Purification and characterization of a co(II)-sensitive alpha-mannosidase from Ginkgo biloba seeds

An alpha-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified alpha-mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo alpha-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro-Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The alpha-mannosidase activity for Man(5)GlcNAc(1) was enhanced by the addition of Co(2+), but the addition of Zn(2+), Ca(2+), or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man(6)GlcNAc(1) was significantly faster than that for pyridylaminated Man(6)GlcNAc(2), suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an alpha-mannosidase, which is activated by Co(2+) and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.     

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.     

Structural heterogeneity in the Man8-13GlcNAc oligosaccharides from log-phase Saccharomyces yeast: a one- and two-dimensional 1H NMR spectroscopic study.

Previously, Man8-14GlcNAc oligosaccharides were isolated from highly purified Saccharomyces cerevisiae invertase and shown by one-dimensional 1H NMR spectroscopy and alpha 1,2-linkage-specific mannosidase digestion to constitute a homologous series of nearly homogeneous compounds, which appeared to define the intermediates in oligosaccharide core synthesis in yeast (Trimble, R.B. and Atkinson, P.H. (1986) J. Biol. Chem., 261, 9815-9824). To evaluate whether invertase oligosaccharides reflected global core processing of yeast glycans, the soluble glycoprotein pool of disrupted log-phase cells was digested with endo-beta-N-acetyl-glucosaminidase H and Man8-13GlcNAc were isolated by Bio-Gel P-4 chromatography. Although analysis of each size class by one-dimensional 400 MHz and two-dimensional 500 MHz phase-sensitive COSY 1H NMR spectroscopy revealed considerable structural heterogeneity in all but Man8GlcNAc, the major positional isomer in Man9-13GlcNAc (approximately 50%) was identical to that previously elucidated on invertase. The heterogeneity resided in four families of oligosaccharides: (i) Glc3Man9GlcNAc----Man8 GlcNAc trimming intermediates; (ii) alpha-mannosidase degradation products of the principal isomers; (iii) mannan elongation intermediates; (iv) core structures with the alpha 1,2-linked mannose usually removed by the processing alpha-mannosidase. The potential for the vacuolar alpha-mannosidase (AMS1 gene product) to generate heterogeneity in vitro was confirmed by isolating oligosaccharides from AMS1 and ams1 yeast strains in the presence of a Man13GlcNAc[3H]-ol marker (where GlcNAc[3H]-ol is N-acetylglucosamin [1-3H]itol). Degradation of the Man13GlcNAc[3H]-ol to Man9-12GlcNAc[3H]-ol occurred in the former, but not in the latter. A role for the vacuolar alpha-mannosidase in generating at least some heterogeneity in vivo was inferred from the 1H NMR spectrum of the AMS1 Man11GlcNAc pool, which showed more structural isomerism than seen in the spectrum of a comparable ams1 Man11GlcNAc preparation. Thus, the principal biosynthetic pathway of inner core mannan in Saccharomyces is defined by the Man8-13GlcNAc oligosaccharides found on external invertase, while structural heterogeneity in these size classes results from precursor processing in the endoplasmic reticulum, core extension in the Golgi and metabolic degradation in the vacuole.     

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).     

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.     

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.     

Studies on the substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct by using sugar chains from glycoproteins.

The substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co2+ hydrolyzed the Man alpha 1-3 and Man alpha 1-6 bonds from the non-reducing termini of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (M5A), but hardly hydrolyzed the Man alpha 1-2 bonds of Man9GlcNAc2. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Man beta 1-4GlcNAc2. For the substrates with the GlcNAc structure at their reducing ends (Man5GlcNAc, Man6GlcNAc and Man9GlcNAc), the hydrolysis rate was remarkably increased: Man5GlcNAc was hydrolyzed 16 times faster than M5A, and Man2GlcNAc 40 times faster than Man9GlcNAc2. The enzyme did not hydrolyze Man alpha 1-2 residue(s) linked to Man alpha 1-3Man beta 1-4GlcNAc. The end products were as follows: [formula; see text] These results suggest that oligomannose-type sugar chains with the GlcNAc structure at their reducing ends seem to be native substrates for neutral alpha-mannosidase and the enzyme seems to hydrolyze endo-beta-N-acetylgucosaminidase digests of oligomannose-type sugar chains in the cytosol.     

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).     

Topology of ER processing alpha-mannosidase of Saccharomyces cerevisiae.

The yeast specific alpha-mannosidase which converts Man9GlcNAc to a single isomer of Man8GlcNAc is involved in N-linked oligosaccharide processing in the endoplasmic reticulum (ER). Sequence analysis of the structural gene for this enzyme suggested that it is a type II transmembrane protein (Camirand et al., 1991). To firmly establish its membrane topology, the gene was transcribed in vitro and translation was performed in a reticulocyte lysate with and without dog pancreas microsomal membranes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of [35S]methionine-labelled products showed that the largest band formed corresponded in size to the 63 kDa peptide expected from the alpha-mannosidase gene product. It was transformed into a 4 kDa larger endoglycosidase H-sensitive band in the presence of microsomal membranes. This glycosylated translation product was completely protected from proteinase K digestion in the absence of detergent. These results demonstrate that the yeast ER alpha-mannosidase is a type II membrane protein, like Golgi enzymes involved in N-linked glycosylation.     

Production in yeast of alpha-galactosidase A,a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease

A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human alpha-galactosidase A (alpha-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant alpha-GalA contained both neutral (Man(8)GlcNAc(2)) and acidic ([Man-P](1-2)Man(8)GlcNAc(2)) sugar chains. Because an efficient incorporation of alpha-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the alpha-GalA by a newly isolated bacterial alpha-mannosidase. Treatment of the alpha-GalA with the alpha-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose. The treated alpha-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease. The uptake was three to four times higher than that of the nontreated alpha-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated alpha-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days. This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease.     

Post-translational protein modification in the endoplasmic reticulum. Demonstration of fatty acylase and deoxymannojirimycin-sensitive alpha-mannosidase activities

We have previously described a hybrid protein, GHHA, that contains a fragment of the influenza hemagglutinin joined to the C terminus of a nearly complete rat growth hormone (Rizzolo, L.J., Finidori, J., Gonzalez, A., Arpin, M., Ivanov, I.E., Adesnik, M., and Sabatini, D.D. (1985) J. Cell Biol. 101, 1351-1362). GHHA was transported from the rough endoplasmic reticulum (ER) to a smooth cisterna, continuous with the rough ER, but proximal to the Golgi apparatus. We have now labeled GHHA with [3H]palmitate, demonstrating that fatty acylation can occur in the ER. As expected for a thioester linkage, the label was released from GHHA by hydroxylamine and identified as palmitic acid by thin-layer chromatography. In a second study, we analyzed the structure of the N-linked carbohydrate chain of GHHA. The N-linked oligosaccharides, all high-mannose type, were released by endoglycosidase H and size-fractionated by high pressure liquid chromatography. The predominant structures were Glc1Man8GlcNAc and Man8GlcNAc, indicating that only 2 or 3 glucose and 1 mannose residues were removed from the original Glc3Man9GlcNAc2. Determination of the structure by acetolysis fragmentation indicated that a single Man8GlcNAc isomer was formed by a deoxymannojirimycin-sensitive alpha-mannosidase. This contrasts with a previously characterized ER alpha-mannosidase (Bischoff, J., Liscum, L., and Kornfeld, R. (1986) J. Biol. Chem. 261, 4766-4774) that generates the same isomer, but is deoxymannojirimycin-resistant. These data suggest the possibility that different enzymes are partitioned within the ER.     

Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene.

A gene encoding an alpha-1,2-mannosyltransferase from Saccharomyces cerevisiae was cloned and sequenced. The alpha-1,2-mannosyltransferase which utilizes alpha-methylmannoside as acceptor of mannose from GDP-mannose was purified. The enzyme activity was shown to correspond to a 41 kDa protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This protein band was digested in situ with trypsin and amino acid sequence information was obtained from four peptides. Degenerate oligonucleotide primers corresponding to the amino acid sequences were designed and used for polymerase chain reactions on yeast genomic DNA. A specific reaction product was used to screen a genomic library of S.cerevisiae. A fragment of approximately 5.7 kb was isolated, of which a 2.9 kb fragment was sequenced. It contained a 1329 base pair open reading frame encoding the peptide sequences of the purified alpha-1,2-mannosyltransferase. The gene, designated MNT1, is located on the right arm of chromosome 4. It encodes a 442 amino acid polypeptide with a calculated mol. wt of 51.4 kDa. The corresponding mRNA has a length of approximately 1.6 kb. Overexpression of the MNT1 gene increased this alpha-1,2-mannosyltransferase activity approximately 2.5-fold. The protein was shown to be modified with N-linked carbohydrate chains and its sequence contains one N-glycosylation site. The enzyme contains a putative membrane-spanning domain near its N-terminus and its topology is thus similar to that of mammalian Golgi glycosyltransferases. This is the first report of the cloning and sequencing of a yeast Golgi mannosyltransferase.     

A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.     

Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum alpha-mannosidase

We have isolated a cDNA encoding an endoplasmic reticulum alpha-mannosidase, an asparagine-linked oligosaccharide processing enzyme, from a rat liver lambda gt11 library. Two degenerate oligonucleotides, based on amino acid sequence data from the purified enzyme, were used as primers in the polymerase chain reaction with liver cDNA as a template to generate an unambiguous cDNA probe. The cDNA fragment (524 base pair) obtained was then used to isolate cDNA clones by hybridization. We isolated two overlapping clones which were used to construct a full-length cDNA of 3392 base pairs. A single open reading frame of 1040 amino acids encodes a protein with a molecular mass of 116 kilodaltons containing the six known peptide sequences. The deduced amino acid sequence revealed no classical signal sequence or membrane-spanning domain. The alpha-mannosidase encoding cDNA can be expressed transiently in COS cells using the mammalian expression vector pXM, causing a 400-fold increase in alpha-mannosidase activity as well as a dramatic increase in immunoreactive polypeptide. The rat liver endoplasmic reticulum alpha-mannosidase bears striking homology to the vacuolar alpha-mannosidase from Saccharomyces cerevisiae.     

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.     

Characterization of Schizosaccharomyces pombe ER alpha-mannosidase: a reevaluation of the role of the enzyme on ER-associated degradation

It has been postulated that creation of Man8GlcNAc2 isomer B (M8B) by endoplasmic reticulum (ER) alpha-mannosidase I constitutes a signal for driving irreparably misfolded glycoproteins to proteasomal degradation. Contrary to a previous report, we were able to detect in vivo (but not in vitro) an extremely feeble ER alpha-mannosidase activity in Schizosaccharomyces pombe. The enzyme yielded M8B on degradation of Man9GlcNAc2 and was inhibited by kifunensin. Live S. pombe cells showed an extremely limited capacity to demannosylate Man9GlcNAc2 present in misfolded glycoproteins even after a long residence in the ER. In addition, no preferential degradation of M8B-bearing species was detected. Nevertheless, disruption of the alpha-mannosidase encoding gene almost totally prevented degradation of a misfolded glycoprotein. This and other conflicting reports may be best explained by assuming that the role of ER mannosidase on glycoprotein degradation is independent of its enzymatic activity. The enzyme, behaving as a lectin binding polymannose glycans of varied structures, would belong together with its enzymatically inactive homologue Htm1p/Mnl1p/EDEM, to a transport chain responsible for delivering irreparably misfolded glycoproteins to proteasomes. Kifunensin and 1-deoxymannojirimycin, being mannose homologues, would behave as inhibitors of the ER mannosidase or/and Htm1p/Mnl1p/EDEM putative lectin properties.     

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.     

The N-glycans of jack bean alpha-mannosidase. Structure, topology and function.

The acid hydrolase alpha-mannosidase, which accumulates in plant vacuoles and probably is involved in the catabolism and turnover of N-linked glycoproteins, is itself a glycoprotein with at least one high-mannose-type and one complex-type N-glycan. The puzzling finding that alpha-mannosidase stably carries its own substrate suggests that the N-glycans have unique topologies, and important functions in protein folding, oligomerization or enzyme activity. As a first step towards the elucidation of this enigma, we purified the N-glycans of jack bean alpha-mannosidase and determined their structures by sugar composition analysis, mass spectrometry and 1H-NMR. The structures of two N-glycans were identified in an approximate ratio of one-to-one: a glucose-containing high-mannose-type glycan (Glc1Man9GlcNAc2) and a small xylose- and fucose-containing complex-type glycan (Xyl1Man1Fuc1GlcNAc2). Isolation and sequencing of glycopeptides strongly suggests that one high-mannose-type and one complex-type glycan are linked to specific glycosylation sites of the large alpha-mannosidase subunit. The high-mannose-type glycan, which is a good substrate of the endoglycosidase (endo-H), can only be removed from the enzyme after denaturation and cleavage of disulfide bonds by a reducing agent, suggesting that this glycan is buried within the folded polypeptide and, thus, protected from its hydrolytic activity. Denaturation and reduction of the native enzyme led to a marked decrease in alpha-mannosidase activity. However, the activity could largely be recovered by renaturation in an appropriate renaturation buffer. In contrast, recovery of alpha-mannosidase activity failed when the high-mannose-type glycan was removed by endo-H prior to renaturation, indicating that this glycan appears to be important for enzyme activity.     

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

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