1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes

1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor.     

Comparison between 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin as inhibitors of oligosaccharide processing in intestinal epithelial cells.

The alpha-glucosidase inhibitor N-methyl-1-deoxynojirimycin (MDJN) inhibits the synthesis of N-linked complex oligosaccharides in rat intestinal epithelial cells to the same extent as reported previously for 1-deoxynojirimycin (DJN) [Saunier, Kilker, Tkacz, Quaroni & Herscovics (1982) J. Biol. Chem. 257, 14155-14161]. Analysis of each of the endo-beta-N-acetylglucosaminidase H (endo H)-sensitive oligosaccharides separated by h.p.l.c. with yeast glucosidase I, which specifically removes the terminal glucose residue from oligosaccharides containing three glucose residues, and with jack-bean (Canavalia ensiformis) alpha-mannosidase, indicates that both inhibitors cause the accumulation of a mixture of glucosylated oligosaccharides containing one to three glucose residues and seven to nine, and even possibly six, mannose residues. About 70% of the endo H-sensitive oligosaccharides formed in the presence of MDJN contain three glucose residues, compared with only about 20% of the corresponding oligosaccharides of the DJN treated cells. It is concluded that both compounds inhibit the formation of N-linked complex oligosaccharides by interfering with the processing glucosidases. These compounds are valuable in the study of the role of oligosaccharides in glycoproteins.     

Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.     

Inhibition of yeast exoglucanases by glucosidase inhibitors

Castanospermine, 1-deoxynojirimycin, and N-methyl-1-deoxynojirimycin, three well-characterized inhibitors of the glucosidases involved in the processing of N-linked oligosaccharides, did not affect the biosynthesis or the secretion of exoglucanases (EC 3.2.1.58) from Saccharomyces cerevisiae and Candida albicans but inhibited the activity itself. Regardless of the substrate used, laminarin or p-nitrophenyl beta-D-glucoside (pNPG), all three inhibitors proved to act in a competitive manner. Castanospermine was the most potent inhibitor, with Ki values ranging from 0.16 to 0.5 microM for three different purified yeast exoglucanases. The inhibition caused by 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin was poorer, but still significant. By contrast, the glucosidase inhibitors did not show any action on a partially purified endoglucanase (EC 3.2.1.39) Candida albicans. A purified exoglucanase from Basidiomycete QM 806, which was specific for laminarin, was unaffected by castanospermine but it was still inhibited in an uncompetitive manner by 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin. The presence of castanospermine in the culture medium of growing yeasts did not have any effect on yeast growth in spite of the fact that, under the conditions used, the external exoglucanase was fully inhibited. None of the yeast exoglucanases hydrolyzed the glucan synthesized in vitro by membrane preparations derived from either yeast. These results support the concept that yeast exoglucanases are glucosidases that also attack laminarin, rather than glucanases capable of attacking pNPG.     

Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2-PP-dolichol in intestinal epithelial cells in culture

The lipid-linked oligosaccharides synthesized in the presence of the alpha-glucosidase inhibitors, 1-deoxynojirimycin (DJN) and N-methyl-1-deoxynojirimycin (MDJN), were compared in IEC-6 intestinal epithelial cells in culture. HPLC analysis of the oligosaccharides obtained before and after exhaustive jack bean alpha-mannosidase digestion indicates that control and MDJN-treated cells synthesize similar amounts of Glc3Man9GlcNAc2-PP-dolichol. In contrast, the formation of this compound is greatly reduced in DJN-treated cells, the major lipid-linked oligosaccharide found being Man9GlcNAc2-PP-dolichol. The decreased availability of the preferred donor for protein glycosylation may account for the impaired glycosylation and secretion of certain glycoproteins in the presence of DJN.     

Inhibition of myoblast fusion by the glucosidase inhibitor N-methyl-1-deoxynojirimycin, but not by the mannosidase inhibitor 1-deoxymannojirimycin.

The effects of N-linked-oligosaccharide-processing inhibitors on the fusion of rat L6 myoblasts to form myotubes were examined. The glucosidase inhibitor N-methyl-1-deoxynojirimycin (MDJN) greatly inhibited fusion, whereas the mannosidase inhibitor 1-deoxymannojirimycin (ManDJN) had relatively little effect, although both compounds prevented the formation of N-linked complex oligosaccharides. These results indicate that complex oligosaccharides on glycoproteins do not play a role in myoblast fusion. With MDJN, high-mannose oligosaccharides containing three glucose residues and seven to eight mannose residues were found at the cell surface, whereas with ManDJN, non-glucosylated high-mannose oligosaccharides with seven to nine mannose residues were obtained. These results indicate that the persistence of glucose residues on high-mannose oligosaccharides may be responsible for the inhibition of fusion. It is suggested that glucose either masks the cell-surface recognition process leading to fusion or prevents the cell-surface expression of specific glycoprotein(s) essential to the fusion process.     

Chemical modification of the glucosidase inhibitor 1-deoxynojirimycin. Structure-activity relationships.

The ability of the glucosidase inhibitor 1-deoxynojirimycin (dNM) and a series of N-alkylated dNM derivatives to interfere with biosynthesis, transport, and maturation of the glycoprotein alpha 1-antitrypsin in HepG2 cells was investigated. Inhibition of endoplasmic reticulum glucosidase I and II by dNM and its derivatives resulted in an intracellular accumulation of alpha 1-antitrypsin with glucose-containing high mannose type oligosaccharides (precursor). N-alkylation of dNM increased its potency in inhibiting endoplasmic reticulum glucosidases, as determined from the concentration required for half maximal inhibition. N-Alkylated derivatives of dNM were better able to inhibit glucosidase I than glucosidase II (deduced from the number of glucose residues retained in Endo H-releasable oligosaccharides). The inhibition of glucosidase activity imposed by alkylated dNM derivatives was less easily reversed than that by dNM, an effect most pronounced for N-methyl-dNM. Branching of the alkyl group of dNM derivatives decreased the inhibitory potency. Although dNM and its derivatives interfered strongly with intracellular oligosaccharide processing, they did not completely block N-glycan maturation of alpha 1-antitrypsin even at the highest concentrations tested.     

Incompletely processed LH molecules synthesized by rat gonadotrophs treated with inhibitors of oligosaccharide processing

Inhibitors of N-linked oligosaccharide processing are useful tools for studies on the biological function of the oligosaccharide structures in glycoprotein hormones. We have synthesized molecules of lutropin (LH) containing high-mannose- and hybrid-type oligosaccharides using rat gonadotroph-enriched primary cultures in the presence of castanospermine (a glucosidase I inhibitor) or swainsonine (a mannosidase II inhibitor), in order to compare the actions of these molecules with that of the hormone containing complex-type oligosaccharides in the activation of the receptor-adenylate cyclase system. Treatment of gonadotrophs with the above inhibitors caused an increase in the synthesis of highly basic LH molecules (pI 9.6-10.0), because addition of charged carbohydrate moieties to these molecules was prevented. Characterization of the oligosaccharide structure performed by enzymatic treatment (endoglycosidase H and neuraminidase) and the use of immobilized lectins (wheat germ agglutinin and Ricinus communis agglutinin-120) showed that these inhibitor-synthesized LH molecules contained high-mannose- and hybrid-type (asialo and sialylated) oligosaccharides. Their immunological properties were similar to that of complex-type oligosaccharide LH, but they had significantly higher receptor-binding ability in comparison with a sialylated complex-type oligosaccharide LH (about 12-fold) and an asialo complex-type oligosaccharide LH (about 3-fold). It was noted that the incompletely processed molecules were less potent than complex-type oligosaccharide LH in the activation of adenylate cyclase of Leydig cells, showing about 40-60% of the activity induced by the sialylated complex-type oligosaccharide molecule. The present data indicate that the inhibition of terminal processing of N-linked oligosaccharides by castanospermine and swainsonine impairs the full hormonal function of rat LH.     

Deglycosylation of alpha 1-proteinase inhibitor by endo-beta-N-acetylglucosaminidase F

The glycosidase endo-beta-N-acetylglucosaminidase F (endo F) from Flavobacterium meningosepticum was used for the deglycosylation of rat alpha 1-proteinase inhibitor (alpha 1 PI). alpha 1 PI containing three oligosaccharide side chains of the complex type was isolated from rat serum or from the medium of rat hepatocyte primary cultures. High-mannose-type alpha 1 PI or hybrid-type alpha 1 PI was isolated from the media of hepatocytes treated with 1-deoxymannojirimycin or swainsonine, respectively. The susceptibility of complex-type alpha 1 PI to endo F was studied in the presence of various detergents. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate and octyl glucopyranoside turned out to be most effective. In the absence of detergents, digestion of alpha 1 PI with high concentrations of endo F and/or long times of incubation led to the formation of alpha 1 PI with one and two oligosaccharide side chains. In the presence of 0.5% octyl glucopyranoside, the major cleavage products were unglycosylated alpha 1 PI and alpha 1 PI carrying one carbohydrate side chain. In contrast to the complex-type alpha 1 PI, the high-mannose type can be totally deglycosylated by endo F even in the absence of detergents. The susceptibility of the hybrid-type alpha 1 PI to endo F is between that of the complex and the high-mannose types.     

Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing.

Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.     

The effects of inhibiting oligosaccharide trimming by 1-deoxynojirimycin on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor has a subunit stoichiometry of alpha 2 beta gamma delta; all 5 subunits contain N-linked oligosaccharides. We investigated what role trimming of the oligosaccharides played in the post-translational processing of the subunits and assembly of the receptor by examining the receptor synthesized in the presence of an inhibitor of oligosaccharide trimming, 1-deoxynojirimycin. BC3H-1 cells express one-third fewer receptors when grown in the presence of 1-deoxynojirimycin. The receptor subunits that are expressed have decreased mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating an inhibition of oligosaccharide trimming. In control cells, 40% of the translated alpha subunit acquires the capacity to bind alpha-bungarotoxin with a half-time of 40 min before assembly with the other subunits; the rest is rapidly degraded. In 1-deoxynojirimycin-treated cells approximately the same amount of alpha subunit is translated as in control cells, but that alpha subunit is degraded more rapidly, and only 25% acquires the capacity to bind alpha-bungarotoxin. From these results, we conclude that oligosaccharide processing either may aid in protecting the alpha subunit primary translation product from degradation or may be required for the conformational change or other post-translational modification(s) necessary for formation of the alpha-bungarotoxin binding form of the alpha subunit, which is then protected from proteolytic degradation. The cell surface receptor that is expressed in the presence of 1-deoxynojirimycin, however, is not altered in its affinity for cholinergic ligands. Thus, we conclude that differential N-linked oligosaccharide trimming of the 2 alpha subunits does not appear to play a part in the differences in affinities of the 2 alpha subunits for cholinergic ligands.     

The effect of castanospermine on the oligosaccharide structures of glycoproteins from lymphoma cell lines.

The effect of castanospermine on the processing of N-linked oligosaccharides was examined in the parent mouse lymphoma cell line and in a mutant cell line that lacks glucosidase II. When the parent cell line was grown in the presence of castanospermine at 100 micrograms/ml, glucose-containing high-mannose oligosaccharides were obtained that were not found in the absence of inhibitor. These oligosaccharides bound tightly to concanavalin A-Sepharose and were eluted in the same position as oligosaccharides from the mutant cells grown in the absence or presence of the alkaloid. The castanospermine-induced oligosaccharides were characterized by gel filtration on Bio-Gel P-4, by h.p.l.c. analysis, by enzymic digestions and by methylation analysis of [3H]mannose-labelled and [3H]galactose-labelled oligosaccharides. The major oligosaccharide released by endoglucosaminidase H in either parent or mutant cells grown in castanospermine was a Glc3Man7GlcNAc, with smaller amounts of Glc3Man8GlcNAc and Glc3Man9GlcNAc. On the other hand, in the absence of castanospermine the mutant produces mostly Glc2Man7GlcNAc. In addition to the above oligosaccharides, castanospermine stimulated the formation of an endoglucosaminidase H-resistant oligosaccharide in both cell lines. This oligosaccharide was characterized as a Glc2Man5GlcNAc2 (i.e., Glc(1,2)Glc(1,3)Man(1,2)Man(1,2)Man(1,3)[Man(1,6)]Man-GlcNAc-GlcNAc). Castanospermine was tested directly on glucosidase I and glucosidase II in lymphoma cell extracts by using [Glc-3H]Glc3Man9GlcNAc and [Glc-3H]Glc2Man9GlcNAc as substrates. Castanospermine was a potent inhibitor of both activities, but glucosidase I appeared to be more sensitive to inhibition.     

Demonstration that Golgi endo-alpha-D-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins

Studies on N-linked oligosaccharide processing were undertaken in HepG2 cells and calf thyroid slices to explore the possibility that the recently described Golgi endo-alpha-D-mannosidase (Lubas, W.A., and Spiro, R.G. (1987) J. Biol. Chem. 262, 3775-3781) is responsible for the frequently noted failure of glucosidase inhibitors to achieve complete cessation of complex carbohydrate unit synthesis. We have found that in the presence of the glucosidase inhibitors, castanospermine (CST) or 1-deoxynojirimycin, there is a substantial production of the glucosylated mannose saccharides (Glc3Man, Glc2Man, and Glc1Man) which are the characteristic products of endomannosidase action. Furthermore, in HepG2 cells, a secretion of these components into the medium could be demonstrated. Characterization of the N-linked polymannose oligosaccharides produced by HepG2 cells in the presence of CST (as well as 1-deoxymannojirimycin to prevent processing by alpha-mannosidase I) indicated the occurrence, in addition to the expected glucosylated species, of substantial amounts of Man8GlcNAc and Man7GlcNAc. Since Man9GlcNAc was almost completely absent and the Man8GlcNAc isomer was shown to be identical with that formed by the in vitro action of endomannosidase on glucosylated polymannose oligosaccharides, we concluded that this enzyme was actively functioning in the intact cells and could provide a pathway for circumventing the glucosidase blockade. Indeed, quantitative studies in HepG2 cells supported this contention as the continued formation of complex carbohydrate units (50% of control) during CST inhibition could be accounted for by the deglucosylation effected by endomannosidase.     

Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase

alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.     

Post-translational addition of chondroitin sulfate glycosaminoglycans. Role of N-linked oligosaccharide addition, trimming, and processing

A melanoma proteoglycan model system has been used to examine the role of core protein asparagine-linked (N-linked) oligosaccharides in the transport and assembly of proteoglycan molecules. The use of agents which block discrete steps in the trimming and processing of core oligosaccharides (castanospermine, 1-deoxynojirimycin, N-methyldeoxynojirimycin, 1-deoxymannojirimycin, and swainsonine) demonstrates that removal of glucose residues from the N-linked oligosaccharides is required for the cell surface expression of a melanoma proteoglycan core protein and for the conversion of the core protein to a chondroitin sulfate proteoglycan. However, complete maturation of the oligosaccharides to a "complex" form is not required for these events. Treatment of M21 human melanoma cells with the glucosidase inhibitors castanospermine, 1-deoxynojirimycin, or N-methyldeoxynojirimycin results in a dose-dependent inhibition of glycosaminoglycan (GAG) addition to the melanoma antigen recognized by monoclonal antibody 9.2.27. In contrast, treatment with the mannosidase inhibitors 1-deoxymannojirimycin and swainsonine does not effect GAG addition. Identical results are obtained when the major histocompatibility complex class II antigen gamma chain proteoglycan is examined in inhibitor-treated melanoma and B-lymphoblastoid cells. These data, in conjunction with the known effects of the glucosidase and mannosidase inhibitors on the transport and secretion of other glycoproteins support the hypothesis that the addition, trimming, and processing of N-linked oligosaccharides is involved in the transport of certain proteoglycan core proteins to the site of GAG addition and to the cell surface.     

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., H?chli, 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.     

Pleiotropic resistance to glycoprotein processing inhibitors in Chinese hamster ovary cells. The role of a novel mutation in the asparagine-linked glycosylation pathway

In order to obtain a better understanding of the control mechanisms involved in asparagine-linked glycosylation, we developed conditions under which the glucosidase I and II inhibitor castanospermine and the mannosidase II inhibitor swainsonine were toxic to Chinese hamster ovary (CHO) cells when cultured in the presence of low concentrations of the plant lectin concanavalin A. Cells resistant to castanospermine (CsR cells) and swainsonine (SwR cells) were obtained by gradual stepwise selections. These cells had normal levels of glucosidase II and mannosidase II and appeared to have no major structural alterations in their surface asparagine-linked oligosaccharides. Interestingly, the CsR and SwR cells were each pleiotropically resistant to castanospermine, swainsonine, and deoxymannojirimycin, an inhibitor of mannosidase I. This resistance was not due to the multiple-drug resistance phenomenon. Both the CsR and SwR cell populations synthesized Man5GlcNAc2 in place of Glc3Man9GlcNAc2 as the major dolichol-linked oligosaccharide. This defect was not due to a loss of mannosylphosphoryldolichol synthetase. Furthermore, the Man5GlcNAc2 oligosaccharide was transferred to protein and appeared to give rise to normal mature oligosaccharides. Thus, the CsR and SwR cells achieved resistance to castanospermine, swainsonine, and deoxymannojirimycin by synthesizing altered dolichol-linked oligosaccharides that reduced or eliminated the requirements for glucosidases I and II and mannosidases I and II during the production of normal asparagine-linked oligosaccharides. We propose that this phenotype be termed PIR, for processing inhibitor resistance.     

Transforming growth factor beta 1: importance of glycosylation and acidic proteases for processing and secretion

The role of glycosylation of the transforming growth factor-beta 1 (TGF-beta 1) precursor was investigated by treating a transfected Chinese hamster ovary (CHO) cell line expressing high levels of recombinant TGF-beta 1 (TGF-beta 3-2000 cells) with a series of glycosylational inhibitors. Tunicamycin, a nucleoside antibiotic which prevents the formation of the dolichol intermediate necessary for oligosaccharide addition of the nascent polypeptide chain, appeared to block secretory exit and led to an increase in the cellular associated, nonglycosylated pro-TGF-beta 1 form. 1-Deoxymannojirimycin and swainsonine, inhibitors of the mannosidases I and II, respectively, blocked complete glycoprotein processing of the TGF-beta 1 precursor as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by sensitivity to glycosidases. However, the abnormal TGF-beta 1 polypeptides containing the altered carbohydrate side chains were secreted readily by the CHO cells. In contrast, inhibitors of the glucosidases at the first step in glycoprotein remodeling, 1-deoxynojirimycin and castanospermine, markedly inhibited secretion of the TGF-beta 1 polypeptides from transfected CHO cells. In all cases, these inhibitors did not appear to affect proteolytic processing of the TGF-beta 1 polypeptides. Furthermore, inhibitor treatment did not affect mannose-6-phosphorylation of the TGF-beta 1 polypeptides. These results suggest that glycosylation and early stage remodeling of oligosaccharide side chains are necessary for secretion of TGF-beta 1. Treatment of the transfected CHO cells with weak bases (NH4Cl and chloroquine), or a monovalent ionophore (monensin), prevented proteolytic processing of the TGF-beta 1 precursor indicating that cleavage occurs by proteases in an acidic cellular compartment.     

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.     

Swainsonine, an inhibitor of mannosidase II during glycoprotein processing, enhances concanavalin A-induced T cell proliferation and interleukin 2 receptor expression exclusively via the T cell receptor complex.

The T cell receptor (TCR) is a disulfide-linked heterodimer consisting of both complex and high-mannose types of N-linked oligosaccharides. The objective of the present investigation was to examine the effect of altered oligosaccharide structure on the expression and function of the TCR. Human mononuclear lymphocytes (MNL) were treated with castanospermine (CAST) or swainsonine (SW), inhibitors of glucosidase I or mannosidase II, respectively. Treatment with these inhibitors does not prevent glycosylation, but results in synthesis of glycoproteins with high-mannose or hybrid types of oligosaccharides. Treatment of MNL with CAST (1000-10 microM) or SW (100-1 microM) for up to 72 hr had no effect on cell surface expression of of the TCR. SW potentiated Con A-induced T cell proliferation without effecting anti-CD3 (OKT3) or alloantigen-induced proliferation. CAST had no effect on Con A, anti-CD3, or alloantigen-induced T cell proliferation. The T cell proliferative response to Con A in the presence of SW was completely eliminated in the presence of monoclonal anti-TCR antibodies. Monoclonal anti-CD2, -CD3, -CD4, -CD8, or isotypic control monoclonal antibodies had no effect on SW enhancement of T cell proliferation. SW treatment potentiated Con A-induced MNL expression of both the alpha and beta subunits of the IL 2R. This effect was also specifically blocked by anti-TCR monoclonal antibodies. These results demonstrate that selective changes in the glycosylation state of the TCR complex can alter mitogen recognition and subsequent cellular activation.