Publications

• Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human alpha-1,2-mannosidase gene involved in N-glycan maturation.

  Tremblay L.O., Campbell-Dyke N., Herscovics A.

  Class I alpha1,2-mannosidases play an essential role in the elaboration of complex and hybrid N -glycans in mammalian cells. Using degenerate primers based on amino acid sequences conserved in all members of this enzyme family for RT-PCR, two distinct PCR products were obtained from placenta and l ... >> More

  Class I alpha1,2-mannosidases play an essential role in the elaboration of complex and hybrid N -glycans in mammalian cells. Using degenerate primers based on amino acid sequences conserved in all members of this enzyme family for RT-PCR, two distinct PCR products were obtained from placenta and lymphocyte cDNAs. One of these was related to the previously cloned human and murine alpha1, 2-mannosidase IA whereas the other was very similar to murine alpha1, 2-mannosidase IB. Northern blot analysis of human tissues with these two alpha1,2-mannosidase probes revealed very different patterns of tissue-specific expression. Similar tissue-specific expression of alpha1,2-mannosidase IA and IB was also observed on Northern blots of adult mouse tissues. A human placenta cDNA library was screened and PCR of brain, placenta, and lymphocyte cDNAs was performed in order to isolate the human alpha1,2-mannosidase IB cDNA. This cDNA encodes a type II membrane protein of 73 kDa that is 94% identical in amino acid sequence to the murine alpha1,2-mannosidase IB (Herscovics et al., 1994, J. Biol. Chem., 269, 9864-9871). A truncated soluble form of the human alpha1,2-mannosidase IB lacking its N -terminal transmembrane domain was expressed as a secreted protein in Pichia pastoris . The recombinant enzyme was incubated with [3H]Man9GlcNAc and [3H]Man8GlcNAc (isomer B), and high performance liquid chromatography analysis of the products showed that [3H]Man9GlcNAc was readily converted to [3H]Man6GlcNAc and much more slowly to [3H]Man5GlcNAc, whereas [3H]Man8GlcNAc was rapidly trimmed to [3H]Man5GlcNAc. The human alpha1,2-mannosidase IB gene was isolated from a P1 human genomic library and shown to be at least 60 kb in size and to contain at least 13 exons. The gene was localized by fluorescence in situ hybridization to human chromosome 1p13, a region that undergoes many aberrations in various types of human cancers. These results show that there are at least two Class I alpha1,2-mannosidases in the human and murine genomes with very distinct transcriptional regulation in different tissues. << Less

  Glycobiology 8:585-595(1998) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Characterization of a cDNA encoding a novel human Golgi alpha 1,2-mannosidase involved in N-glycan biosynthesis.

  Tremblay L.O., Herscovics A.

  A human cDNA encoding a 70.9-kDa type II membrane protein with sequence similarity to class I alpha1,2-mannosidases was isolated. The enzymatic properties of the novel alpha1,2-mannosidase IC were studied by expressing its catalytic domain in Pichia pastoris as a secreted glycoprotein. alpha1,2-Ma ... >> More

  A human cDNA encoding a 70.9-kDa type II membrane protein with sequence similarity to class I alpha1,2-mannosidases was isolated. The enzymatic properties of the novel alpha1,2-mannosidase IC were studied by expressing its catalytic domain in Pichia pastoris as a secreted glycoprotein. alpha1,2-Mannosidase IC sequentially hydrolyzes the alpha1,2-linked mannose residues of [(3)H]mannose-labeled Man(9)GlcNAc to form [(3)H]Man(6)GlcNAc and a small amount of [(3)H]Man(5)GlcNAc. The enzyme requires calcium for activity and is inhibited by both 1-deoxymannojirimycin and kifunensine. The order of mannose removal was determined by separating oligosaccharide isomers formed from pyridylaminated Man(9)GlcNAc(2) by high performance liquid chromatography. The terminal alpha1,2-linked mannose residue from the middle branch is the last mannose removed by the enzyme. This residue is the mannose cleaved from Man(9)GlcNAc(2) by the endoplasmic reticulum alpha1, 2-mannosidase I to form Man(8)GlcNAc(2) isomer B. The order of mannose hydrolysis from either pyridylaminated Man(9)GlcNAc(2) or Man(8)GlcNAc(2) isomer B differs from that previously reported for mammalian Golgi alpha1,2-mannosidases IA and IB. The full-length alpha1,2-mannosidase IC was localized to the Golgi of MDBK and MDCK cells by indirect immunofluorescence. Northern blot analysis showed tissue-specific expression of a major transcript of 3.8 kilobase pairs. The expression pattern is different from that of human Golgi alpha1,2-mannosidases IA and IB. Therefore, the human genome contains at least three differentially regulated Golgi alpha1, 2-mannosidase genes encoding enzymes with similar, but not identical specificities. << Less

  J. Biol. Chem. 275:31655-31660(2000) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

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

  Lal A., Pang P., Kalelkar S., Romero P.A., Herscovics A., Moremen K.W.

  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, ... >> More

  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. << Less

  Glycobiology 8:981-995(1998) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• 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.

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

  Tabas I., Kornfeld S.

  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 ... >> More

  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. << Less

  J Biol Chem 254:11655-11663(1979) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• alpha-D-Mannosidases of rat liver Golgi membranes. Mannosidase II is the GlcNAcMAN5-cleaving enzyme in glycoprotein biosynthesis and mannosidases Ia and IB are the enzymes converting Man9 precursors to Man5 intermediates.

  Tulsiani D.R., Hubbard S.C., Robbins P.W., Touster O.

  Current evidence indicates that the trimming of mannosyl residues from intermediates in the biosynthesis of the N-linked oligosaccharides of glycoproteins occurs in the Golgi complex. We now present evidence that mannosidase II (Tulsiani, D. R. P., Opheim, D. J., and Touster, O. (1977) J. Biol Che ... >> More

  Current evidence indicates that the trimming of mannosyl residues from intermediates in the biosynthesis of the N-linked oligosaccharides of glycoproteins occurs in the Golgi complex. We now present evidence that mannosidase II (Tulsiani, D. R. P., Opheim, D. J., and Touster, O. (1977) J. Biol Chem. 252, 3227-3233) is the Golgi enzyme that converts GlcNAc Man5 species to GlcNAcMan3 species in completing the mannosyl trimming process required in the biosynthesis of complex type glycoproteins. GlcNAc([3H]Man)5GlcNAc-mannosidase and p-nitrophenyl alpha-D-mannosidase activities copurify throughout the preparative procedure and show the same properties. In addition to mannosidase IA (Tabas, I., and Kornfeld, S. (1979) J. Biol. Chem. 254, 11655-11663), a second alpha-1,2-mannosidase (mannosidase IB) can be prepared from Golgi membranes which is effective in converting Man9GlcNAc to Man5GlcNAc. The two alpha-1,2-mannosidases are very similar in catalytic properties, but they are also distinguishable by several criteria. Although these two enzymes have not been extensively purified, several lines of evidence lead to the tentative conclusion that they are distinct enzymes. They appear to be present in comparable activities in the Golgi membranes and together account for the alpha-1,2-mannosidase activity of these membranes. The particular role of each alpha-1,2-mannosidase remains to be determined. << Less

  J Biol Chem 257:3660-3668(1982) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Man9-mannosidase from human kidney is expressed in COS cells as a Golgi-resident type II transmembrane N-glycoprotein.

  Bieberich E., Bause E.

  Man9-mannosidase, an alpha 1,2-specific exo-enzyme involved in N-linked oligosaccharide processing, has been cloned recently from a human kidney cDNA library [Bause, E., Bieberich, E., Rolfs, A., Völker, C. & Schmidt, B. (1993) Eur. J. Biochem. 217, 533-540]. Transient expression in COS 1 cells of ... >> More

  Man9-mannosidase, an alpha 1,2-specific exo-enzyme involved in N-linked oligosaccharide processing, has been cloned recently from a human kidney cDNA library [Bause, E., Bieberich, E., Rolfs, A., Völker, C. & Schmidt, B. (1993) Eur. J. Biochem. 217, 533-540]. Transient expression in COS 1 cells of the enzyme resulted in a more than 20-fold increase of a catalytic activity cleaving specifically alpha 1,2-mannosidic linkages in [14C]Man9-GlcNAc2 or [14C]Man5-GlcNAc2. Man9-mannosidase is expressed as a N-glycoprotein with a molecular mass of 73 kDa. Its enzymic activity is metal ion dependent and inhibited strongly by 1-deoxymannojirimycin (50% at 100 microM). Proteolytic studies with the membrane-associated form of Man9-mannosidase support the view that the enzyme is a type II transmembrane protein as predicted from its cDNA sequence. Several lines of evidence suggest that Man9-mannosidase, as expressed, is N-glycosylated at one of three potential Asn-Xaa-Thr/Ser/Cys acceptor sites. Approximately 50% of the N-linked oligosaccharide chains are removed by endoglycosidase H treatment, whereas complete deglycosylation of the enzyme is observed, when transfected cells were cultured in the presence of the Golgi mannosidase II inhibitor swainsonine, indicating that the sugar moiety of Man9-mannosidase is processed partially by Golgi-resident enzymes. This observation is consistent with the results of indirect immunofluorescence studies, pointing to a localization of the Man9-mannosidase predominantly in the juxtanuclear Golgi region. This localization clearly differs from that of pig liver Man9-mannosidase which appears to be located in the endoplasmic reticulum and transient vesicles. << Less

  Eur J Biochem 233:644-649(1995) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.