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.     

Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases

Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER alpha1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N-glycans. Here we demonstrate that overexpression of Golgi alpha1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human alpha1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi alpha1,2-mannosidases can also contribute to NHK degradation.     

Structure of Penicillium citrinum alpha 1,2-mannosidase reveals the basis for differences in specificity of the endoplasmic reticulum and Golgi class I enzymes.

Class I alpha1,2-mannosidases (glycosylhydrolase family 47) are key enzymes in the maturation of N-glycans. This protein family includes two distinct enzymatically active subgroups. Subgroup 1 includes the yeast and human endoplasmic reticulum (ER) alpha1,2-mannosidases that primarily trim Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomer B whereas subgroup 2 includes mammalian Golgi alpha1,2-mannosidases IA, IB, and IC that trim Man(9)GlcNAc(2) to Man(5)GlcNAc(2) via Man(8)GlcNAc(2) isomers A and C. The structure of the catalytic domain of the subgroup 2 alpha1,2-mannosidase from Penicillium citrinum has been determined by molecular replacement at 2.2-A resolution. The fungal alpha1,2-mannosidase is an (alphaalpha)(7)-helix barrel, very similar to the subgroup 1 yeast (Vallée, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588) and human (Vallée, F., Karaveg, K., Herscovics, A., Moremen, K. W., and Howell, P. L. (2000) J. Biol. Chem. 275, 41287-41298) ER enzymes. The location of the conserved acidic residues of the catalytic site and the binding of the inhibitors, kifunensine and 1-deoxymannojirimycin, to the essential calcium ion are conserved in the fungal enzyme. However, there are major structural differences in the oligosaccharide binding site between the two alpha1,2-mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal alpha1,2-mannosidase this arginine is replaced by glycine. This replacement and other sequence variations result in a more spacious carbohydrate binding site. Modeling studies of interactions between the yeast, human and fungal enzymes with different Man(8)GlcNAc(2) isomers indicate that there is a greater degree of freedom to bind the oligosaccharide in the active site of the fungal enzyme than in the yeast and human ER alpha1,2-mannosidases.     

The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.     

The processing alpha1,2-mannosidase of Saccharomyces cerevisiae depends on Rer1p for its localization in the endoplasmic reticulum.

The yeast alpha1,2-mannosidase Mns1p is involved in N-linked oligosaccharide processing in Saccharomyces cerevisiae by converting Man9GlcNAc2 to a single isomer of Man8GlcNAc2. alpha1,2-Mannosidase is a 63 kDa type II resident membrane protein of the endoplasmic reticulum that has none of the known endoplasmic reticulum localization signals (HDEL/KDEL, KKXX, or RRXX). Using antibodies against recombinant alpha1,2-mannosidase, indirect immunofluorescence showed that alpha1,2-mannosidase localization is abnormal in rer1 cells and that the alpha1,2-mannosidase localizes in the vacuoles of rer1/deltapep4 cells whereas in wild-type and deltapep4 cells it is found in the endoplasmic reticulum. 35S-labeled cell extracts were subjected to double immunoprecipitation, first with antibodies to alpha1,2-mannosidase, then with either alpha1,2-mannosidase antibodies or antibodies to alpha1,6-mannose residues added in the Golgi. The labeled proteins were examined by autoradiography after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A significant proportion of the labeled alpha1,2-mannosidase was immunoprecipitated by alpha1,6-mannose antibodies in wild-type, deltapep4 and rer1/deltapep4 cells with endogenous levels of alpha1,2-mannosidase, and in wild-type, deltapep4, rer1 and rer1/deltapep4 cells overexpressing alpha1,2-mannosidase. The alpha1,2-mannosidase of rer1/deltapep4 cells had a slower mobility on the gels than alpha1,2-mannosidase precipitated from wild-type or deltapep4 cells, indicating increased glycosylation due to transport through the Golgi to the vacuoles. It is concluded that the endoplasmic reticulum localization of alpha1,2-mannosidase in wild-type cells depends on Rer1p for retrieval from an early Golgi compartment.     

N-Glycan processing by a lepidopteran insect alpha1,2-mannosidase

Protein glycosylation pathways are relatively poorly characterized in insect cells. As part of an overall effort to address this problem, we previously isolated a cDNA from Sf9 cells that encodes an insect alpha1,2-mannosidase (SfManI) which requires calcium and is inhibited by 1-deoxymannojirimycin. In the present study, we have characterized the substrate specificity of SfManI. A recombinant baculovirus was used to express a GST-tagged secreted form of SfManI which was purified from the medium using an immobilized glutathione column. The purified SfManI was then incubated with oligosaccharide substrates and the resulting products were analyzed by HPLC. These analyses showed that SfManI rapidly converts Man(9)GlcNAc(2)to Man(6)Glc-NAc(2)isomer C, then more slowly converts Man(6)GlcNAc(2)isomer C to Man(5)GlcNAc(2). The slow step in the processing of Man(9)GlcNAc(2)to Man(5)GlcNAc(2)by SfManI is removal of the alpha1,2-linked mannose on the middle arm of Man(9)GlcNAc(2). In this respect, SfManI is similar to mammalian alpha1,2-mannosidases IA and IB. However, additional HPLC and(1)H-NMR analyses demonstrated that SfManI converts Man(9)GlcNAc(2)to Man(5)GlcNAc(2)primarily through Man(7)GlcNAc(2)isomer C, the archetypal Man(9)GlcNAc(2)missing the lower arm alpha1,2-linked mannose residues. In this respect, SfManI differs from mammalian alpha1,2-mannosidases IA and IB, and is the first alpha1,2-mannosidase directly shown to produce Man(7)GlcNAc(2)isomer C as a major processing intermediate.     

Structure and function of Class I alpha 1,2-mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control

Class I alpha 1,2-mannosidases (glycosylhydrolase family 47) are conserved through eukaryotic evolution. This protein family comprises three subgroups distinguished by their enzymatic properties. The first subgroup includes yeast (Saccharomyces cerevisiae) and human alpha 1,2-mannosidases of the endoplasmic reticulum that primarily form Man(8)GlcNAc(2) isomer B from Man(9)GlcNAc(2). The second subgroup includes mammalian Golgi alpha 1,2-mannosidases, as well as enzymes from insect cells and from filamentous fungi, that trim Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomers A and/or C intermediates toward the formation of Man(5)GlcNAc(2). Yeast and mammalian proteins of the third subgroup have no enzyme activity with Man(9)GlcNAc(2) as substrate. The members of subgroups 1 and 3 participate in endoplasmic reticulum quality control and promote proteasomal degradation of misfolded glycoproteins. The yeast endoplasmic reticulum alpha 1,2-mannosidase has served as a model for structure-function studies of this family. Its structure was determined by X-ray crystallography as an enzyme-product complex. It consists of a novel (alpha alpha)(7) barrel containing the active site that includes essential acidic residues and calcium. The structures of the subgroup 1 human endoplasmic reticulum alpha 1,2-mannosidase and of a subgroup 2 fungal alpha 1,2-mannosidase were determined by molecular replacement. Comparison of the enzyme structures is providing some insight into the reasons for their different specificities.     

Characterization of MOPC 315 IgA oligosaccharide processing intermediates

The structures of alpha 1,2-mannose containing partially processed asparagine-linked oligosaccharides on the alpha-chain of MOPC 315 IgA were characterized using specific glycosidases and acetolysis. Man6GlcNAc2, a substrate for a Golgi alpha 1,2-mannosidase, was found to be a single isomeric structure. Likewise, Man7-9GlcNAc2 were single isomers indicating an ordered sequence of removal of alpha 1,2-linked mannose residues on this murine immunoglobulin heavy chain.     

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.     

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.      

Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei

We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man9GlcNAc2 from Man9GlcNAc2-PP-Dol while others receive Man5GlcNA(2 from Man5GlcNAc2-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the alpha-mannosyltransferase that converts Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man9GlcNAc2 and processed no further than Man5GlcNAc2 in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man5GlcNAc2-PP-Dol is the only route to complex N-glycans in T. brucei and that Man9GlcNAc2-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man5GlcNAc2 or Man9GlcNAc2 transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.     

Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity

The processing of asparagine-linked oligosaccharides on the alpha- chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N- acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.     

Biosynthesis of human-type N-glycans in heterologous systems

Insects, yeasts and plants generate widely different N-glycans, the structures of which differ significantly from those produced by mammals. The processing of the initial Glc2Man9GlcNAc2 oligosaccharide to Man8GlcNAc2 in the endoplasmic reticulum shows significant similarities among these species and with mammals, whereas very different processing events occur in the Golgi compartments. For example, yeasts can add 50 or even more Man residues to Man(8-9)GlcNAc2, whereas insect cells typically remove most or all Man residues to generate paucimannosidic Man(3-1)GlcNAc2N-glycans. Plant cells also remove Man residues to yield Man(4-5)GlcNAc2, with occasional complex GlcNAc or Gal modifications, but often add potentially allergenic beta(1,2)-linked Xyl and, together with insect cells, core alpha(1,3)-linked Fuc residues. However, genomic efforts, such as expression of exogenous glycosyltransferases, have revealed more complex processing capabilities in these hosts that are not usually observed in native cell lines. In addition, metabolic engineering efforts undertaken to modify insect, yeast and plant N-glycan processing pathways have yielded sialylated complex-type N-glycans in insect cells, and galactosylated N-glycans in yeasts and plants, indicating that cell lines can be engineered to produce mammalian-like glycoproteins of potential therapeutic value.     

Alpha-mannosidases involved in N-glycan processing show cell specificity and distinct subcompartmentalization within the Golgi apparatus of cells in the testis and epididymis.

The Golgi apparatus is enriched in specific enzymes involved in the maturation of carbohydrates of glycoproteins. Among them, alpha-mannosidases IA, IB and II are type II transmembrane Golgi-resident enzymes that remove mannose residues at different stages of N-glycan maturation. alpha-Mannosidases IA and IB trim Man9GlcNAc2 to Man5GlcNAc2, while alpha-mannosidase II acts after GlcNAc transferase I to remove two mannose residues from GlcNAcMan5GlcNAc2 to form GlcNAcMan3GlcNAc2 prior to extension into complex N-glycans by Golgi glycosyltransferases. The objective of this study is to examine the expression as well as the subcellular localization of these Golgi enzymes in the various cells of the male rat reproductive system. Our results show distinct cell-and region-specific expression of the three mannosidases examined. In the testis, only alpha-mannosidase IA and II were detectable in the Golgi apparatus of Sertoli and Leydig cells, and while alpha-mannosidase IB was present in the Golgi apparatus of all germ cells, only the Golgi apparatus of steps 1-7 spermatids was reactive for alpha-mannosidase IA. In the epididymis, principal cells were unreactive for alpha-mannosidase II, but they expressed alpha-mannosidase IB in the initial segment and caput regions, and alpha-mannosidase IA in the corpus and cauda regions. Clear cells expressed alpha-mannosidase II in all epididymal regions, and alpha-mannosidase IB only in the caput and corpus regions. Ultrastructurally, alpha-mannosidase IB was localized mainly over cis saccules, alpha-mannosidase IA was distributed mainly over trans saccules, and alpha-mannosidase II was localized mainly over medial saccules of the Golgi stack. Thus, the cell-specific expression and distinct Golgi subcompartmental localization suggest that these three alpha-mannosidases play different roles during N-glycan maturation.     

The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase.     

Biosynthesis and subcellular localization of a lepidopteran insect alpha 1,2-mannosidase

Like lower and higher eucaryotes, insects have alpha 1,2-mannosidases which function in the processing of N-glycans. We previously cloned and characterized an insect alpha 1,2-mannosidase cDNA and demonstrated that it encodes a member of a family of N-glycan processing alpha 1,2-mannosidases (Kawar, Z., Herscovics, A., Jarvis, D.L., 1997. Isolation and characterisation of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology 7, 433-443). These enzymes have similar protein sequences, require calcium for their activities, and are sensitive to 1-deoxymannojirimycin, but can have different substrate specificities and intracellular distributions. We recently determined the substrate specificity of the insect alpha 1,2-mannosidase, SfManI (Kawar, Z., Romero, P., Herscovics, A., Jarvis, D.L., 2000. N-glycan processing by a lepidopteran insect and 1,2-mannosidase. Glycobiology 10, 347-355). Now, we have examined the biosynthesis and subcellular localization of SfManI. We found that SfManI is partially N-glycosylated and that N-glycosylation is dramatically enhanced if the wild type sequon is changed to one that is highly utilized in a mammalian system. We also found that an SfManI-GFP fusion protein had a punctate cytoplasmic distribution in insect cells. Colocalization studies indicated that this fusion protein is localized in the Golgi apparatus, not in the endoplasmic reticulum or lysosomes. Finally, N-glycosylation had no influence over the substrate specificity or subcellular localization of SfManI.     

Characterization of a novel alpha-D-mannosidase from rat brain microsomes

A new alpha-D-mannosidase has been identified in rat brain microsomes. The enzyme was purified 70-100-fold over the microsomal fraction by solubilization with Triton X-100, followed by ion exchange, concanavalin A-Sepharose, and hydroxylapatite chromatography. The purified enzyme is very active towards mannose-containing oligosaccharides and has a pH optimum of 6.0. Unlike rat liver endoplasmic reticulum alpha-D-mannosidase and both Golgi mannosidases IA and IB, which have substantial activity only towards alpha 1,2-linked mannosyl residues, the brain enzyme readily cleaves alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannosyl residues present in high mannose oligosaccharides. The brain enzyme is also different from liver Golgi mannosidase II in that it hydrolyzes (Man)5GlcNAc and (Man)4GlcNAc without their prior N-acetylglucosaminylation. Moreover, the facts that the ability of the enzyme to cleave GlcNAc(Man)5GlcNAc, the biological substrate for Golgi mannosidase II, is not inhibited by swainsonine, and that p-nitrophenyl alpha-D-mannoside is a poor substrate provide further evidence for major differences between the brain enzyme and mannosidase II. Inactivation studies and the co-purification of activities towards various substrates suggest that a single enzyme is responsible for all the activities found. In view of these results, it seems possible that, in rat brain, a single mannosidase cleaves asparagine-linked high mannose oligosaccharide to form the core Man3GlcNAc2 moiety, which would then be modified by various glycosyl transferases to form complex type glycoproteins.