Transfer of nonglucosylated oligosaccharide from lipid to protein in a mammalian cell

We have previously shown that the glucosidase inhibitor, N-methyl-1-deoxynojirimycin (MedJN), only partially inhibited N-linked complex oligosaccharide biosynthesis in F9 teratocarcinoma cells whereas the alpha-mannosidase I inhibitor, manno-1-deoxynojirimycin, completely prevented this synthesis (Romero, P. A. and Herscovics, A. (1986) Carbohydr. Res. 151, 21-28). In order to determine whether a pathway independent of processing glucosidases can occur, F9 cells were pulse-labeled for 2 min with D-[2-3H]mannose in the presence or absence of 2 mM MedJN. In control cells, Man7GlcNAc was identified in the protein-bound oligosaccharides released with endo-beta-N-acetylglucosaminidase H, in addition to the expected Glc1-3Man9GlcNAc and Man9GlcNAc arising from processing of Glc3Man9GlcNAc. MedJN completely prevented the removal of glucose residues from Glc3Man9GlcNAc, but did not greatly affect the appearance of Man7GlcNAc associated with protein. Labeled Man7GlcNAc was also found in the lipid-linked oligosaccharides of both control and treated cells. The 2-min pulse-labeled Man7GlcNAc obtained from both the lipid and protein fractions were shown to have identical structures by concanavalin A-Sepharose chromatography and by acetolysis and were clearly different from the Man7GlcNAc obtained from the usual processing pathway. These results demonstrate that transfer of a nonglucosylated oligosaccharide (Man7GlcNAc2) from dolichyl pyrophosphate to protein occurs in F9 cells.     

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.     

Evaluation of the role of rat liver Golgi endo-alpha-D-mannosidase in processing N-linked oligosaccharides

Golgi membranes from rat liver have been shown to contain an endo-alpha-D-mannosidase which can convert Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1----3Man (Lubas, W. A., and Spiro, R. G. (1987) J. Biol. Chem. 262, 3775-3781). We now report that this enzyme has the capacity to cleave the alpha 1----2 linkage between the glucose-substituted mannose residue and the remainder of the polymannose branch in a wide range of oligosaccharides (Glc3Man9GlcNAc to Glc1Man4GlcNAc) as well as glycopeptides and oligosaccharide-lipids. Whereas the tri- and diglucosylated species (Glc3Man9GlcNAc and Glc2Man9GlcNAc), which yielded Glc3Man and Glc2Man, respectively, were processed more slowly than Glc1Man9GlcNAc, the monoglucosylated components with truncated mannose chains (Glc1Man8GlcNAc to Glc1Man4GlcNAc) were trimmed at an increased rate which was inversely related to the number of mannose residues present. The endomannosidase was not inhibited by a number of agents which are known to interfere with N-linked oligosaccharide processing by exoglycosidases, including 1-deoxynojirimycin, castanospermine, bromoconduritol, 1-deoxymannojirimycin, swainsonine, and EDTA. However, Tris and other buffers containing primary hydroxyl groups substantially decreased its activity. After Triton solubilization, the endomannosidase was observed to be bound to immobilized wheat germ agglutinin, indicating the presence of a type of carbohydrate unit consistent with Golgi localization of the enzyme. The Man8GlcNAc isomer produced by endomannosidase action was found to be processed by Golgi enzymes through a different sequence of intermediates than the rough endoplasmic reticulum-generated Man8GlcNAc variant, in which the terminal mannose of the middle branch is absent. Whereas the latter oligosaccharide is converted to Man5GlcNAc via Man7GlcNAc and Man6GlcNAc at an even rate, the processing of the endomannosidase-derived Man8GlcNAc stalls at the Man6GlcNAc stage due to the apparent resistance to Golgi mannosidase I of the alpha 1,2-linked mannose of the middle branch. The results of our study suggest that the Golgi endomannosidase takes part in a processing route for N-linked oligosaccharides which have retained glucose beyond the rough endoplasmic reticulum; the distinctive nature of this pathway may influence the ultimate structure of the resulting carbohydrate units.     

alpha-Glucosidase II-deficient cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.

The kinetics of N-linked oligosaccharide processing and the structures of the processing intermediates have been examined in normal parental BW5147 mouse lymphoma cells and the alpha-glucosidase II-deficient PHAR2.7 mutant cells. The mutant cells accumulated glucosylated intermediates but were able to deglucosylate and process about 40% of their oligosaccharides to complex-type. This processing was not due to residual alpha-glucosidase II activity since the alpha-glucosidase inhibitors 1-deoxynojirimycin (DNJ) and N-butyl-DNJ did not prevent it. Parent cells also showed alpha-glucosidase II-independent processing in the presence of DNJ and N-butyl-DNJ. Membrane preparations from both parent and mutant cells had endo alpha-mannosidase activity, that is, split Glc1,2Man9GlcNAc to Glc1,2Man plus Man8GlcNAc, indicating that this was a candidate for an alternate route to complex oligosaccharide formation in the mutant cells. A balance study in which the cellular glycoproteins, intracellular water soluble saccharides, and saccharides secreted into the medium were isolated and analyzed from [2-3H]mannose-labeled mutant cells showed that the cells formed the di- and trisaccharides Glc1Man and Glc2Man in amounts equivalent to the deglucosylated oligosaccharides found in the cellular glycoproteins. This result shows unequivocally that the alpha-glucosidase II-deficient mutant cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.     

Glycoprotein biosynthesis. Rat liver microsomal glucosidases which process oligosaccharides.

Asparagine-linked oligosaccharides of glycoproteins undergo extensive modification or "processing" following their attachment to protein. A key step in post-glycosylation processing is the sequential removal of glucose residues from the protein-linked oligosaccharide. We have studied rat liver preparations which catalyze removal of glucose from Glc3Man9GlcNAc, Glc2Man9GlcNAc, and Glc1Man9GlcNAc. Detergent solubilization studies, inhibitor studies, and temperature-activity profiles indicate that at least two distinct glucosidases are present in the membranes. One of these glucosidases removes the distal glucose from Glc3Man9GlcNAc, and the other glucosidase sequentially removes glucose from Glc2Man9GlcNAc and Glc1Man9GlcNAc. The latter glucosidase has been solubilized from the microsomal memrbranes and purified 12-fold. The glucosidases, which are integral membrane proteins, are localized in the rough and smooth microsomes and appear to be located on the cisternal surface of the microsomal vesicles. These glucosidases are suggested to be of biological importance in catalyzing the initial events in the post-glycosylation processing of cellular glycoprotein.     

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.     

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.     

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.     

Effects of manno-1-deoxynojirimycin and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine on N-linked oligosaccharide processing in intestinal epithelial cells

The effects of manno-1-deoxynojirimycin (ManDJN) and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) were compared in IEC-6 intestinal epithelial cells in culture. ManDJN caused complete inhibition of N-linked complex oligosaccharide synthesis whereas a maximum of 80% inhibition was obtained with DMDP. HPLC showed similar endo H-sensitive oligosaccharides for control and treated cells. ManDJN caused a large increase in the levels of labeled Man7-9 GlcNAc and a decrease in Man5GlcNAc. DMDP produced similar changes except that the increase in Man7-9GlcNAc was less pronounced and some increase in glucosylated oligosaccharides was observed. Since the major oligosaccharides found in DMDP-treated cells were non-glucosylated, its primary effect on complex oligosaccharide synthesis is not due to inhibition of glucosidases, in contrast to what has been reported for influenza virus-infected MDCK cells [(1984) J. Biol. Chem. 259, 12409-12413].     

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.     

Effect of castanospermine on the structure and secretion of glycoprotein enzymes in Aspergillus fumigatus.

Aspergillus fumigatus secretes a number of glycosidases into the culture medium when the cells are grown in a mineral salts medium containing guar flour (a galactomannan) as the carbon source. At least some of these glycosidases have been reported to be glycoproteins having N-linked oligosaccharides. In this study, we examined the effect of the glycoprotein processing inhibitor, castanospermine, on the structures of the N-linked oligosaccharides and on the secretion of various glycosidases. Cells were grown in the presence of various amounts of castanospermine; at different times of growth, samples of the media were removed for the measurement of enzymatic activity. Of the three glycosidases assayed, beta-hexosaminidase was most sensitive to castanospermine; and its activity was depressed 30 to 40% at 100 micrograms of alkaloid per ml and even more at higher alkaloid concentrations. On the other hand, beta-galactosidase activity was hardly diminished at castanospermine levels of up to 1 mg/ml, but significant inhibition was observed at 2 mg/ml. beta-Galactosidase was intermediate in sensitivity. Cells were grown in the presence or absence of castanospermine and labeled with [2-3H]mannose, [6-3H]glucosamine, or [1-3H]galactose to label the sugar portion of the glycoproteins. The secreted glycoproteins were digested with pronase to obtain glycopeptides, and these were identified on Bio-Gel P-4 (Bio-Rad Laboratories). The glycopeptides were then digested with endoglucosaminidase H to release the peptide portion of susceptible structures, and the released oligosaccharides were reisolated and identified on Bio-Gel P-4. The oligosaccharides from control and castanospermine-grown cells were identified by a combination of enzymatic and chemical studies. In control cells, the oligosaccharide appeared to be mostly Man8GlcNAc and Man9GlcNAc, whereas in the presence of alkaloid, the major structures were Glc3Man7GlcNAc and Glc3Man8GlcNAc. These data fit previous observations that castanospermine inhibits glucosidase I.     

Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.

Studies on N-linked oligosaccharide processing in the mouse lymphoma glucosidase II-deficient mutant cell line (PHAR2.7) as well as the parent BW5147 cells indicated that the former maintain their capacity to synthesize complex carbohydrate units through the use of the deglucosylation mechanism provided by endomannosidase. The in vivo activity of this enzyme was evident in the mutant cells from their production of substantial amounts of glucosylated mannose saccharides, predominantly Glc2Man; moreover, in the presence of 1-deoxymannojirimycin or kifunensine to prevent processing by mannosidase I, N-linked Man8GlcNAc2 was observed entirely in the form of the characteristic isomer in which the terminal mannose of the alpha 1,3-linked branch is missing (isomer A). In contrast, parent lymphoma cells, as well as HepG2 cells in the presence of 1-deoxymannojirimycin accumulated Man9GlcNAc2 as the primary deglucosylated N-linked oligosaccharide and contained only about 16% of their Man8GlcNAc2 as isomer A. In the presence of the glucosidase inhibitor castanospermine the mutant released Glc3Man instead of Glc2Man, and the parent cells converted their deglucosylation machinery to the endomannosidase route. Despite the mutant's capacity to accommodate a large traffic through this pathway no increase in the in vitro determined endomannosidase activity was evident. The exclusive utilization of endomannosidase by the mutant for the deglucosylation of its predominant N-linked Glc2Man9GlcNAc2 permitted an exploration of the in vivo site of this enzyme's action. Pulse-chase studies utilizing sucrose-D2O density gradient centrifugation indicated that the Glc2Man9GlcNAc2 to Man8GlcNAc2 conversion is a relatively late event that is temporally separated from the endoplasmic reticulum-situated processing of Glc3Man9GlcNAc2 to Glc2Man9GlcNAc2 and in contrast to the latter takes place in the Golgi compartment.     

The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding.

Glucosylated oligomannose N-linked oligosaccharides (Glc(x)Man9GlcNAc2 where x = 1-3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N-glycosylation and chaperone recognition and (ii) substrates in the initial steps of N-glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the alpha-glucosidase inhibitor N-butyl-deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well-defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.     

Processing of N-linked oligosaccharides in soybean cultured cells

Evidence, based on both in vivo and in vitro studies with suspension-cultured soybean cells, is presented to demonstrate the processing of the oligosaccharide chain of plant N-linked glycoproteins. Following a 1-h incubation of soybean cells with [2-3H]mannose, the predominant glycopeptide obtained by pronase digestion of the membrane fraction was a Man7- or Man8GlcNAc2-Asn (GlcNAc, N-acetylglucosamine). However, the major oligosaccharide isolated from the lipid-linked oligosaccharides of these cells was a Glc2- or Glc3Man9GlcNAc2. Soybean cells were incubated with [2-3H]mannose and the incorporation of mannose into Pronase-released glycopeptides was followed during a 2-h chase. During the first 10 min of labeling, the radioactivity was mostly in a large-sized glycopeptide that appeared to be a Glc1Man9GlcNAc2-peptide. During the next 60 to 90 min of chase, this radioactivity was shifted to smaller and smaller-sized glycopeptides indicating that removal of sugars (i.e., processing) had occurred. Both glucosidase and mannosidase activity was detected in membrane preparations of soybean cells. Nine different glycopeptides were isolated from Pronase digests of soybean cell membrane fractions. These glycopeptides were purified by repeated gel filtration on columns of Bio-Gel P-4. Partial characterization of these glycopeptides by endoglucosaminidase H and alpha-mannosidase digestion, and by analysis of the products, suggested the following glycopeptides: Glc1Man9GlcNAc2-Asn, Man8GlcNAc2-Asn, Man7GlcNAc2-Asn, Man6GlcNAc2-Asn, and Man5GlcNAc2-Asn.     

Protein glycosylation in Trypanosoma cruzi. The mechanism of glycosylation and structure of protein-bound oligosaccharides

As reported previously (Parodi, A.J., and Cazzulo, J.J. (1982) J. Biol. Chem. 257, 7641-7645), label was incorporated first to the glucose residues of protein-bound Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 when Trypanosoma cruzi cells, the causative agent of Chagas disease, were incubated with [U-14C]glucose. It is now reported that the glucose residues are removed from the oligosaccharides after a chase period. The relative proportion of Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, and Man6GlcNAc2 appeared to be the same after 120 and 180 min of chase, thus indicating that these compounds were the fully processed protein-bound oligosaccharides. No complex type protein-bound oligosaccharides were detected. Evidence is presented indicating that Glc1Man7GlcNAc2 was formed mainly by glucosylation of Man7GlcNAc2 and not by demannosylation of Glc1Man9GlcNAc2. Man9GlcNAc2 was the first oligosaccharide to be labeled when cells were incubated with [2-3H]mannose. Based on these and previous results, the overall mechanism of protein N-glycosylation appeared to be: (formula; see text) The structure of the oligosaccharides appeared to be similar to some of those present in human glycoproteins. T. cruzi cells isolated from distant locations in South America were found to share a common mechanism of protein glycosylation.     

Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices

Incubations of thyroid microsomes with radiolabeled dolichyl pyrophosphoryl oligosaccharide (Glc3Man9-GlcNAc2) under conditions optimal for the N-glycosylation of protein resulted in the release, by apparently independent enzymatic reactions, of two types of neutral glucosylated polymannose oligosaccharides which differed from each other by terminating either in an N-acetylglucosamine residue (Glc3Man9GlcNAc1) or a di-N-acetylchitobiose moiety (Glc3Man9GlcNAc2). The first mentioned oligosaccharide, which was released in a steady and slow process unaffected by the addition of EDTA, appeared to be primarily the product of endo-beta-N-acetylglucosaminidase action on newly synthesized glycoprotein and such an enzyme with a neutral pH optimum capable of hydrolyzing exogenous glycopeptides and oligosaccharides (Km = 18 microM) was found in the thyroid microsomal fraction. The Glc3Man9GlcNAc2 oligosaccharide, in contrast, appeared to originate from the oligosaccharide-lipid by a rapid hydrolysis reaction which closely paralleled the N-glycosylation step, progressing as long as oligosaccharide transfer to protein occurred and terminating when carbohydrate attachment ceased either due to limitation of lipid-saccharide donor or addition of EDTA. There was a striking similarity between oligosaccharide release and transfer to protein with lipid-linked Glc3Man9GlcNAc2 serving as a 10-fold better substrate for both reactions than lipid-linked Man9-8GlcNAc2. The coincidence of transferase and hydrolase activities suggest the possibility of the existence of one enzyme with both functions. The physiological relevance of oligosaccharide release was indicated by the formation of such molecules in thyroid slices radiolabeled with [2-3H]mannose. Large oligosaccharides predominated (12 nmol/g) and consisted of two families of components; one group terminating in N-acetylglucosamine, ranged from Glc1Man9GlcNAc1 to Man5GlcNAc1 while the other contained the di-N-acetylchitobiose sequence and included Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2.     

Regulation of asparagine-linked oligosaccharide processing. Oligosaccharide processing in Aedes albopictus mosquito cells

We have examined the synthesis and processing of asparagine-linked oligosaccharides from Aedes albopictus C6/36 mosquito cells. These cells synthesized a glucose-containing lipid-linked oligosaccharide with properties identical to that of Glc3Man9GlcNAc2-PP-dolichol. Results of brief pulse label experiments with [3H]mannose were consistent with the transfer of Glc3Man9GlcNAc2 to protein followed by the rapid removal of glucose residues. Pulse-chase experiments established that further processing of oligosaccharides in C6/36 cells resulted in the removal of up to six alpha-linked mannose residues yielding Man3GlcNAc2 whose structure is identical to that of the trimannosyl "core" of N-linked oligosaccharides of vertebrate cells and yeast. Complex-type oligosaccharides were not observed in C6/36 cells. When Sindbis virus was grown in mosquito cells, Man3GlcNAc2 glycans were preferentially located at the two glycosylation sites which were previously shown to have complex glycans in virus grown in vertebrate cells. These Man3GlcNAc2 structures are the most extensively processed oligosaccharides in A. albopictus, and as such, are analogous to the complex glycans of vertebrate cells. We suggest that determinants of oligosaccharide processing which reside in the polypeptide are universally recognized despite evolutionary divergence of the oligosaccharide-processing pathway between insects and vertebrates.     

Effects of castanospermine and 1-deoxynojirimycin on insulin receptor biogenesis. Evidence for a role of glucose removal from core oligosaccharides

The insulin proreceptor is a 190-kDa glycoprotein that is processed to mature alpha (135-kDa) and beta (95-kDa) subunits. In order to determine the role of carbohydrate chain processing in insulin receptor biogenesis, we investigated the effect of inhibiting glucose removal from core oligosaccharides of the insulin proreceptor with glucosidase inhibitors, castanospermine and 1-deoxynojirimycin. Cultured IM-9 lymphocytes treated with inhibitors had 50% reduction in surface insulin receptors as demonstrated by ligand binding, affinity cross-linking with 125I-insulin, and lactoperoxidase/Na 125I labeling studies. Degradation rates of surface labeled receptors were similar in both control and inhibitor-treated cells (t1/2 = 5 h); thus, accelerated receptor degradation could not account for this reduction. Biosynthetic labeling experiments with [3H]leucine and [3H]mannose identified an apparently higher molecular size proreceptor (approximately 205 kDa) that failed to show the characteristic decline with time as seen in the normal 190-kDa proreceptor. Along with this finding, the biosynthetic label appearing in the mature subunits was reduced in these inhibitor-treated cells. Endoglycosidase H treatment of both precursors produced identical 170-kDa bands. Carbohydrate chains released from the 205-kDa precursor by endoglycosidase H migrated in the same position as the Glc2-3Man9GlcNAc standards when separated by high performance liquid chromatography, whereas the 190-kDa proreceptor oligosaccharides migrated similar to the Man7-9GlcNAc chains. Although the mature subunits of control and inhibitor-treated cells demonstrated equal electrophoretic mobility, the endoglycosidase H-sensitive oligosaccharides of the mature subunits in treated cells also contained residues that migrated similar to the Glc2-3Man9GlcNAc standards. Thus, glucose removal from core oligosaccharides is apparently not necessary for the cleavage of the insulin proreceptor, but does delay processing of this precursor, which probably accounts for the reduction in cell-surface receptors.     

The importance of trimming reactions on asparagine-linked oligosaccharides for protein quality control

Although the biochemistry of early trimming reactions by glucosidases and ER mannosidases occurring on asparagine-linked oligosaccharides has been known for a long time, their involvement in quality control of protein folding has become apparent only more recently. Here we review the evidence for the involvement of specific oligosaccharide trimming intermediates such as Glc(1)Man(9)GlcNAc(2) and Man(8)GlcNAc(2) B isomer in this fundamental cellular process and the subcellular distribution of components of the protein quality control machinery which indicates the involvement of both the ER and pre-Golgi intermediates in this process. In addition, recent studies on the subcellular distribution of endomannosidase in conjunction with previously obtained biochemical data will be reviewed which demonstrate that an alternative deglucosylation pathway exists in pre-Golgi intermediates and the Golgi apparatus.     

A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues

We have isolated and characterized a new yeast mutation in the glucosylation steps of lipid-linked oligosaccharide biosynthesis, alg8-1. Cells carrying the alg8-1 mutation accumulate Glc1Man9GlcNAc2-lipid both in vivo and in vitro. We present evidence showing that the alg8-1 mutation blocks addition of the second alpha 1,3-linked glucose. alg8-1 cells transfer Glc1Man9GlcNAc2 to protein instead of the wild type oligosaccharide, Glc3Man9GlcNAc2. Pulse-chase studies indicate that the Glc1Man9GlcNAc2 transferred is processed more slowly than the wild type oligosaccharide. The yeast mutation gls1-1 lacks glucosidase I activity (Esmon, B., Esmon, P.C., and Schekman, R. (1984) J. Biol. Chem. 259, 10322-10327), the enzyme responsible for removing the alpha 1,2-linked glucose residues from protein-linked oligosaccharides. We demonstrate that gls1-1 cells contain glucosidase II activity (which removes alpha 1,3-linked glucose residues) and have constructed the alg8-1 gls1-1 haploid double mutant. The Glc1Man9GlcNAc2 oligosaccharide was trimmed normally in these cells, demonstrating that the alg8-1 oligosaccharide contained an alpha 1,3-linked glucose residue. A novel Glc2 compound was probably produced by the action of the biosynthetic enzyme that normally adds the alpha 1,2-linked glucose to lipid-linked Glc2Man9GlcNAc2. This enzyme may be able to slowly add alpha 1,2-linked glucose residue to protein-bound Glc1Man9GlcNAc2. The relevance of these findings to similar observations in other systems where glucose residues are added to asparagine-linked oligosaccharides and the possible significance of the reduced rate of oligosaccharide trimming in the alg mutants are discussed.