Importance of glycosidases in mammalian glycoprotein biosynthesis

Processing glycosidases play an important role in N-glycan biosynthesis in mammalian cells by trimming Glc(3)Man(9)GlcNAc(2) and thus providing the substrates for the formation of complex and hybrid structures by Golgi glycosyltransferases. Processing glycosidases also play a role in the folding of newly formed glycoproteins and in endoplasmic reticulum quality control. The properties and molecular nature of mammalian processing glycosidases are described in this review. Membrane-bound alpha-glucosidase I and soluble alpha-glucosidase II of the endoplasmic reticulum remove the alpha1,2-glucose and alpha1,3-glucose residues, respectively, beginning immediately following transfer of Glc(3)Man(9)GlcNAc(2) to nascent polypeptides. The alpha-glucosidases participate in glycoprotein folding mediated by calnexin and calreticulin by forming the monoglucosylated high mannose oligosaccharides required for the interaction with the chaperones. In some mammalian cells, Golgi endo alpha-mannosidase provides an alternative pathway for removal of glucose residues. Removal of alpha1,2-linked mannose residues begins in the endoplasmic reticulum where trimming of mannose residues in the endoplasmic reticulum has been implicated in the targeting of malfolded glycoproteins for degradation. Removal of mannose residues continues in the Golgi with the action of alpha1, 2-mannosidases IA and IB that can form Man(5)GlcNAc(2) and of alpha-mannosidase II that removes the alpha1,3- and alpha1,6-linked mannose from GlcNAcMan(5)GlcNAc(2) to form GlcNAcMan(3)GlcNAc(2). These membrane-bound Golgi enzymes have been cloned and shown to have very distinct patterns of tissue-specific expression. There are also broad specificity alpha-mannosidases that can trim Man(4-9)GlcNAc(2) to Man(3)GlcNAc(2), and provide an alternative pathway toward complex oligosaccharide formation. Cloning of the remaining alpha-mannosidases will be required to evaluate their specific functions in glycoprotein maturation.     

Insect cells encode a class II alpha-mannosidase with unique properties

Previously, we cloned and characterized an insect (Sf9) cell cDNA encoding a class II alpha-mannosidase with amino acid sequence and biochemical similarities to mammalian Golgi alpha-mannosidase II. Since then, it has been demonstrated that other mammalian class II alpha-mannosidases can participate in N-glycan processing. Thus, the present study was performed to evaluate the catalytic properties of the Sf9 class II alpha-mannosidase and to more clearly determine its relationship to mammalian Golgi alpha-mannosidase II. The results showed that the Sf9 enzyme is cobalt-dependent and can hydrolyze Man(5)GlcNAc(2) to Man(3)GlcNAc(2), but it cannot hydrolyze GlcNAcMan(5)GlcNAc(2). These data establish that the Sf9 enzyme is distinct from Golgi alpha-mannosidase II. This enzyme is not a lysosomal alpha-mannosidase because it is not active at acidic pH and it is localized in the Golgi apparatus. In fact, its sensitivity to swainsonine distinguishes the Sf9 enzyme from all other known mammalian class II alpha-mannosidases that can hydrolyze Man(5)GlcNAc(2). Based on these properties, we designated this enzyme Sf9 alpha-mannosidase III and concluded that it probably provides an alternate N-glycan processing pathway in Sf9 cells.     

Characterization of a soluble class I alpha-mannosidase in human serum.

Class I alpha-mannosidases are thought to exist exclusively as integral membrane proteins that play intracellulary an essential role in the N-glycan biosynthesis. Using [3H]Man9GlcNAc2 as a substrate, we were able to identify a soluble alpha-mannosidase in human serum that trims the substrate Man9GlcNAc2 to Man(5-8)GlcNAc2 with Man6GlcNAc2 being the major product. This serum mannosidase is Ca2+-dependent, sensitive to 1-deoxymannojirimycin but insensitive to the class II inhibitor swainsonine and, hence, belongs to class I mannosidases. The enzymatic properties of the serum class I mannosidase are similar to that of the membrane bound class I mannosidases Golgi-mannosidase IA and IB and Man9-mannosidase.     

Substrate specificities of recombinant murine Golgi alpha1, 2- mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1,2-mannosidases

The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris . Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase. Recombinant mannosidases IA and IB were used to cleave Man9GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy. Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man5GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha1, 2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major isomer. In contrast, rat liver Golgi membranes rapidly cleaved Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA. These immunodepleted membranes were enriched in a Man9GlcNAc2to Man8GlcNAc2- cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man9GlcNAc2.      

Structure of Golgi alpha-mannosidase II: a target for inhibition of growth and metastasis of cancer cells.

Golgi alpha-mannosidase II, a key enzyme in N-glycan processing, is a target in the development of anti- cancer therapies. The crystal structure of Drosophila Golgi alpha-mannosidase II in the absence and presence of the anti-cancer agent swainsonine and the inhibitor deoxymannojirimycin reveals a novel protein fold with an active site zinc intricately involved both in the substrate specificity of the enzyme and directly in the catalytic mechanism. Identification of a putative GlcNAc binding pocket in the vicinity of the active site cavity provides a model for the binding of the GlcNAcMan(5)GlcNAc(2) substrate and the consecutive hydrolysis of the alpha1,6- and alpha1,3-linked mannose residues. The enzyme-inhibitor interactions observed provide insight into the catalytic mechanism, opening the door to the design of novel inhibitors of alpha-mannosidase II.     

A deletion in the golgi alpha-mannosidase II gene of Caenorhabditis elegans results in unexpected non-wild-type N-glycan structures

The processing of N-linked oligosaccharides by alpha-mannosidases in the endoplasmic reticulum and Golgi is a process conserved in plants and animals. After the transfer of a GlcNAc residue to Asn-bound Man(5)GlcNAc(2) by N-acetylglucosaminyltransferase I, an alpha-mannosidase (EC 3.2.1.114) removes one alpha1,3-linked and one alpha1,6-linked mannose residue. In this study, we have identified the relevant alpha-mannosidase II gene (aman-2; F58H1.1) from Caenorhabditis elegans and have detected its activity in both native and recombinant forms. For comparative studies, the two other cDNAs encoding class II mannosidases aman-1 (F55D10.1) and aman-3 (F48C1.1) were cloned; the corresponding enzymes are, respectively, a putative lysosomal alpha-mannosidase and a Co(II)-activated alpha-mannosidase. The analysis of the N-glycan structures of an aman-2 mutant strain demonstrates that the absence of alpha-mannosidase II activity results in a shift to structures not seen in wild-type worms (e.g. N-glycans with the composition Hex(5-7)HexNAc(2-3)Fuc(2)Me) and an accumulation of hybrid oligosaccharides. Paucimannosidic glycans are almost absent from aman-2 worms, indicative also of a general lack of alpha-mannosidase III activity. We hypothesize that there is a tremendous flexibility in the glycosylation pathway of C. elegans that does not impinge, under standard laboratory conditions, on the viability of worms with glycotypes very unlike the wild-type pattern.     

Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver

An alpha-mannosidase activity has been identified in a preparation of rat liver endoplasmic reticulum and shown to be distinct from the previously described Golgi alpha-mannosidases I and II and the lysosomal alpha-mannosidase. The enzyme was solubilized with deoxycholate and separated from other alpha-mannosidases by passage over concanavalin A-Sepharose to which it does not bind. The endoplasmic reticulum alpha-mannosidase cleaves alpha-1,2-linked mannoses from high mannose oligosaccharides and, unlike Golgi alpha-mannosidase I, is active against p-nitrophenyl-alpha-D-mannoside (Km = 0.17 mM). It has no activity toward GlcNAc-Man5GlcNAc2 peptide, the specific substrate of the Golgi alpha-mannosidase II. The endoplasmic reticulum alpha-mannosidase activity toward p-nitrophenyl-alpha-D-mannoside is relatively insensitive to swainsonine, an inhibitor of both the lysosomal alpha-mannosidase and Golgi alpha-mannosidase II. We propose that the endoplasmic reticulum alpha-mannosidase is responsible for the removal of mannose residues from asparagine-linked high mannose type oligosaccharides prior to their entry into the Golgi.     

Biosynthesis and processing of Spodoptera frugiperda alpha-mannosidase III

We previously cloned a lepidopteran insect cell cDNA that encodes a class II alpha-mannosidase that is localized in the Golgi apparatus but is cobalt-dependent, has a neutral pH optimum, hydrolyzes Man(5)GlcNAc(2) to Man(3)GlcNAc(2), and cannot hydrolyze GlcNAcMan(5)GlcNAc(2). This enzyme was designated SfManIII to distinguish it from Golgi alpha-mannosidase II and indicate its derivation from the fall armyworm Spodoptera frugiperda. In the present study, we prepared a polyclonal antibody and used it to study the biosynthesis and processing of SfManIII. The results showed that Sf9 cells produce at least three different forms of SfManIII. SfManIII is initially synthesized as a precursor glycoprotein, which is slowly converted to two smaller end products with at least some endoglycosidase H-resistant N-glycans. The smallest form of SfManIII is the only one of these two products that accumulates in the extracellular fraction. Tunicamycin blocked the production of SfManIII activity and the secretion of SfManIII protein and activity. Castanospermine blocked production of the larger SfManIII product, retarded production of the smaller, increased intracellular SfManIII activity, and decreased extracellular SfManIII activity. Together, these results indicate that SfManIII is initially synthesized as a high-mannose glycoprotein precursor, its N-glycans are trimmed as it is transported to the Golgi apparatus, and a subpopulation, which appears to be proteolytically cleaved, is secreted in enzymatically active form. N-glycosylation is required for the production of active SfManIII, and N-glycosylation and N-glycan trimming are both required for the efficient secretion of an active form of this protein.     

Overexpression of the Golgi-localized enzyme alpha-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway.

Golgi alpha-mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn(1)M(5)Gn(2) to Gn(1)M(3)Gn(2) during biosynthesis of N-glycans. Previously, we isolated a cDNA encoding a protein homologous to alpha-mannosidase II and designated it alpha-mannosidase IIx. Here, we show by immunocytochemistry that alpha-mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with alpha-mannosidase II, alpha-mannosidase IIx colocalizes with alpha-mannosidase II in COS cells. A protein A fusion of the catalytic domain of alpha-mannosidase IIx hydrolyzes a synthetic substrate, 4-umbelliferyl-alpha-D-mannoside, and this activity is inhibited by swainsonine. [(3)H]glucosamine-labeled Chinese hamster ovary cells overexpressing alpha-mannosidase IIx show a reduction of M(6)Gn(2) and an accumulation of M(4)Gn(2). Structural analysis identified M(4)Gn(2) to be Man alpha 1-->6(Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc. The results suggest that alpha-mannosidase IIx hydrolyzes two peripheral Man alpha 1-->6 and Man alpha 1-->3 residues from [(Man alpha 1-->6)(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc, during N-glycan processing.     

Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line.

The most frequent type of N-glycan synthesized by lepidopteran Sf9 cells appears to be fucosylated Man3GlcNAc2,and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in cultured insect cells. The current knowledge of the protein glycosylation pathway derived from structural studies on recombinant glyco-proteins expressed by using baculovirus vectors. In this work we provide more direct evidence for the sequential events occurring in the processing of endogenous N-glycoproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the presence of processing inhibitors (castanospermine and swainsonine) and in the presence of an intracellular trafficking inhibitor (monensin). We thus demonstrated that from the glycan precursor Glc3Man9GlcNAc2 to GlcNAcMan5(Fuc)GlcNAc2 intermediate, the processing pathway in Sf9 cells paralleled the one demonstrated in mammalian cells. By using monensin, we demonstrated the formation of Man3(Fuc)GlcNAc2 from GlcNAcMan3(Fuc)GlcNAc2, a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological relevance to the glycosylation pathway and is Golgi located.     

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.     

Mutation of Arg(273) to Leu alters the specificity of the yeast N-glycan processing class I alpha1,2-mannosidase

Class I alpha1,2-mannosidases (glycosyl hydrolase family 47) involved in the processing of N-glycans during glycoprotein maturation have different specificities. Enzymes in the endoplasmic reticulum of yeast and mammalian cells remove a single mannose from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2) isomer B (lacking the alpha1, 2-mannose residue of the middle alpha1, 3-arm), whereas other alpha1,2-mannosidases, including Golgi alpha1,2-mannosidases IA and IB, can convert Man(9)GlcNAc(2) to Man(5)GlcNAc(2). In the present work, it is demonstrated that with a single mutation in its catalytic domain (Arg(273) --> Leu) the yeast endoplasmic reticulum alpha1,2-mannosidase acquires the ability to transform Man(9)GlcNAc to Man(5)GlcNAc. High resolution proton nuclear magnetic resonance analysis of the products shows that the order of removal of mannose from Man(9)GlcNAc is different from that of other alpha1, 2-mannosidases that remove four mannose from Man(9)GlcNAc. These results demonstrate that Arg(273) is in part responsible for the specificity of the endoplasmic reticulum alpha1,2-mannosidase and that small differences in non-conserved amino acids interacting with the oligosaccharide substrate in the active site of class I alpha1, 2-mannosidases are responsible for the different specificities of these enzymes.     

Purification to homogeneity and properties of mannosidase II from mung bean seedlings

Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.     

Developmental regulation of asparagine-linked oligosaccharide synthesis in Dictyostelium discoideum

In the preceding report we demonstrated that the expression of two developmentally regulated alpha-mannosidase activities is induced in Dictyostelium discoideum during its differentiation from single-cell amoebae to multicellular organism (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18477-18484). These activities, designated membrane alpha-mannosidase I (MI) and membrane alpha-mannosidase II (MII), were shown to have several properties in common with rat liver Golgi alpha-mannosidases I and II, respectively, suggesting that MI and MII may play a role in the processing of asparagine-linked oligosaccharides in developing D. discoideum. In this study we analyzed the structures of the asparagine-linked oligosaccharides synthesized by D. discoideum at various stages of development to determine the timing and extent of asparagine-linked oligosaccharide processing. Cells were labeled with [2-3H] mannose, and then total cellular glycoproteins were digested with Pronase to generate glycopeptides that were fractionated on concanavalin A-Sepharose. Glycopeptides from each fraction were digested with endoglycosidase H, both before and after desulfation by solvolysis, and the released, neutral oligosaccharides were sized by high pressure liquid chromatography. At early stages of development, D. discoideum contain predominantly large high mannose-type oligosaccharides (Man9GlcNAc and Man8GlcNAc). Some of these are modified by GlcNAc residues attached beta 1-4 to the mannose-linked alpha 1-6 to the beta-linked core mannose (the "intersecting" position), as well as by fucose, sulfate, and phosphate. In contrast, the oligosaccharides found at late stages of development (18-24 h) have an array of sizes from Man9GlcNAc to Man3GlcNAc. These are still modified by GlcNAc, fucose, sulfate, and phosphate, but the percent of larger high mannose oligosaccharides that are modified with GlcNAc in the intersecting position decreases after 6 h of development, in parallel with the decrease in the intersecting GlcNAc transferase activity. Similarly, the changes in the size of asparagine-linked oligosaccharides synthesized during development correlate well with the appearance of MI and MII activities and suggest that these developmentally regulated alpha-mannosidase activities function in the processing of these oligosaccharides. This is supported further by the observation that oligosaccharide processing was inhibited in late stage cells labeled in the presence of either deoxymannojirimycin, an inhibitor of MI, or swainsonine, an inhibitor of MII.     

Processing of the Man(10)GlcNAc (M(10)) oligosaccharide by alpha-mannosidases from Candida albicans

The hydrolysis of Man(10)GlcNAc (M(10)) by purified alpha-mannosidases and its further processing by a mixed membrane preparation from Candida albicans were studied. Incubation of the oligosaccharide with purified alpha-mannosidases I (E-I) or II (E-II) from C. albicans released 1 and 2 mol of mannose per mol of M(10), respectively. This treatment converted M(10) into an acceptor substrate of further mannose residues from GDP-Man as catalyzed by membrane-bound mannosyltransferases. Elongation of E-I- or E-II-trimmed M(10) yielded a low molecular mass product (14-17 mannose residues added), and in the case of E-II, a minor amount of an additional product of a higher molecular mass. Our results indicate that purified alpha-mannosidases participate in N-glycan processing in C. albicans.     

The purification and characterization of mannosidase IA from rat liver Golgi membranes

Rat liver Golgi membranes contain two alpha 1,2-specific mannosidases (IA and IB) (Tulsiani, D. R. P., Hubbard, S. C., Robbins, P. W., and Touster, O. (1982) J. Biol. Chem. 257, 3660-3668). Mannosidase IA has now been purified to apparent homogeneity by detergent extraction and (NH4)2SO4 precipitation, followed by Sephacryl S-300, ion-exchange, and hydroxylapatite chromatography. The enzyme was homogeneous by nondenaturing polyacrylamide gel electrophoresis with different gel concentrations, and Ferguson plot analysis indicated an Mr of 230,000 for the native enzyme. Although electrophoresis under denaturing conditions generally gave a subunit Mr of 57,000, electrophoresis of less than 1 microgram of protein yielded a faint doublet of Mr 57,000 and 58,000. Thus, the enzyme appears to be a tetramer with four very similar subunits. The enzyme bound to concanavalin A-Sepharose 4B only when it was kept in contact with the lectin for 16 h. Endoglycosidase H treatment resulted in loss of its binding to the lectin, without leading to a detectable change in the size of the enzyme subunit. On electrophoretic gels, the enzyme gave a faint positive stain with periodic acid-Schiff's base. The enzyme contained about 0.9% hexose by direct analysis. It did not bind to affinity resins specific for neuraminic acid, galactose, or N-acetylglucosamine. All these studies suggest that the enzyme is a glycoprotein containing only one or two clusters of high mannose oligosaccharide. Mannosidase IA is active toward oligosaccharides containing alpha 1,2-linked mannosyl residues. [3H]Man9GlcNAc, [3H] Man8GlcNAc, [3H]Man7GlcNAc, and [3H]Man6GlcNAc are good substrates. Man9GlcNAc, the best substrate, yields Man8, Man7, and Man6 derivatives with structures suggesting that the sequence of release of mannose residues is rather specific. Immunoprecipitation studies using polyclonal antibody (IgG) prepared against homogeneous mannosidase IA cross-reacted with mannosidase IB, a result suggesting that these two enzymes share antigenic determinants. However, no cross-reactivity was observed with rat liver cytosolic and lysosomal alpha-D-mannosidases or with Golgi mannosidase II.     

Developmental regulation of processing alpha-mannosidases and "intersecting" N-acetylglucosaminyltransferase in Dictyostelium discoideum.

We have identified three developmentally regulated oligosaccharide-processing enzyme activities in Dictyostelium discoideum. Two different alpha-mannosidase activities present at extremely low levels in vegetative cells are expressed during development. The first of these activities (MI) rises sharply from 6 to 12 h of development whereas the second activity (MII) rises sharply from 12 to 18 h of development. MI acts on Man9GlcNAc, which it can degrade to Man5GlcNAc but is inactive toward p-nitrophenyl-alpha-D-mannoside (pnpMan). MII acts on pnpMan but not Man9GlcNAc. These activities are distinct from each other and from lysosomal alpha-mannosidase activity as demonstrated by pH optima, substrate specificity, sensitivity to inhibitors and divalent cations, developmental profiles, and solubility. The characteristics of these developmentally regulated alpha-mannosidase activities are similar to those of Golgi alpha-mannosidases I and II from higher eucaryotes, and they appear to catalyze the in vivo formation of processed asparagine-linked oligosaccharides by developed cells. In addition, developed cells have very low levels of a soluble alpha-mannosidase activity, which is the predominant activity in vegetative cells. This soluble vegetative alpha-mannosidase activity has properties that are reminiscent of the endoplasmic reticulum alpha-mannosidase from rat liver. The intersecting N-acetylglucosaminyltransferase activity that we have described recently in vegetative cells of D. discoideum (Sharkey, D. J., and Kornfeld, R. (1989) J. Biol. Chem. 264, 10411-10419) has a developmental profile that is distinct from that of either of the alpha-mannosidase activities. It has maximum activity at 6 h of development and decreases sharply to its minimum level by 12 h of development. The changes that occur in the levels of these three processing enzymes with development correlate well with the different arrays of asparagine-linked oligosaccharides found in early and late stages of development (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18485-18497).     

Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2

It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)n (GlcNAc)1. No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-beta-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series--series 1: oligosaccharides processed from Manalpha1-2Manalpha1-6 (Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3) Manbeta1-4GlcNAc (M9A') and Manalpha1-2Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M8A') by digestion with cytosolic alpha-mannosidase; series 2: oligosaccharides processed with Golgi alpha-mannosidases in addition to endoplasmic reticulum (ER) and cytosolic alpha-mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic alpha-mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic alpha-mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic alpha-mannosidase, and Manalpha1-6(Manalpha1- 2Manalpha1-3)Manbeta1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic alpha-mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi alpha-mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc1M5B' were maintained at relatively high concentrations in the cytosol.     

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.