Publications

• Intact alpha-1,2-endomannosidase is a typical type II membrane protein.

  Hamilton S.R., Li H., Wischnewski H., Prasad A., Kerley-Hamilton J.S., Mitchell T., Walling A.J., Davidson R.C., Wildt S., Gerngross T.U.

  Rat endomannosidase is a glycosidic enzyme that catalyzes the cleavage of di-, tri-, or tetrasaccharides (Glc(1-3)Man), from N-glycosylation intermediates with terminal glucose residues. To date it is the only characterized member of this class of endomannosidic enzymes. Although this protein has ... >> More

  Rat endomannosidase is a glycosidic enzyme that catalyzes the cleavage of di-, tri-, or tetrasaccharides (Glc(1-3)Man), from N-glycosylation intermediates with terminal glucose residues. To date it is the only characterized member of this class of endomannosidic enzymes. Although this protein has been demonstrated to localize to the Golgi lumenal membrane, the mechanism by which this occurs has not yet been determined. Using the rat endomannosidase sequence, we identified three homologs, one each in the human, mouse, and rat genomes. Alignment of the four encoded protein sequences demonstrated that the newly identified sequences are highly conserved but differed significantly at the N-terminus from the previously reported protein. In this study we have cloned two novel endomannosidase sequences from rat and human cDNA libraries, but were unable to amplify the open reading frame of the previously reported rat sequence. Analysis of the rat genome confirmed that the 59- and 39-termini of the previously reported sequence were in fact located on different chromosomes. This, in combination with our inability to amplify the previously reported sequence, indicated that the N-terminus of the rat endomannosidase sequence previously published was likely in error (a cloning artifact), and that the sequences reported in the current study encode the intact proteins. Furthermore, unlike the previous sequence, the three ORFs identified in this study encode proteins containing a single N-terminal transmembrane domain. Here we demonstrate that this region is responsible for Golgi localization and in doing so confirm that endomannosidase is a type II membrane protein, like the majority of other secretory pathway glycosylation enzymes. << Less

  Glycobiology 15:615-624(2005) [PubMed] [EuropePMC]

• Golgi endo-alpha-D-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme.

  Lubas W.A., Spiro R.G.

  An enzyme has been found in Triton-treated rat liver Golgi membranes which trims Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1-3Man. By removing a glucosylmannose disaccharide and yielding only one Man8GlcNAc isomer, this endo-alpha-D-mannosidase provides a processing route alternat ... >> More

  An enzyme has been found in Triton-treated rat liver Golgi membranes which trims Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1-3Man. By removing a glucosylmannose disaccharide and yielding only one Man8GlcNAc isomer, this endo-alpha-D-mannosidase provides a processing route alternative to the sequential actions of alpha-glucosidase II and alpha-mannosidase I. The endomannosidase was fully active in the presence of 1-deoxynojirimycin and EDTA which inhibited exoglycosidase release of glucose and mannose, respectively, and these agents were, therefore, included in the standard assay. The specific activity of the endomannosidase was found to be 69-fold greater in Golgi than in rough endoplasmic reticulum (RER) membranes, and Golgi-RER mixing experiments excluded the possibility that the low activity in the RER was the result of some inhibitor present in this fraction. The neutral pH optimum (approximately 7.0) of the enzyme was consistent with a role in N-linked oligosaccharide processing. The existence of an endo-alpha-D-mannosidase pathway for glucose removal could provide an explanation for the incomplete block in oligosaccharide processing which is observed in cells with inhibited or deficient alpha-glucosidase. << Less

  J Biol Chem 262:3775-3781(1987) [PubMed] [EuropePMC]

• Ligand affinity chromatographic purification of rat liver Golgi endomannosidase.

  Hiraizumi S., Spohr U., Spiro R.G.

  In order to achieve isolation of endo-alpha-D-mannosidase, a Golgi-located processing enzyme that accomplishes deglucosylation of glycoproteins with N-linked carbohydrate units by cleaving the linkage between the glucose-substituted mannose residue and the remainder of the oligosaccharide, we have ... >> More

  In order to achieve isolation of endo-alpha-D-mannosidase, a Golgi-located processing enzyme that accomplishes deglucosylation of glycoproteins with N-linked carbohydrate units by cleaving the linkage between the glucose-substituted mannose residue and the remainder of the oligosaccharide, we have prepared an affinity matrix (Glc alpha 1-->3Man-O-(CH2)8CONH-Affi-Gel 102) containing the derivative of the characteristic disaccharide product of this enzyme. Chromatography of a Triton extract of rat liver Golgi membranes on a column of this gel in the presence of castanospermine to prevent binding of alpha-glucosidases permitted a rapid purification of the endomannosidase (70,000-fold over the homogenate) with a 12% yield. This purified enzyme was free of other processing glycosidases and was completely inhibited by Glc alpha 1-->3(1-deoxy)mannojirimycin. Examination of the endomannosidase by SDS-polyacrylamide gel electrophoresis revealed a doublet (M(r) 60,000 and 56,000) with the bands being of approximately equal density. Gel permeation high performance liquid chromatography indicated that in its native form the enzyme has an oligomeric structure (M(r) approximately 560,000) consisting of eight to ten subunits. << Less

  J Biol Chem 269:4697-4700(1994) [PubMed] [EuropePMC]

• Asparagine-linked glycoprotein biosynthesis in rat brain: identification of glucosidase I, glucosidase II, and and endomannosidase (glucosyl mannosidase).

  Tulsiani D.R., Coleman V.D., Touster O.

  Previous studies from this laboratory provided evidence, largely based upon the presence of a novel alpha-D-mannosidase, suggesting that the biosynthesis of N-linked glycoproteins may be different in brain as compared to other tissues (Tulsiani, D. R. P., and Touster, O. (1985) J. Biol. Chem. 260, ... >> More

  Previous studies from this laboratory provided evidence, largely based upon the presence of a novel alpha-D-mannosidase, suggesting that the biosynthesis of N-linked glycoproteins may be different in brain as compared to other tissues (Tulsiani, D. R. P., and Touster, O. (1985) J. Biol. Chem. 260, 13,081-13,087). In the present report we describe studies on the enzymes involved in early processing reactions. These studies indicate that the brain, like other tissues, contains glucosidases I and II. The two glucosidases were separated as distinct activities with some overlapping by chromatography on a DE-52 column. The differential inhibition studies and substrate specificity studies support our conclusion that, as in other tissues, rat brain glucosidase I cleaves alpha 1,2-linked terminal glucosyl residues, whereas glucosidase II prefers alpha 1,3-linked glucosyl residues. In addition to these two processing glucosidases, we have characterized an endo enzyme (glucosyl mannosidase) in rat brain. The endomannosidase cleaves a disaccharide (glucosyl alpha 1,3-mannose) from monoglucosylated oligosaccharides (GlcMan7-9GlcNAc). Little or no activity was observed when di- or triglucosylated oligosaccharide was used as a substrate. The pH optimum of the glucosyl mannosidase is 6.2-6.8. The enzyme appears to be an intrinsic microsomal membrane component, since washing of the microsomal membranes with salt solution did not release the enzyme in soluble form. A mixture of Triton X-100 and sodium deoxycholate is required for complete solubilization of the enzyme. The solubilized enzyme is eluted from a Bio-Gel A-1.5m column as a single peak with an apparent molecular weight of 380,000. << Less

  Arch Biochem Biophys 277:114-121(1990) [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.

• Molecular cloning and expression of rat liver endo-alpha-mannosidase, an N-linked oligosaccharide processing enzyme.

  Spiro M.J., Bhoyroo V.D., Spiro R.G.

  A clone containing the open reading frame of endo-alpha-D-mannosidase, an enzyme involved in early N-linked oligosaccharide processing, has been isolated from a rat liver lambdagt11 cDNA library. This was accomplished by a strategy that involved purification of the endomannosidase from rat liver G ... >> More

  A clone containing the open reading frame of endo-alpha-D-mannosidase, an enzyme involved in early N-linked oligosaccharide processing, has been isolated from a rat liver lambdagt11 cDNA library. This was accomplished by a strategy that involved purification of the endomannosidase from rat liver Golgi by ligand affinity chromatography (Hiraizumi, S., Spohr, U., and Spiro, R. G. (1994) J. Biol. Chem. 269, 4697-4700) and preparative electrophoresis, followed by sequence determinations of tryptic peptides. Using degenerate primers based on these sequences, the polymerase chain reaction with rat liver cDNA as a template yielded a 470-base pair product suitable for library screening as well as Northern blot hybridization. EcoRI digestion of the purified lambda DNA released a 5.4-kilobase fragment that was amplified in Bluescript II SK(-) vector. Sequence analysis indicated that the deduced open reading frame of the endomannosidase extended from nucleotides 89 to 1441, encoding a protein of 451 amino acids and corresponding to a molecular mass of 52 kDa. Data base searches revealed no homology with any other known protein. When a vector coding for this protein fused to an NH2-terminal peptide containing a polyhistidine region was introduced into Escherichia coli, high levels of the enzyme were expressed upon induction with isopropyl-beta-D-thiogalactoside. Purification of the endomannosidase to electrophoretic homogeneity from E. coli lysates was accomplished by Ni2+-chelate and Glcalpha1-->3Man-O-(CH2)8CONH-Affi-Gel ligand chromatographies. Polyclonal antibodies raised against this protein reacted with Golgi endomannosidase. By both immunoblotting and silver staining, the purified E. coli-expressed enzyme was approximately 8 kDa smaller than anticipated from the open reading frame; timed induction studies indicated that this was due to scission of the enzyme's COOH-terminal end by host cell proteases. All rat tissues examined demonstrated mRNA levels (4.9-kilobase message) for the endomannosidase that correlated well with their enzyme activity. << Less

  J. Biol. Chem. 272:29356-29363(1997) [PubMed] [EuropePMC]

• Human endo-alpha1,2-mannosidase is a Golgi-resident type II membrane protein.

  Hardt B., Voelker C., Mundt S., Salska-Navarro M., Hauptmann M., Bause E.

  The cDNA for human endo-alpha1,2-mannosidase was reconstructed using two independent EST-clones and its properties characterized. The 2837 bp cDNA construct contained a 1389 bp open reading frame (ORF) encoding for 462 amino acids and an approximately 53.6 kDa protein, respectively. Hydrophobicity ... >> More

  The cDNA for human endo-alpha1,2-mannosidase was reconstructed using two independent EST-clones and its properties characterized. The 2837 bp cDNA construct contained a 1389 bp open reading frame (ORF) encoding for 462 amino acids and an approximately 53.6 kDa protein, respectively. Hydrophobicity analysis of this amino acid sequence, as well as proteolytic degradation studies, indicate that the enzyme is a type II protein, anchored in the membrane via a 19 amino-acid long apolar sequence close to the N-terminus. Human endo-alpha1,2-mannosidase displays a high degree of sequence identity with the catalytic domain of the homologous rat liver endo-enzyme, but differs substantially in the N-terminal peptide region, which includes the transmembrane domain. No sequence similarity exists with other processing alpha-glycosidases. Based on sequence information provided by the 2837 bp construct, the cDNA consisting of the complete 1389 bp ORF was amplified by RT-PCR using human fibroblast RNA. Incubation of E. coli lysates with this cDNA, previously modified for boost translation by codon optimization, resulted in the synthesis of an approximately 52 kDa protein which degraded [(14)C]Glc(3)-Man(9)-GlcNAc(2) efficiently, indicating that the catalytic domain of the enzyme folds correctly under cell-free conditions. Transfection of the endo-alpha1,2-mannosidase wild-type cDNA into COS 1 cells resulted in a moderate (approximately 1.5-fold) but reproducible increase of activity compared with control cells, whereas >18-fold increase in activity was measured after expression of a chimera containing green-fluorescent-protein (GFP) attached to the N-terminus of the endo-alpha1,2-mannosidase polypeptide. This, together with the observation that GFP-endo-alpha1,2-mannosidase is expressed as a Golgi-resident type II protein, points to enzyme-specific parameters directing folding and membrane anchoring, as well as Golgi-targeting, not being affected by fusion of GFP to the endo-alpha1,2-mannosidase N-terminus. << Less

  Biochimie 87:169-179(2005) [PubMed] [EuropePMC]