Inhibitors Of Glycoprotein Processing Research Articles

Human guanylate binding proteins: nanomachines orchestrating host defense.

Disease-causing microorganisms not only breach anatomical barriers and invade tissues but also frequently enter host cells, nutrient-enriched environments amenable to support parasitic microbial growth. Protection from many infectious diseases is therefore reliant on the ability of individual host cells to combat intracellular infections through the execution of cell-autonomous defense programs. Central players in human cell-autonomous immunity are members of the family of dynamin-related guanylate binding proteins (GBPs). The importance of these interferon-inducible GTPases in host defense to viral, bacterial, and protozoan pathogens has been established for some time; only recently, cell biological and biochemical studies that largely focused on the prenylated paralogs GBP1, GBP2, and GBP5 have provided us with robust molecular frameworks for GBP-mediated immunity. Specifically, the recent characterization of GBP1 as a bona fide pattern recognition receptor for bacterial lipopolysaccharide (LPS) disrupting the integrity of bacterial outer membranes through LPS aggregation, the discovery of a link between hydrolysis-induced GMP production by GBP1 and inflammasome activation, and the classification of GBP2 and GBP5 as inhibitors of viral envelope glycoprotein processing via suppression of the host endoprotease furin have paved the way for a vastly improved conceptual understanding of the molecular mechanisms by which GBP nanomachines execute cell-autonomous immunity. The herein discussed models incorporate our current knowledge of the antimicrobial, proinflammatory, and biochemical properties of human GBPs and thereby provide testable hypotheses that will guide future studies into the intricacies of GBP-controlled host defense and their role in human disease.

Antiviral Activity of a Small-Molecule Inhibitor of Arenavirus Glycoprotein Processing by the Cellular Site 1 Protease

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.

Probing Glycans With the Copper(I)‐Catalyzed [3+2] Azide–Alkyne Cycloaddition

AbstractThe imposing structural complexity and heterogeneity of glycans has made the study of their precise biological roles challenging. However, recently developed synthetic and screening techniques have enabled the study of many aspects of glycan activity that were previously inaccessible. Among these new methods, the Copper(I)‐catalyzed Azide–Alkyne [3+2] Cycloaddition (CuAAC) has provided a means to mimic multivalent glycan display, manipulate cellular glycans in vivo for visualization and isolation, and generate useful inhibitors of glycoprotein processing.

Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells.

Skeletal alkaline phosphatase (sALP) is a glycoprotein- approximately 20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular-extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin-which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis-decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P <0.001 for each). In contrast to the effects of tunicamycin on N-linked glycosylation, the effects of mannosamine, which inhibits GPI-anchor glycosylation/formation, included (1) an increase in cell layer protein; (2) decreases in sALP specific activity, in the cells and in the CM; and (3) increases in the percentages of both anchorless and wheat germ agglutinin (WGA)-soluble sALP in the medium, but not in the cells (P <0.005 for each). These effects of mannosamine were, presumably, a consequence of inhibiting the insertion/attachment of sALP to the outside of the plasma membrane surface. Neither mannosammine nor tunicamycin had any effect on the reaction kinetics of sALP or on the apparent affinity (the value of KM) for the phosphoryl substrate.

Purification and biochemical characterisation of a membrane-bound α-glucosidase from the parasite Entamoeba histolytica

An α-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-α- d-glucopyranoside (MUαGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 °C. Although the enzyme preferentially hydrolysed nigerose (α1,3-linked), it also cleaved kojibiose (α1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (α1,4), trehalose (α1,1) and isomaltose (α1,6). Activity on α1,3- and α1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy- d-glucose and 6-deoxy- d-glucose were strong inhibitors of activity, whereas 2-deoxy- d-glucose and other monosaccharides had no effect. Enzyme activity on MUαGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)- N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.

Effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells.

We have investigated the effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells to elucidate the role of carbohydrate in the triggering of T cell function. The inhibitors of glycoprotein processing, especially mannnosidase inhibitors, enhanced the anti CD3-induced production of interleukin-2 (IL-2), which is a cytokine without the linkage sequence of N-linked oligosaccharides. On the other hand, N-methyl-1-deoxynojirimycin (NMdNM, an inhibitor of processing glucosidase 1), 1-deoxynojirimycin (dNM, an inhibitor of processing glucosidase I and II) and bromoconduritol (BCD, an inhibitor of processing glucosidase II) inhibited the secretion of interleukin-4 (IL-4), interferon-gamma (IFN-gamma), or interleukin-5 (IL-5) into culture supernatants of anti CD3-stimulated T cells, which had N-linked oligosaccharides. Mannosidase inhibitors, 1-deoxymannojirimycin (dMAN, an inhibitor of processing mannosidase I) and swainsonine (SWN, an inhibitor of processing mannosidase II) did not inhibit the secretion or production of IL-4, IFN-gamma and IL-5. To confirm the inhibition of N-linked oligosaccharide processing in the cytokines by the above inhibitors, the binding of IFN-gamma to lectins with various sugar-binding specificities was investigated. All inhibitors reduced the binding of IFN-gamma to PHA E4, which had a high affinity to bi- or tri-antennary complex type N-linked oligosaccharides with bisecting N-acetylglucosamine. Similarly, all inhibitors reduced the binding of IFN-gamma to PHA L4, which had high affinity to tri- or tetra-antennary complex type N-linked oligosaccharides with beta1-6-linked branching. SWN and dMAN increased the binding of IFN-gamma to concanavalin A (ConA), which had a high affinity to bi-antennary complex type, hybrid type and high-mannose type N-linked oligosaccharides. These results suggest that the processing inhibitors used here inhibit the N-linked oligosaccharide processing of cytokines, and the inhibition of processing enzyme glucosidases I and II induces a decreased secretion of cytokines with N4-linked oligosaccharides.

Increased degradation of newly synthesized interferon-gamma (IFN-gamma) in anti CD3-stimulated lymphocytes treated with glycoprotein processing inhibitors.

As described previously (Kosuge T., Toyoshima S., Biol. Pharm. Bull., 23, 1-5 (2000)), inhibitors of the glycoprotein processing enzymes, glucosidase I and II, induce decreased secretion of interferon-gamma (IFN-gamma) into culture supernatants of anti CD3-stimulated lymphocytes, and in the present study the mechanism has been investigated in further detail. The processing inhibitors did not affect intracellular levels of IFN-gamma but enhanced the degradation of newly synthesized IFN-gamma in anti CD3-stimulated lymphocytes. Furthermore, since the stability of N-glycosylated proteins is known to be regulated by lectin family chaperones, such as calnexin, a type I transmembrane protein located in the endoplasmic reticulum (ER), and calreticulin, a soluble protein in the ER lumen, the effect of the processing inhibitors on the interaction of IFN-gamma with calnexin and calreticulin was investigated. It was found that IFN-gamma formed complexes with calnexin and calreticulin in anti CD3-stimulated lymphocytes. Total binding of IFN-gamma to calnexin was not affected but that to calreticulin was increased in anti CD3-stimulated lymphocytes treated with the processing inhibitors. However, binding of newly synthesized IFN-gamma to calreticulin was decreased in the lymphocytes under the same conditions as above. These results suggest that these glycoprotein processing inhibitors block the release of IFN-gamma from already formed calreticulin complexes, which prevents the binding of newly synthesized IFN-gamma to calreticulin and results in the enhancement of IFN-gamma degradation.

Transfer of Two Oligosaccharides to Protein in a Chinese Hamster Ovary Cell B211 Which Utilizes Polyprenol for Its N-Linked Glycosylation Intermediates

B211, a glycosylation mutant isolated from Chinese hamster ovary cells, synthesizes 10- to 15-fold less Glc3Man9GlcNAc2-P-P-lipid, the substrate used by the oligosaccharide transferase in the synthesis of asparagine-linked glycoproteins. B211 cells are also 10- to 15-fold deficient in the glucosylation of oligosaccharide-lipid. Despite these properties, protein glycosylation in B211 cells proceeds at a level similar to (50% of) parental cells. We asked whether the near wild-type level of glycosylation was due to the transfer of alternative oligosaccharide structures to protein in B211 cells. The aberrant size of [35S]methionine-labeled VSV G protein and the increased percentage of endoglycosidase H-resistant tryptic peptides as compared to parental cells supported this hypothesis. B211 cells were labeled with [2-3H]mannose either for 1 min or for 1 h in the presence of glycoprotein-processing inhibitors so that the oligosaccharides initially transferred to protein could be analyzed. In addition to Glc3Man9GlcNAc2, a second, endoglycosidase H-resistant oligosaccharide was transferred whose structure was determined by α-mannosidase digestion, gel filtration chromatography, and HPLC to be Glc0,1Man5GlcNAc2. Finally, since the synthesis of reduced amounts of Glc3Man9GlcNAc2-P-P-lipid was also a phenotype seen in another glycosylation mutant, Lec9, we analyzed the long-chain prenol in B211 cells. B211 cells synthesized and utilized polyprenol rather than dolichol for all N-linked glycosylation intermediates as determined by HPLC analysis of [3H]mevalonate-labeled lipids. Cell fusions analyzed by similar techniques indicated that B211, originally isolated as a concanavalin A-resistant cell line, is in the Lec9 complementation group.

Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors in glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated beta-subunits and not of non-glycosylated beta-subunits. Transport of the H+,K+-ATPase beta-subunit to the plasma membrane but not its ability to assemble with the alpha-subunit dependent on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cellcell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1α, LFA-1β, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W 3 13 ), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1α on lymphoid cells in the thymus, lymph node, spleen and heart allografts, ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W 3 13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1α on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecules which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1α-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

Tunicamycin potentiates drug cytotoxicity and vincristine retention in multidrug resistant cell lines.

Tunicamycin (TM), an inhibitor of glycoprotein processing, was investigated for its potential to reverse the multiple drug resistance (MDR) phenotype. When TM was added in vitro to drug-resistant NIH-3T3-MDR and KB-8-5-11 cells, they developed an increased sensitivity to doxorubicin, epirubicin, vincristine and colchicine. Similarly, the sensitivity of NIH-3T3-MDR cells to cisplatin was also enhanced by TM. In the presence of TM, drug-sensitive NIH-3T3-parental cells exhibited greater susceptibility to the toxic effects of doxorubicin, epirubicin, vincristine (marginally significant), and colchicine, but not to cisplatin. Tunicamycin-treated drug-sensitive KB-3-1 cells showed an increased response to vincristine, but not to the other anticancer drugs. Pretreatment with TM inhibited glycoprotein synthesis in all the cell lines. Neither prior exposure to, nor co-incubation with TM, influenced the uptake of vincristine (VCR) in the various cell lines. However, NIH-3T3-MDR cells accumulated less VCR than their drug-sensitive controls and also exhibited reduced efflux of the drug when treated with TM. There were no significant differences in the levels of intracellular VCR uptake between drug-sensitive KB-3-1 and KB-8-5-11 cells. Tunicamycin increased intracellular VCR retention in KB-8-5-11 and NIH-3T3-MDR cells, but not in NIH-3T3-parental cells. However, drug-sensitive KB-3-1 cells expressed reduced VCR retention in response to TM exposure, indicating that correlations between VCR toxicity and its retention in the presence of TM should be made with caution. The results suggest that the enhancement of intracellular VCR retention in MDR cells lines caused by TM is likely to be the result of inhibition of VCR efflux. Inhibition of glycoprotein synthesis during TM exposure may account for the changes in VCR efflux and retention observed in the MDR cell lines. The enhancement of cisplatin cytotoxicity in NIH-3T3-MDR cells after exposure to TM is an interesting observation, since it is generally believed that agents which modify the MDR phenotype do not show a sensitising effect to cisplatin. These findings may have applications in the reversal of drug resistance.

Rapid monitoring of site-specific glycosylation microheterogeneity of recombinant human interferon-gamma.

An analytical system is presented for rapid assessment of site-specific microheterogeneity of the two potential N-linked glycosylation sites of recombinant human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary cell culture. The target protein is first purified from culture supernatant by immunoaffinity chromatography, and the acidic eluent is neutralized via an in-line mixing tee. On-line proteolysis is rapidly performed by an immobilized trypsin cartridge, and reversed-phase chromatography isolates the two pools of glycopeptides representing the potential glycosylation sites. Following off-line analysis by matrix-assisted laser-desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry, observed mass shifts of glycopeptides relative to the known masses of their amino acid portions are correlated to site-specific oligosaccharide structures. Desialylation of glycopeptides by sialidase treatment on the MALDI sample plate allows for quantitative estimations of asialoglycan structures by MALDI/TOF. This methodology permits glycoprotein microheterogeneity to be evaluated in a time frame of approximately 2 h, utilizing as little as 0.5 microgram (25 pmol) of product. Results of monitoring a batch culture are presented as well as analysis of a culture containing deoxymannojirimycin, an inhibitor of glycoprotein processing.

Uptake and metabolism of BuCast: a glycoprotein processing inhibitor and a potential anti-HIV drug.

We have previously shown (Sunkara et al., 1989; Taylor et al., 1991) that 6-o-butanoyl castanospermine (BuCast) was a 30-50-fold better inhibitor of HIV syncytia formation than castanospermine (Cast). Radiolabeled Cast and BuCast were used to study the uptake and metabolism of these compounds in cultured cells and in mice. BuCast was preferentially taken up by cells compared to Cast. Approximately 30-50-fold higher radioactivity was found in cells treated with BuCast compared to cells treated with Cast during the initial 4-6 h of labeling. HPLC analysis showed that once BuCast was taken up by cells, it was rapidly converted to Cast. Mice given oral doses of BuCast had 5-10-fold higher levels of Cast in the plasma and tissues as compared to Cast treated mice. However, when the compounds were given i.v., the levels of plasma and tissue radioactivity obtained from Cast of BuCast were equivalent suggesting rapid conversion of BuCast to Cast in the blood. In mice orally treated with BuCast, HPLC analysis suggested that only Cast was found in the plasma and tissues. With multiple dosing of mice, additive results were obtained, suggesting that multiple doses may be used to obtain higher concentrations of the compound in the target cells. These data suggest that the lipophilic properties of butanoyl side chain on the Cast ring makes BuCast significantly better absorbed, and this may help to alleviate some of the gut toxicity associated with Cast treatment.

Flexible Enantiodivergent Synthesis and Biological Activity of Mannostatin Analogues, New Cyclitol Glycosidase Inhibitors.

Mannostatin A (1) is a new cyclitol inhibitor of glycoprotein processing. 2-Epimannostatin A (12) and its enantiomer (13) as well as their positional isomers (14, 15) were designed for probing structure-activity relationships in this class of glycosidase inhibitors. The analogues have been synthesized from (S)-4-((tert-butyldimethylsilyl)oxy)-2-cyclopentenone by an enantiodivergent strategy in a totally stereospecific fashion. Compound 13 showed inhibition against almond beta-glucosidase and is shown to be a topographical analogue of beta-D-glucopyranoside.

Antiviral Activity and Metabolism of the Castanospermine Derivative MDL 28,574, in Cells Infected with Herpes Simplex Virus Type 2

The 6-O-butanoyl derivative of castanospermine (MDL 28,574: BUCAST), an inhibitor of glycoprotein processing, blocked the growth of herpes simplex virus type-2 with the effect markedly enhanced by exposure of cells to the compound pre- as well as post-infection. The effectiveness of the derivative corresponded to an increased uptake with greatest accumulation after virus infection. Gas chromatography/mass spectrometry identified the predominant component in MDL 28,574 treated cells as castanospermine, an inhibitor of α-glucosidase 1. The effects of this compound on the synthesis of viral glycoprotein, gB, was determined with the increased molecular weight of the mannose-rich precursor evidence for the modulation of glycoprotein processing.

Effects of the anti‐inflammatory compounds castanospermine, mannose‐6‐phosphate and fucoidan on allograft rejection and elicited peritoneal exudates

The glycoprotein processing inhibitor castanospermine (CS) and the monosaccharide mannose-6-phosphate (M6P), as well as some sulfated polysaccharides (SPS), have been shown to inhibit inflammation in rat models of experimental autoimmune encephalomyelitis and adjuvant-induced arthritis. Here, the anti-inflammatory effects of these agents have been further explored in murine models of allograft rejection and elicitation of peritoneal exudates. CS, M6P and the SPS, fucoidan, partially inhibited rejection of permanently accepted thyroid allografts induced by the i.p. injection of donor strain (H-2d) spleen cells with a reduction in leucocyte infiltration of 25-36%. However none of these agents reduced the more extensive leucocyte infiltration induced by the i.p. injection of P815 (H-2d) unless recipient mice were pretreated with the immunosuppressant, cyclosporin A (CsA). Elicitation of peritoneal exudates by thioglycollate was inhibited by CS, M6P and fucoidan with sustained leucopenia being induced by CS. In contrast, CS and fucoidan, but not M6P, inhibited antigen-elicited peritoneal exudates. These results suggest that CS, M6P and the SPS fucoidan exhibit subtle differences in their anti-inflammatory activity but probably inhibit inflammation at the level of leucocyte extravasation.

ChemInform Abstract: Synthetic Studies on Mannostatin A and Its Derivatives: A New Family of Glycoprotein Processing Inhibitors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthetic Studies on Mannostatin A and Its Derivatives: A New Family of Glycoprotein Processing Inhibitors

Mannostatin A (1) is a naturally-occurring α-mannosidase inhibitor whose carbocyclic structure represents a significant departure from known alkaloid-based glycosidase inhibitors. The total synthesis of 1 [(1R,2R,3R,4R,5S)-1-(methylthio)-2,3,4-trihydroxy-5-aminocyclopentane], as well as those of several analogs and derivatives, was devised in order to probe structure-activity relationships in this family of N-linked glycoprotein biosynthesis inhibitors. The synthetic strategy features an asymmetric hetero Diels-Alder reaction and a highly syn-stereoselective alkene dihydroxylation using OsO 4 . Both N-benzylmannostatin and mannostatin sulfone exhibit good competitive inhibition of jack bean N-mannosidase (K 1 =380±81 and 126±16 nM, respectively), although not as potent as that of 1. Interestingly, enantiomerically pure 3,4-bis-epi-mannostatin is also a modest competitive inhibitor of jack bean α-mannosidase (K 1 of 16 ± 2μM), comparable in activity to 1-deoxymannojirimycin. Synthetic samples of both diastereomeric sulfoxides of 1 exhibit activity comparable to that of 1. Finally, epoxide 41, the 3,4-anhydro derivative of 1, was synthesized an shown to inactive jack bean α-mannosidase in a time-dependent manner (K 1 =153 ± 26μM; K inact /K 1 =60min -1 M -1

Reversible phosphorylation of eukaryotic initiation factor 2 alpha in response to endoplasmic reticular signaling.

Agents, such as EGTA, thapsigargin, and ionophore A23187, that mobilize sequestered Ca2+ from the endoplasmic reticulum (ER) or dithiothreitol (DTT) that compromises the oxidizing environment of the organelle, disrupt early protein processing and inhibit translational initiation. Increased phosphorylation of eIF-2 alpha (5-fold) and inhibition of eIF-2B activity (50%) occur in intact GH3 cells exposed to these agents for 15 min (Prostko et al. J. Biol. Chem. 267:16751-16754, 1992). Alterations in eIF-2 alpha phosphorylation and translational activity in response to EGTA were reversed by addition of Ca2+ in excess of chelator while responses to DTT were reversible by washing. Exposure for 3 h to either A23187 or DTT, previously shown to induce transcription-dependent translational recovery, resulted in dephosphorylation of eIF-2 alpha in a manner blocked by actinomycin D. Phosphorylation of eIF-2 alpha in response to A23187 or DTT was not prevented by conventional inhibitors of translation including cycloheximide, pactamycin, puromycin, or verrucarin. Prolonged inhibition of protein synthesis to deplete the ER of substrates for protein processing resulted in increased eIF-2 alpha phosphorylation, decreased eIF-2B activity, and reduced monosome content that were indicative of time-dependent blockade; these inhibitors did not abolish polysomal content. Superphosphorylation of eIF-2 alpha occurred upon exposure of these preparations to either A23187 or DTT. Tunicamycin, an inhibitor of co-translational transfer of core oligosaccharide, provoked rapid phosphorylation of eIF-2 alpha and inhibition of translational initiation whereas sugar analog inhibitors of glycoprotein processing did neither. A flow of processible protein to the ER does not appear to be required for the phosphorylation of eIF-2 alpha in response to ER perturbants. We hypothesize that perturbation of the translocon, rather than suppression of protein processing, initiates the signal emanating from the ER culminating in eIF-2 alpha phosphorylation and translational repression.

Accumulation of pentamannose oligosaccharides in human mononuclear leukocytes by action of swainsonine, an inhibitor of glycoprotein processing

Swainsonine, a known inhibitor of the α-mannosidase II involved in processing of asparagine-linked glycoproteins, causes accumulation of hybrid-type oligosaccharide-containing glycoproteins in mammalian cells. Swainsonine augments lymphokine-activated, killer-cell induction at supoptimal doses of interlekin-2; the amount needed to increase LAK activity is 100–1000 fold higher than required to completely inhibit mannosidase II. Human mononuclear lymphocytes, when treated with these relatively high (58 μM) concentrations of swainsonine showed a 3–4 fold increase in d-[ 3H]mannose incorporation into the glycan as compared to glycans of untreated cells. Analysis indicated accumulation of high-mannose type, free oligosaccharides in the soluble fractions of the cell. Chromatographic analysis of glycan obtained by d-[2- 3H]mannose labeling of human mononuclear lymphocytes showed synthesis of a new oligosaccharide, at 58 μM of swainsonine, that contained 36% of the total radioactivity incorporated into the glycan (oligosaccharide pool). This oligosaccharide fraction was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, O and Nglycanase, but was susceptible to cleavage by Jack bean α-mannosidase and was bound > 90% to concanavalin A-Sepharose. A similar chromatographic elution profile was obtained from glycans labeled with d-[2- 3H]mannose from mouse B 16F 10 melanoma and baby hamster kidney cells subsequent to sswainsonine treatment. Methylation analysis of free oligosaccharides obtained from MNL revealed th presence of a pentamannose. These results indicate the accumulation of a free high-mannose oligosaccharide rather than expected hybrid-type structure on treatment of cells with relatively high concentrations of swainsonine.