Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.     

The soluble form of rat liver alpha-mannosidase is immunologically related to the endoplasmic reticulum membrane alpha-mannosidase

The soluble alpha-mannosidase of rat liver, originally described as a cytoplasmic alpha-mannosidase, has been purified to homogeneity by conventional techniques. The purified enzyme has an apparent molecular weight of 350,000 and is composed of 107-kDa subunits. The soluble alpha-mannosidase has the same enzymatic properties as the endoplasmic reticulum (ER) membrane alpha-mannosidase of rat liver (Bischoff, J., and Kornfeld, R. (1983) J. Biol. Chem. 258, 7909-7910) which is believed to play a role in oligosaccharide processing in the rough ER. Like the membrane-bound ER alpha-mannosidase, the soluble alpha-mannosidase can hydrolyze alpha-linked mannose from both p-nitrophenyl alpha-mannoside (Km = 0.14 mM) and high mannose oligosaccharides, is not inhibited by the mannose analogues swainsonine and 1-deoxymannojirimycin, is stabilized by MnCl2 or CoCl2, and does not bind to concanavalin A-Sepharose. A goat polyclonal antibody raised against the purified soluble alpha-mannosidase specifically recognizes the rat liver membrane-bound ER alpha-mannosidase, leading us to propose that they are two forms of the same enzyme and that the soluble form is derived from the ER membrane alpha-mannosidase by proteolysis. The antibody also cross-reacts with both the soluble and membrane-bound forms of ER alpha-mannosidase activity in cultured Chinese hamster ovary cells and rat H35 hepatoma cells. Since the ER alpha-mannosidase is presumed to be involved in the early steps of oligosaccharide processing, the action of the purified soluble form of the enzyme on high mannose oligosaccharides was examined. Surprisingly, the enzyme released free mannose from oligosaccharides ranging in size from Glc1Man9GlcNAc to Man5GlcNAc with almost equal efficiency. However, a long term incubation of the enzyme with Man9GlcNAc led to the accumulation of Man7GlcNAc and produced only small amounts of Man6GlcNAc and Man5GlcNAc. Structural analysis of these reaction products indicated that the purified soluble form of ER alpha-mannosidase shows little specificity for which mannose residues it removes from Man9GlcNAc. In contrast, as shown in the accompanying paper, the intracellular action of ER alpha-mannosidase on glycoprotein-bound Man9GlcNAc2 is highly specific.     

The initial stages of processing of protein-bound oligosaccharides in vitro.

Following the rapid enzymatic transfer of an oligosaccharide (GlcNAc2Man9Glc3) from a lipid carrier to endogenous protein acceptors in membrane preparations from NIL fibroblasts, the transferred oligosaccharide chain undergoes processing. Protein-bound oligosaccharides, released from the polypeptide backbone by treatment with endo-beta-N-acetylglucosaminidase H, were analyzed by gel filtration and by susceptibility to alpha-mannosidase digestion. The initial stages of this processing in vitro consist of sequential excision of 3 glucose residues prior to the removal of mannose residues. The array of oligosaccharides generated in vitro by membrane preparations from NIL cells appears to be identical with processed oligosaccharides derived in vivo in intact NIL cells.     

Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.

The relationship between maturation of lipoprotein lipase (LPL) and its translocation from the endoplasmic reticulum (ER) to the Golgi complex was determined by measuring lipolytic activity under conditions preventing transport of the enzyme from the ER to the Golgi compartment. In the presence of brefeldin A, a reagent that inhibits movement of proteins from the ER and causes the disassembly of the Golgi complex, pro-5 Chinese hamster ovary cells accumulated catalytically active LPL, while secretion of the enzyme was effectively blocked. LPL retained intracellularly by brefeldin A treatment possessed oligosaccharide chains that were processed to the complex form by the Golgi enzymes redistributed into the ER. At 16 degrees C, a condition disrupting protein transport to the cis-Golgi, the retained enzyme again remained catalytically active although the oligosaccharides remained in the high mannose form. Lastly, attachment of the specific ER retention signal KDEL (Lys-Asp-Glu-Leu) to the carboxyl terminus of LPL also resulted in intracellularly retained enzyme that was fully active. The importance of oligosaccharide processing for attainment of LPL catalytic activity in vitro was also determined. LPL was active and secreted when trimming of the mannose residues was inhibited by deoxymannojirimycin and when addition of complex sugars was blocked using Chinese hamster ovary mutants (lec1 and lec2), indicating that these processing events are not necessary for the expression of a functional enzyme. However, blocking glucose removal by glucosidase inhibitors (castanospermine and N-methyl-deoxynojirimycin) resulted in a significant reduction in LPL specific activity and secretion. Thus, glucose trimming of LPL oligosaccharides is essential for enzyme activation; however, further oligosaccharide processing or translocation of the enzyme to the cis-Golgi is not required for full expression of lipolytic activity in vitro.     

Analysis of the role of glycosylation of the human fibronectin receptor

1-Deoxymannojirimycin (MNJ), an inhibitor of Golgi alpha-mannosidase IA and IB, was used to assess the possible roles of asparagine-linked oligosaccharides in the structure and function of the integrin fibronectin receptor from cultured human fibroblasts. These cells normally attach well to fibronectin substrates and have only mature forms of the fibronectin receptor on their surfaces. MNJ inhibits the intracellular trimming of high mannose oligosaccharides, and cells treated with 0.2 mg/ml MNJ synthesize only immature precursor forms of both the alpha and beta subunits of the fibronectin receptor. The immature receptor polypeptides were found to be nonfunctional by two criteria: 1) cells treated with MNJ attached poorly to fibronectin substrates; and 2) receptor from the treated cells was defective in binding to fibronectin affinity columns. The precursor forms of the fibronectin receptor subunits were found on the surfaces of cells treated with MNJ, demonstrating that processing of receptor carbohydrates to mature forms was not necessary for receptor insertion into the plasma membrane. A monoclonal antibody that specifically bound the alpha subunit of the fibronectin receptor immunoprecipitated both alpha and beta subunit polypeptides from both control cells and cells treated with MNJ. Similarly, a monoclonal antibody that specifically bound only the beta subunit also immunoprecipitated both alpha and beta subunit polypeptides of the receptor from extracts of both control and MNJ-treated cells. These results indicate that receptor assembly can occur in the absence of complete oligosaccharide processing. Thus, oligosaccharide processing to the mature form of the fibronectin receptor is important for its binding function but not for receptor assembly or insertion into the plasma membrane.     

Regulation of glycosylation. The influence of protein structure on N-linked oligosaccharide processing

The Sindbis virus glycoproteins, E1 and E2, comprise a useful model system for evaluating the effects of local protein structure on the processing of N-linked oligosaccharides by Golgi enzymes. The conversion of oligomannose to N-acetyllactosamine (complex) oligosaccharides is hindered to different extents at the four glycosylation sites, so that the complex/oligomannose ratio decreases in the order E1-Asn139 greater than E2-Asn196 greater than E1-Asn245 greater than E2-Asn318. The processing steps most susceptible to interference were deduced from the oligosaccharide compositions at hindered sites in virus from baby hamster kidney cells (BHK), chick embryo fibroblasts (CEF), and normal and hamster sarcoma virus (HSV)-transformed hamster fibroblasts (Nil-8). Persistence of Man6-9GlcNAc2 was taken to indicate interference with alpha 2-mannosidase(s) I (alpha-mannosidase I), Man5GlcNAc2, with UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase I (GlcNAc transferase I), and unbisected hybrid glycans, with GlcNAc transferase I-dependent alpha 3(alpha 6)-mannosidase (alpha-mannosidase II). Taken together, the results indicate that all four sites acquire a precursor oligosaccharide with equally high efficiency, but alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are all impeded at E2-Asn318 and, to a lesser extent, at E1-Asn245. In contrast, sialic acid and galactose transfer to hybrid glycans (in BHK cells) is virtually quantitative even at E2-Asn318. E2-Asn318 carried no complex oligosaccharides, but the structures of those at E1-Asn245 indicate almost complete GlcNAc transfer by UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase II (GlcNAc transferase II), galactosylation, and sialylation. Because the E2-Asn318 and E1-Asn245 glycans have previously been shown to be less accessible to a steric probe than those at E2-Asn196 or E1-Asn139, a simple explanation for these results would be that alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are more susceptible to steric hindrance than are the later processing steps examined. Finally, in addition to these site-specific effects, the overall extent of viral oligosaccharide processing varied with host and cellular growth status. For example, alpha-mannosidase I processing is more complete in BHK cells compared to CEF, and in confluent Nil-8 cells compared to subconfluent or HSV-transformed Nil-8 cells.     

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.     

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-³H]mannose or [³⁵S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-β-N-acetylglucosaminidase H, which specifically cleaves the ‘high-mannose’ class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing.This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a ‘high-mannose’ oligosaccharide precursor and a ‘complex’ carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (M_r 2100) comparable to (Man)₉GlcNAc (M_r 2080), and was sensitive to endo-β-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistent to endo-β-N-acetylglucosaminadase H cleavage. The final ‘complex’ or processed oligosaccharide structure contained approximately two-thirds less associated with the mature glycoprotein. They also indicate that the ‘complex’ structure is synthesized as a ‘high-mannose’ intermediate which is processed by the removal of mannose.     

N-Linked oligosaccharides of the H-2Dk histocompatibility protein heavy chain influence its transport and cellular distribution

The H-2K and H-2D proteins encoded by the K and D region of the major histocompatibility complex of the mouse were isolated by immunoprecipitation with specific antisera and resolved by two-dimensional gel electrophoresis. Of these two polypeptides, the H-2Dk glycoproteins isolated from macrophages of C3H/HeHa mice exhibit distinct cell surface and cytoplasmic forms although they share a strong degree of homology in the polypeptide backbone. Structurally they differ in their oligosaccharide structures. The structure of the oligosaccharides on the intracellular forms is of the high mannose type while the same structures on the cell surface forms are of the complex type. In the absence of all three oligosaccharide side chains, the unglycosylated polypeptides are expressed on the cell surface. In contrast, polypeptides containing one, two, or all three oligosaccharide side chains of the high mannose type are not transported to the cell surface. Cell surface expression of these glycoproteins requires processing of the oligosaccharide side chains from the high mannose form to the complex type. However, not all oligosaccharide antennae have to be terminally modified since H-2Dk glycoproteins synthesized in the presence of oligosaccharide-processing enzyme inhibitors such as swainsonine or monensin are also transported to the cell surface. H-2Dk glycoproteins containing oligosaccharide structures of the complex type but lacking terminal sialic acids are found on the cell surface, suggesting that sialylation is not required for transport. These results indicate that the oligosaccharide structures of the H-2Dk glycoproteins act to influence their cellular distribution.     

Purification and characterization of a rat liver Golgi alpha-mannosidase capable of processing asparagine-linked oligosaccharides.

Studies in intact cells have shown the following processing reaction to occur during Asn-linked oligosaccharide biosynthesis (M, mannose; GlcNAc, N-acetylglucosamine): Formula: (See Text) We have identified a rat liver Golgi enzyme which catalyzes this reaction in vitro. This alpha-mannosidase has been purified 3,000 to 6,000-fold by subcellular fractionation, Triton X-100 solubilization, and ion exchange and hydroxylapatite chromatography. The purified enzyme has a pH optimum between 6.0 and 6.5 and a Km between 17 and 100 microM for a processing intermediate. The enzyme shows specificity for alpha 1,2-linked mannose residues. Structural analysis of the in vitro reaction products reveal that specific intermediates are formed in the conversion of the (Man)9GlcNAc oligosaccharide to the (Man)5GlcNAc oligosaccharide. Heat inactivation studies are consistent with the possibility that one enzyme activity is responsible for this conversion. The alpha 1,2-specific mannosidase described here appears to be distinct from two other rat liver Golgi alpha-mannosidase activities based on differential substrate specificity, inhibitor susceptibility, and detergent extractability.     

Characterization of a specific alpha-mannosidase involved in oligosaccharide processing in Saccharomyces cerevisiae

Fractionation of a crude extract from Saccharomyces cerevisiae X-2180 on Sepharose 6B in the presence of 0.5% Triton X-100 resolves two enzyme fractions containing alpha-mannosidase activity. Fraction I which is excluded from the gel contains alpha-mannosidase activity toward both p-nitrophenyl-alpha-D-mannopyranoside and Man9GlcNAc oligosaccharide as substrates, whereas Fraction II which is included in the gel contains only oligosaccharide alpha-mannosidase activity. The latter enzyme is very specific and removes a single mannose residue from Man9GlcNAc, whereas the alpha-mannosidase activity of Fraction I removes several mannose residues from Man9GlcNAc oligosaccharide. High resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the alpha-mannosidase of Fraction II showed only a single isomer with the following structure: (see formula; see text) This specific enzyme is most probably involved in processing of oligosaccharide during biosynthesis of mannoproteins. The mannose analog of 1-deoxynojirimycin (50-500 microM), dideoxy-1,5-imino-D-mannitol, inhibits the oligosaccharide alpha-mannosidase activities of Fractions I and II to about the same extent, but has no effect on the nonspecific alpha-mannosidase which acts on p-nitrophenyl-alpha-D-mannopyranoside.     

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.     

Reduced contact-inhibition and substratum adhesion in epithelial cells expressing GlcNAc-transferase V

Malignant transformation of fibroblast and epithelial cells is accompanied by increased beta 1-6 N-acetylglucosaminyltransferase V (GlcNAc-TV) activity, a Golgi N-linked oligosaccharide processing enzyme. Herein, we report that expression of GlcNAc-TV in Mv1Lu cells, an immortalized lung epithelial cell line results in loss of contact- inhibition of cell growth, an effect that was blocked by swainsonine, an inhibitor of Golgi processing enzyme alpha-mannosidase II. In serum- deprived and high density monolayer cultures, the GlcNAc-TV transfectants formed foci, maintained microfilaments characteristic of proliferating cells, and also experienced accelerated cell death by apoptosis. Injection of the GlcNAc-TV transfectants into nude mice produced a 50% incidence of benign tumors, and progressively growing tumors in 2:12 mice with a latency of 6 mo, while no growth was observed in mice injected with control cells. In short term adhesion assays, the GlcNAc-TV expressing cells were less adhesive on surfaces coated with fibronectin and collagen type IV, but no changes were observed in levels of cell surface alpha 5 beta 1 or alpha v beta 3 integrins. The larger apparent molecular weights of the LAMP-2 glycoprotein and integrin glycoproteins alpha 5, alpha v and beta 1 in the transfected cells indicates that their oligosaccharide chains are substrates for GlcNAc-TV. The results suggest that beta 1-6GlcNAc branching of N-linked oligosaccharides contributes directly to relaxed growth controls and reduce substratum adhesion in premalignant epithelial cells.     

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.     

Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity

The processing of asparagine-linked oligosaccharides on the alpha- chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N- acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.     

Cell type dependent inhibition of transport of cathepsin D in HepG2 cells and fibroblasts exposed to deoxy-manno-nojirimycin and deoxynojirimycin

The synthesis, transport and processing of lysosomal enzymes was examined in human hepatoma HepG2 cells and in human fibroblasts exposed to the Golgi alpha-mannosidase I inhibitor 1-deoxy-manno-nojirimycin. In HepG2 cells cathepsin D, beta-hexosaminidase and arylsulfatase B synthesized in the presence of 5 mM 1-deoxy-manno-nojirimycin contained exclusively endo-beta-N-acetylglucosaminidase H-cleavable oligosaccharides, indicating that alpha-mannosidase I had been inhibited efficiently. The proteolytic processing of intracellularly retained cathepsin D was retarded and the fraction of secreted cathepsin D was increased two-fold. In fibroblasts neither segregation nor maturation of cathepsin D were affected by 1-deoxy-manno-nojirimycin in spite of the inhibition of oligosaccharide processing. In the presence of the glucosidase I inhibitor 1-deoxynojirimycin, the precursor of cathepsin D (larger by about 1 kDa than the secreted form) accumulated transiently in light membranes in HepG2 cells. Release from the site of accumulation was accompanied by a decrease in size by about 1 kDa. This change was attributed to the removal of glucose residues. In fibroblasts the transient accumulation of larger precursors in the presence of 1-deoxynojirimycin was more pronounced than in HepG2 cells. The differential effects of alpha-mannosidase I and glucosidase I inhibitors on the transport of cathepsin D in HepG2 cells and fibroblasts may indicate that different intermediates in the biosynthetic pathway of asparagine-linked oligosaccharides participate in the transport of lysosomal enzymes in the two cell types.     

Characterization of the oligosaccharides of prolyl hydroxylase, a microsomal glycoprotein

Prolyl hydroxylase is a tetrameric glycoprotein that catalyzes a vital posttranslational modification in the biosynthesis of collagen. The enzyme purified from whole chick embryos (WCE) possesses two nonidentical subunits, alpha and beta, and has been shown by several techniques to reside in the endoplasmic reticulum of chick embryo fibroblasts. The studies described here demonstrate that the larger of the two subunits (alpha) exists in two forms in chick embryo fibroblasts (CEF); these two forms differ in carbohydrate content. The larger alpha subunit, alpha', contains two N-linked high mannose oligosaccharides, each containing eight mannose units; the smaller subunit, alpha, contains a single seven-mannose N-linked oligosaccharide. Both oligosaccharides could be cleaved by endo-beta-N-acetylglucosaminidase H and completely digested with alpha-mannosidase to yield mannosyl-N-acetylglucosamine.     

Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella aerogenes type 54[A3(S1)]

Several strains of bacteriophage have been isolated that induce the formation of a polysaccharide hydrolase after infection of Klebsiella aerogenes type 54 [A3(S1)]. The action of this enzyme on polysaccharide solutions was to decrease their viscosity and increase their reducing value. These effects were associated with the release of two oligosaccharides (O1 and O2) from the polysaccharide. These two substances are not identical with any of the four oligosaccharides isolated from autohydrolysates. The two enzymically isolated fractions have been tentatively identified as tetrasaccharides, and oligosaccharide O2 is probably an acetylated version of oligosaccharide O1. This latter oligosaccharide differs in some way, still unknown, from the tetrasaccharide cellobiosylglucuronosylfucose found in acid hydrolysates of the slime polysaccharide. The enzyme is limited in its activity to the polysaccharide excreted by the A3 strain of K. aerogenes type 54 or by similar strains. It is also active on the polysaccharides altered by acid or alkaline treatment. The enzyme has optimum activity at pH6.5. A study of the products released by enzyme action has shown it to be a fucosidase splitting the fucosylglucose linkages found in the intact polysaccharide.     

Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function

A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required.     

Inhibition of glycoprotein oligosaccharide processing in vitro and in influenza-virus-infected cells by alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene.

The effects of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (MMNT) on mannosidases involved in asparagine-linked oligosaccharide processing were investigated. MMNT was found to inhibit the activity of rat liver Golgi alpha-mannosidase I in a concentration-dependent manner (50% inhibition with 0.18 mM-MMNT), whereas rat liver endoplasmic-reticulum alpha-mannosidase appeared to be resistant (less than 5% inhibition at 1 mM-MMNT). Jack-bean alpha-mannosidase was also sensitive to inhibition by MMNT (50% inhibition with 0.32 mM-MMNT). Treatment of influenza-virus-infected chick-embryo cells with 1 mM-MMNT led to a decrease in the formation of complex-type asparagine-linked oligosaccharides and an accumulation of high-mannose-type oligosaccharides with the composition Man8(GlcNAc)2 and Man7(GlcNAc)2 on the viral glycoproteins. The biological activities of influenza-virus haemagglutinin and neuraminidase synthesized in the presence of 1 mM-MMNT remained unchanged, but the virus was less infectious than the control.