectin resistant phenotype. Although swainsonine inhibited neither tumor cell growth in vitro nor solid tumor growth in situ, the drug dramatically reduced the incidence of lung colonies after i.v. inoculation of both MDAY-D2 and B16F10 melanoma cells. These results, taken together, indicate that certain sialylated Asn-linked oligosaccharides found on metastatic tumor cells are required for expression of the metastatic phenotype.

Inhibition of N-linked oligosaccharide processing does not prevent the secretion of thyroglobulin. A study with swainsonine and deoxynojirimycin.

The effects of two drugs, swainsonine (SW) and deoxynojirimycin (dNM), on synthesis and export of thyroglobulin were studied in folliculized porcine thyroid cells cultured in a serum-free medium. These drugs were expected to alter N-linked glycans in thyroglobulin. Newly synthesized thyroglobulin labeled with [2-3H]mannose or [4,5-3H]leucine was obtained by immunoprecipitation from the follicular contents, culture media and cell extracts; the first two compartments, containing secreted thyroglobulin, were sometimes analyzed together. Leucine incorporation was not inhibited by SW and only slightly by dNM. In contrast dNM strongly decreased mannose incorporation (by up to 50-75% at 1-3 mM). However after 16-h mannose labelings, SW and/or dNM at 2.5 microM and 3 mM respectively did not significantly modify the relative proportions of radioactive thyroglobulin in the above-mentioned compartments. Pronase glycopeptides prepared from these thyroglobulins were examined with respect to behaviour on concanavalin-A-Sepharose and position on Bio-Gel P-4. Oligosaccharides released by endoglucosaminidase H and with high affinity for the lectin, i.e. high-mannose and certain hybrids, were further characterized by various exoglycosidase treatments. Thyroglobulin from control cells displayed complex and high-mannose glycans comparable in size and proportion to those attributed to tissue-extracted porcine thyroglobulin. After treatment with SW (an inhibitor of alpha-mannosidase II), complex glycans were almost totally replaced by sialylated hybrid glycans. In contrast to this nearly total suppression, dNM (an inhibitor of the trimming glucosidases) caused only a 30% decrease in labeling of complex units and an about 50% increase in high-mannose glycans, covered to some degree by glucose. Finally a [3H]leucine pulse-chase study was performed on thyroglobulin secretion in the absence or presence of both SW and dNM. Though a slowdown was detectable in the first few hours, this study revealed no change in the long-term export of thyroglobulin.

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

Oligosaccharide moieties of cell-surface glycoconjugates are thought to be involved in recognition events associated with tumor metastasis and invasion. Using swainsonine (SW), an inhibitor of Golgi alpha-mannosidase II that results in the formation of hybrid-type oligosaccharides on N-linked glycoproteins, we have tested the hypothesis that specific glycan structures are required for pulmonary colonization by tumor cells. B16-F10 murine melanoma cells were treated with SW in growth medium and then injected intravenously into syngeneic C57BL/6 mice. This treatment resulted in dramatic inhibition of colonization, but it had no effect on B16-F10 viability or on cellular tumorigenicity after subcutaneous implantation. SW-treated radiolabeled B16-F10 cells were cleared from the lungs at a greater rate than control cells, suggesting that one effect of treatment is to alter tumor cell retention in the target organ. Our results implicate specific glycan structures in pulmonary colonization and offer a potential approach for identification of specific macromolecules involved in tumor cell-organ recognition during metastasis.

Properties of baby-hamster kidney (BHK) cells treated with Swainsonine, an inhibitor of glycoprotein processing. Comparison with ricin-resistant BHK-cell mutants.

Baby-hamster kidney (BHK) cells were grown continuously in long-term monolayer culture in the presence of Swainsonine, an inhibitor of alpha-mannosidase II, a processing enzyme involved in glycoprotein biosynthesis. The asparagine-linked oligosaccharides (N-glycans) were isolated from Pronase-digested cells by gel filtration, ion-exchange chromatography and affinity chromatography on concanavalin A--Sepharose and lentil lectin--Sepharose. The major N-glycans, analysed by 500 MHz 1H-n.m.r. spectroscopy, were identified as hybrid structures containing five mannose residues and neutral high-mannose N-glycans. The major hybrid species contained a core-substituted fucose alpha(1----6) residue and a NeuNAc alpha(2----3)Gal beta(1----4)GlcNAc terminal sequence; smaller amounts of non-sialylated and non-fucosylated hybrid structures were also detected. Swainsonine-treated cells also produced neutral oligosaccharides containing a single reducing N-acetylglucosamine residue substituted with polymannose sequences. The glycopeptide composition of Swainsonine-treated BHK cells resembles closely that of the ricin-resistant BHK cell mutant, RicR21 [P. A. Gleeson, J. Feeney and R. C. Hughes (1985) Biochemistry 24, 493-503], except the hybrid structures of RicR21 cells contain three, not five, mannose residues. Like RicR21 cells, Swainsonine-treated BHK cells showed a greatly increased resistance to ricin cytotoxicity, but not to modeccin, another galactose-binding lectin. These effects were readily reversed on removal of Swainsonine and growth in normal medium.

Abnormal Processing of the Modified Oligosaccharide Side Chains of Phytohemagglutinin in the Presence of Swainsonine and Deoxynojirimycin

Phytohemagglutinin, the glycoprotein lectin of the common bean, Phaseolus vulgaris, has both high-mannose (Man(8-9)GlcNAc(2)) and modified oligosaccharide side chains. The modified side chains have glucosamine, mannose, fucose, and xylose in the molar ratios 2:3.8:0.6:0.5, and are resistant to hydrolysis by endoglycosidase H. Synthesis and processing of side chains in the presence of 1-deoxynojirimycin, an inhibitor of alpha-glucosidase, results in the formation of chains which are all alike. They are sensitive to endoglycosidase H, do not contain fucose, and are largely resistant to alpha-mannosidase. This indicates that they are probably high-mannose chains blocked by terminal glucose residues. Synthesis and processing of side chains in the presence of swainsonine, an inhibitor of alpha-mannosidase II, results in the formation of normal high-mannose chains, and of modified chains which contain fucose residues, are resistant to endoglycosidase H, and can be distinguished from normal modified chains only by the presence of extra mannose residues.Processing of the phytohemagglutinin modified chains of PHA under normal conditions involves the attachment of peripheral N-acetylglucosamine residues in the Golgi complex and their subsequent removal in the protein bodies. The attachment of the N-acetylglucosamine residues is largely inhibited by deoxynojirimycin but still occurs in the presence of swainsonine. The results presented in this work show that processing of the asparagine-linked oligosaccharides is under the control of several glycosidases and glycosyltransferases and involves the formation of intermediate products.

Isolation and Characterization of Swainsonine from Texas Locoweed (Astragalus emoryanus)

Swainsonine (1,2,8-trihydroxyoctahydroindolizine) was isolated from locoweed (Astragalus emoryanus) that grows in Texas. Using a biological assay as a measure of activity and purity, a relatively straightforward purification of the compound is described. The purified material was a potent inhibitor of jack bean alpha-mannosidase and also of glycoprotein processing. The positive ion electron impact mass spectra of this compound was identical to that of authentic swainsonine. Nuclear magnetic resonance analysis also confirmed that the material was swainsonine.

Proteins of the kidney microvillar membrane. Effects of monensin, vinblastine, swainsonine and glucosamine on the processing and assembly of endopeptidase-24.11 and dipeptidyl peptidase IV in pig kidney slices.

The effects of various inhibitors were studied on the biogenesis of endopeptidase-24.11 (EC 3.4.24.11) and dipeptidyl peptidase IV (EC 3.4.14.5) in slices of renal cortex, from piglets of the Yucatan strain, maintained in organ culture. These microvillar peptidases were synthesized within membrane compartments and underwent glycosylation to yield high-mannose and complex forms [the preceding paper, Stewart & Kenny (1984) Biochem. J. 224, 549-558]. Monensin caused very gross ultrastructural changes in the proximal-tubular cells, resulting from distension of the Golgi sacs. It blocked the processing of the high-mannose to the complex glycosylated forms of the peptidases and prevented their assembly in the microvillar membrane. Swainsonine, an inhibitor of alpha-mannosidase II, generated new 'hybrid' forms of the proteins, intermediate in Mr between the high-mannose and the complex forms, but did not prevent assembly of the hybrid forms in microvilli. Vinblastine, an agent that affects microtubules, delayed, but did not abolish, either the processing or the transport to microvilli. Glucosamine interfered with the initial glycosylation reactions and generated heterogeneous sets of partially glycosylated polypeptides of lower Mr than the high-mannose forms. These results are discussed in relation to the site and mechanism of glycosylation and the involvement of the Golgi complex and microtubules in the biogenesis of these membrane peptidases.

Swainsonine and castanospermine blockade of mannose glycoprotein uptake by macrophages. Apparent inhibition of receptor-mediated endocytosis by endogenous ligands.

Receptor-mediated uptake of mannose-terminated glycoproteins by macrophages is blocked by treating the cells with swainsonine, an inhibitor of alpha-mannosidase II, and by castanospermine, an inhibitor of the endoplasmic reticulum processing enzyme alpha-glucosidase. Both inhibitors are known to cause accumulation of unprocessed oligosaccharide chains terminating in mannose. Inhibition of ligand uptake by the drugs was time- and dose-dependent. Swainsonine produced a maximal effect after 2 h; castanospermine required 5-6 h. Following swainsonine treatment, complete recovery of mannose receptor activity required 24 h and was blocked by cycloheximide suggesting that new receptor synthesis was necessary. Tunicamycin, an inhibitor of oligosaccharide assembly, had no effect on uptake of mannosylated ligands, but tunicamycin pretreatment reduced the sensitivity to swainsonine. These effects of swainsonine and castanospermine appear to be specific, other macrophage pinocytosis receptors (e.g. mannose phosphate) or phagocytosis of yeast particles were unaffected. Moreover, swainsonine had no effect on the fibroblast mannose phosphate receptor. The ability of macrophages to process newly synthesized oligosaccharides was blocked following treatment with swainsonine. Normal processing was fully recovered 24 h after removal of the drug. Mannosidase II was partially inactivated by swainsonine treatment and only a portion was recovered after 24 h. Treatment of macrophages with swainsonine also resulted in an increase in net lysosomal enzyme secretion. Inhibition of mannose-specific receptor-mediated endocytosis in macrophages by swainsonine and castanospermine appears to be due to the formation of mannose-terminated membrane glycoproteins which engage the mannose receptor thereby preventing function. These results suggest a novel mechanism for regulation of receptor-mediated endocytosis.

The effect of 1-deoxymannojirimycin on rat liver α-mannosidases

The mannose analogue, 1-deoxymannojirimycin, has been tested for its effect on five alpha-mannosidase activities present in rat liver and shown to be a specific inhibitor of Golgi alpha-mannosidase I at low mumolar concentrations. Golgi alpha-mannosidases I and II were assayed in a highly purified Golgi membrane preparation. Endoplasmic reticulum alpha-mannosidase activity was measured in a rough endoplasmic reticulum detergent extract. A purified soluble alpha-mannosidase activity which we believe is derived from the endoplasmic reticulum during tissue homogenization was also tested. And finally, the lysosomal or acidic alpha-mannosidase was measured in a postnuclear supernatant fraction obtained from rat liver. The results presented here show that 1-deoxymannojirimycin inhibits only Golgi alpha-mannosidase I, which is consistent with its effect on oligosaccharide processing in vivo (Fuhrmann et al. Nature 1984 307:755-758).