Publications

• Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.

  Avezov E., Frenkel Z., Ehrlich M., Herscovics A., Lederkremer G.Z.

  We had previously shown that endoplasmic reticulum (ER)-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of three to four mannose residues from the N-linked oligosaccharide Man(9)GlcNAc(2). A possible candidate for this activity, ER mannosidase I (ERManI), accele ... >> More

  We had previously shown that endoplasmic reticulum (ER)-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of three to four mannose residues from the N-linked oligosaccharide Man(9)GlcNAc(2). A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to four alpha1,2-linked mannose residues. Using small interfering RNA knockdown of ERManI, we show that this enzyme is required for trimming to Man(5-6)GlcNAc(2) and for ERAD in cells in vivo, leading to the accumulation of Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2) on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI is strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knockdown prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation. << Less

  Mol. Biol. Cell 19:216-225(2008) [PubMed] [EuropePMC]

• Identification, expression, and characterization of a cDNA encoding human endoplasmic reticulum mannosidase I, the enzyme that catalyzes the first mannose trimming step in mammalian Asn-linked oligosaccharide biosynthesis.

  Gonzalez D.S., Karaveg K., Vandersall-Nairn A.S., Lal A., Moremen K.W.

  We have isolated a full-length cDNA clone encoding a human alpha1, 2-mannosidase that catalyzes the first mannose trimming step in the processing of mammalian Asn-linked oligosaccharides. This enzyme has been proposed to regulate the timing of quality control glycoprotein degradation in the endopl ... >> More

  We have isolated a full-length cDNA clone encoding a human alpha1, 2-mannosidase that catalyzes the first mannose trimming step in the processing of mammalian Asn-linked oligosaccharides. This enzyme has been proposed to regulate the timing of quality control glycoprotein degradation in the endoplasmic reticulum (ER) of eukaryotic cells. Human expressed sequence tag clones were identified by sequence similarity to mammalian and yeast oligosaccharide-processing mannosidases, and the full-length coding region of the putative mannosidase homolog was isolated by a combination of 5'-rapid amplification of cDNA ends and direct polymerase chain reaction from human placental cDNA. The open reading frame predicted a 663-amino acid type II transmembrane polypeptide with a short cytoplasmic tail (47 amino acids), a single transmembrane domain (22 amino acids), and a large COOH-terminal catalytic domain (594 amino acids). Northern blots detected a transcript of approximately 2.8 kilobase pairs that was ubiquitously expressed in human tissues. Expression of an epitope-tagged full-length form of the human mannosidase homolog in normal rat kidney cells resulted in an ER pattern of localization. When a recombinant protein, consisting of protein A fused to the COOH-terminal luminal domain of the human mannosidase homolog, was expressed in COS cells, the fusion protein was found to cleave only a single alpha1,2-mannose residue from Man(9)GlcNAc(2) to produce a unique Man(8)GlcNAc(2) isomer (Man8B). The mannose cleavage reaction required divalent cations as indicated by inhibition with EDTA or EGTA and reversal of the inhibition by the addition of Ca(2+). The enzyme was also sensitive to inhibition by deoxymannojirimycin and kifunensine, but not swainsonine. The results on the localization, substrate specificity, and inhibitor profiles indicate that the cDNA reported here encodes an enzyme previously designated ER mannosidase I. Enzyme reactions using a combination of human ER mannosidase I and recombinant Golgi mannosidase IA indicated that that these two enzymes are complementary in their cleavage of Man(9)GlcNAc(2) oligosaccharides to Man(5)GlcNAc(2). << Less

  J. Biol. Chem. 274:21375-21386(1999) [PubMed] [EuropePMC]

• The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap MS.

  Prien J.M., Ashline D.J., Lapadula A.J., Zhang H., Reinhold V.N.

  Thirteen high mannose isomers have been structurally characterized within three glycomers, Man(5)GlcNAc(2), Man(7)GlcNAc(2), and Man(8)GlcNAc(2) released from bovine ribonuclease B, six previously unreported. The study was carried out with a single ion trap instrument involving no chromatography. ... >> More

  Thirteen high mannose isomers have been structurally characterized within three glycomers, Man(5)GlcNAc(2), Man(7)GlcNAc(2), and Man(8)GlcNAc(2) released from bovine ribonuclease B, six previously unreported. The study was carried out with a single ion trap instrument involving no chromatography. Three previously characterized isomers from Man(7) and Man(8) (three each) have been identified plus one unreported Man(7) isomer. Incomplete alpha-glucosidase activity on the Man(6) and Man(7) glycoproteins appears to account for two additional isomeric structures. The preeminence of ion traps for detail analysis was further demonstrated by resolving three new isomers within the Man(5) glycomer summing to the six previously unreported structures in this glycoprotein. All reported structures represent a distribution of Golgi processing remnants that fall within the Man(9)GlcNAc(2) footprint. Topologies were defined by ion compositions along a disassembly pathway while linkage and branching were aided by spectral identity in a small oligomer fragment library. Isomers from this glycoprotein appear to represent a distribution of Golgi processing remnants, and an alphanumeric classification scheme has been devised to identify all products. Although numerous analytical strategies have been introduced to identify selected components of structure, it has been the continued focus of this and previous reports to only build upon protocols that can be integrated into a high throughput strategy consistent with automation. Duplication of these and results from comparable standards could bring an important analytical focus to carbohydrate sequencing that is greatly lacking. << Less

  J Am Soc Mass Spectrom 20:539-556(2009) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• The specificity of the yeast and human class I ER alpha 1,2-mannosidases involved in ER quality control is not as strict previously reported.

  Herscovics A., Romero P.A., Tremblay L.O.

  Glycobiology 12:14G-15G(2002) [PubMed] [EuropePMC]

• Glycoprotein biosynthesis in Saccharomyces cerevisiae. Purification of the alpha-mannosidase which removes one specific mannose residue from Man9GlcNAc.

  Jelinek-Kelly S., Herscovics A.

  A soluble form of the specific alpha-mannosidase from Saccharomyces cerevisiae, which catalyzes the following reaction, was purified at least 100,000-fold by conventional chromatography procedures: (Formula: see text). The purified enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel elect ... >> More

  A soluble form of the specific alpha-mannosidase from Saccharomyces cerevisiae, which catalyzes the following reaction, was purified at least 100,000-fold by conventional chromatography procedures: (Formula: see text). The purified enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of about 60 kDa in the absence of reducing agent, and as two bands of about 44.5 kDa and 22.5 kDa in the presence of reducing agent. The apparent molecular weight of the soluble enzyme is about 75,000 by gel filtration on Sephacryl S-200. The specific alpha-mannosidase does not require the addition of divalent cation for activity, but it is inhibited by Tris, EDTA, Mn2+, Co2+, Zn2+, and Mg2+. The inhibition caused by EDTA can be reversed completely by Ca2+ and partially by Mg2+, but not by other divalent cations. The soluble alpha-mannosidase arises from a larger hydrophobic form of the enzyme which is found in the detergent phase during partition in Triton X-114. The formation of the soluble enzyme, which is recovered in the aqueous phase during partition in Triton X-114, is time- and temperature-dependent and is prevented by pepstatin, but not by other protease inhibitors. These results indicate that the purified soluble alpha-mannosidase represents the catalytically active domain of the enzyme which has been proteolytically released from its membrane-bound form. << Less

  J Biol Chem 263:14757-14763(1988) [PubMed] [EuropePMC]

• Class I alpha-mannosidases are required for N-glycan processing and root development in Arabidopsis thaliana.

  Liebminger E., Huttner S., Vavra U., Fischl R., Schoberer J., Grass J., Blaukopf C., Seifert G.J., Altmann F., Mach L., Strasser R.

  In eukaryotes, class I alpha-mannosidases are involved in early N-glycan processing reactions and in N-glycan-dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type alpha-mannosidase I (MNS3) and the two Golgi-alph ... >> More

  In eukaryotes, class I alpha-mannosidases are involved in early N-glycan processing reactions and in N-glycan-dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type alpha-mannosidase I (MNS3) and the two Golgi-alpha-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected alpha1,2-mannose residue from Man(9)GlcNAc(2), MNS1/2 readily removed three alpha1,2-mannose residues from Man(8)GlcNAc(2). Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man(8)GlcNAc(2) accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man(9)GlcNAc(2), demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I alpha-mannosidases in wild-type seedlings resulted in a similar root phenotype. These findings show that class I alpha-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis. << Less

  Plant Cell 21:3850-3867(2009) [PubMed] [EuropePMC]

• Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Man9 processing alpha-mannosidase.

  Ziegler F.D., Trimble R.B.

  Saccharomyces cerevisiae Man9-alpha-mannosidase, responsible for trimming Man9GlcNAc2 in the endoplasmic reticulum to Man8GlcNAc2, the substrate for oligosaccharide elongation, has been purified to homogeneity from stabilized microsomal membranes without employing autolytic digestion. The activity ... >> More

  Saccharomyces cerevisiae Man9-alpha-mannosidase, responsible for trimming Man9GlcNAc2 in the endoplasmic reticulum to Man8GlcNAc2, the substrate for oligosaccharide elongation, has been purified to homogeneity from stabilized microsomal membranes without employing autolytic digestion. The activity was solubilized by the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulphonate (CHAPS), whose presence was necessary for maximal activity. Purification included Q-Sepharose ion-exchange chromatography, preparative isoelectric focusing and HPLC gel filtration on TSK 3000 matrix. Overall purification from post-nuclear supernatants was estimated to be 110,000-fold with a 50% recovery of activity. The purified enzyme hydrolysed Man9GlcNAc1,2 from thyroglobulin or oligosaccharide-lipid, but not invertase Man9GlcNAc, Man1 alpha 2Man1 alpha OCH3 or p-nitrophenyl-alpha-D-mannopyranoside. Conversion of thyroglobulin Man9GlcNAc to Man8GlcNAc was linear with time and enzyme concentration, with an apparent Km of 0.2 mM and a specific activity of 220 IU/mg. Glc3Man9GlcNAc2 from oligosaccharide-lipid was as good a substrate as Man9GlcNAc, but the lipid-linked Man7GlcNAc2 isomer was hydrolysed at only 10% of this rate. Hydrolysis of defined isomers of IgM and bovine thyroglobulin Man6,7,8GlcNAc indicated that, for maximal alpha 1,2-mannosidase activity, only the alpha 1,2-linked terminal mannoses on the alpha 3 branch of the Man9GlcNAc precursor were dispensable. Isomers lacking the terminal alpha 1,2-linked mannose on the alpha 6 branch were hydrolysed at only approximately 10% of the maximal rate. The enzyme exhibited a pI of 5.3 and a pH optimum at 6.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents gave a single sharp band at 66 kDa, while in the presence of beta-mercaptoethanol equimolar amounts of two peptides, one of 44 kDa and one of 23 kDa, were obtained. Sizing on Sephacryl SF300, Superose 12 and TSK 3000 provided a holoenzyme mol. wt of 60-68 kDa, indicating that the isolated active form of the Man9-alpha-mannosidase was composed of one each of the sulphydryl-bonded dissimilar peptides. The enzyme bound to concanavalin A (ConA)-Sepharose and was eluted with alpha-methylmannoside, indicating the presence of high-mannose oligosaccharides. The Man9-alpha-mannosidase required low levels of Ca2+, which could be removed by EGTA. Activity was restored by Ca2+ or Zn2+, but not by Mg2+ or Mn2+. << Less

  Glycobiology 1:605-614(1991) [PubMed] [EuropePMC]