Free oligosaccharides in the cytosol of Caenorhabditis elegans are generated through endoplasmic reticulum-golgi trafficking

Free oligosaccharides (FOSs) in the cytosol of eukaryotic cells are mainly generated during endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded glycoproteins. We analyzed FOS of the nematode Caenorhabditis elegans to elucidate its detailed degradation pathway. The major FOSs were high mannose-type ones bearing 3-9 Man residues. About 94% of the total FOSs had one GlcNAc at their reducing end (FOS-GN1), and the remaining 6% had two GlcNAc (FOS-GN2). A cytosolic endo-beta-N-acetylglucosaminidase mutant (tm1208) accumulated FOS-GN2, indicating involvement of the enzyme in conversion of FOS-GN2 into FOS-GN1. The most abundant FOS in the wild type was Man(5)GlcNAc(1), the M5A' isomer (Manalpha1-3(Manalpha1-6)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAc), which is different from the corresponding M5B' (Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAc) in mammals. Analyses of FOS in worms treated with Golgi alpha-mannosidase I inhibitors revealed decreases in Man(5)GlcNAc(1) and increases in Man(7)GlcNAc(1). These results suggested that Golgi alpha-mannosidase I-like enzyme is involved in the production of Man(5-6)-GlcNAc(1), which is unlike in mammals, in which cytosolic alpha-mannosidase is involved. Thus, we assumed that major FOSs in C. elegans were generated through Golgi trafficking. Analysis of FOSs from a Golgi alpha-mannosidase II mutant (tm1078) supported this idea, because GlcNAc(1)Man(5)GlcNAc(1), which is formed by the Golgi-resident GlcNAc-transferase I, was found as a FOS in the mutant. We concluded that significant amounts of misfolded glycoproteins in C. elegans are trafficked to the Golgi and are directly or indirectly retro-translocated into the cytosol to be degraded.     

Quantitation and isomeric structure analysis of free oligosaccharides present in the cytosol fraction of mouse liver: detection of a free disialobiantennary oligosaccharide and glucosylated oligomannosides.

The amounts and isomeric structures of free oligosaccharides derived from N-linked sugar chains present in the cytosol fraction of perfused mouse liver were analyzed by tagging the reducing end with 2-aminopyridine followed by 2-dimensional HPLC mapping with standard sugar chains. Sixteen pyridylaminated (PA-) oligomannosides terminating with a PA-GlcNAc residue (GN1-type), three glucose-containing oligomannosides, and four oligomannosides terminating with a PA-di-N-acetylchitobiose (GN2-type) were detected. The total contents of the GN1- and GN2-type oligomannosides were 3. 4 and 0.5 nmol, respectively, per gram of wet tissue. Maltooligosaccharides (dimer to pentamer) were also detected, the total content of which was 13 nmol per gram of wet tissue. Besides these oligosaccharides, a PA-disialobiantennary sugar chain-the sole complex-type sugar chain-was also detected. All the oligomannosides identified had partial structures of Glc(3)Man(9)GlNAc(2)-p-p-dolichol, revealing that they were metabolic degradation products. Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)++ +Manbeta1-4GlcNAc (M5B') was the major oligomannoside, suggesting that cytosolic endo-beta-N-acetylglucosaminidase and neutral alpha-mannosidase participate in the degradation, because these enzymes have suitable substrate specificities for the production of M5B'. Degradation by these enzymes seems to be the main pathway by which oligomannosides are degraded in mouse cytosol; however, small amounts of Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4(GlcNAc)1-2 and related oligomannosides together with parts of their structures were also detected, suggesting that there is another minor route by which cytosolic free oligomannosides are produced.     

Co(II)-regulated substrate specificity of cytosolic alpha-mannosidase

Cytosolic neutral alpha-mannosidase is a putative catabolic enzyme that produces cytosolic free oligomannosides. Activation of the enzyme by Co(II) treatment has been reported using pyridylamino derivatives of Man(5)GlcNAc and Man(5)GlcNAc2, and p-nitrophenyl alpha-mannoside as substrates, with the Co(II)-treated enzyme releasing four alpha-mannose residues from Man(9)GlcNAc to give Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc as an end product. When Man(9)GlcNAc, which is considered to be the actual substrate in the cytosol, was used as a substrate, we found that even before treatment with Co(II) the enzyme was able to cleave a single Manalpha1-2 residue from Man(9)GlcNAc to give Manalpha1-6(Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc as the end product. The K(m) value of the Co(II)-treated enzyme for Man(9)GlcNAc was found to be 37 microM, which is one-twelfth that of the non-treated enzyme, while the values were V(max) values were almost the same, indicating that the affinity of the substrate is higher with Co(II). These results indicate that Co(II) regulates the substrate specificity of the enzyme.     

Structural studies of two ovalbumin glycopeptides in relation to the endo-beta-N-acetylglucosaminidase specificity.

Heterogeneities of the two ovalbumin glycopeptides, (Man)5(GlcNAc)2Asn and (Man)6(GlcNAc)2Asn, were revealed by borate paper electrophoresis of oligosaccharide alcohols obtained from the glycopeptides by endo-beta-N-acetylglucosaminidase H digestion and NaB3H4 reduction. The structures of the major components of the oligosaccharides were determined by the combination of methylation analysis, acetolysis, and alpha-mannosidase digestion. Based on the results, the whole structures of the major components of (Man)5(GlcNAc)2Asn and (Man)6(GlcNAc)2Asn were elucidated as Manalpha1 leads to 6[Manalpha1 leads to 3]-Manalpha1 leads to 6[Manalpha1 leads to 3[Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc leads to Asn and Manalpha1 leads to 6[Manalpha1 leads to 3]Manalpha1 leads to 6[Manalpha1 leads to 2Manalpha1 leads to 3]Manbeta1 leads to 4GlcNAcbeta1 leads to GlcNAc leads to Asn, respectively. Since endo-beta-N-acetylglucosamini dase D hydrolyzes (Man)5(GlcNAc)2Asn but not (Man)6(GlcNAc)2Asn, the presence of the unsubstituted alpha-mannosyl residue linked at the C-3 position of the terminal mannose of Manbeta1 leads to 4GlcNAcbeta1 leads to 4 GlcNAcAsn core must be essential for the action of the enzyme.     

Deletion of the glucosidase II gene in Trypanosoma brucei reveals novel N-glycosylation mechanisms in the biosynthesis of variant surface glycoprotein

The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, and Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man(4-3)GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.     

Processing pathway deduced from the structures of N-glycans in Carica papaya

A processing The processing pathway of N-glycans in Carica papaya was deduced from the structures of N-glycans. The N-glycans were liberated by hydrazinolysis followed by N-acetylation. Their reducing-end sugar residues were tagged with 2-aminopyridine and the pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Eleven PA-sugar chains were found, and their structures were analyzed by two-dimensional sugar mapping combined with partial acid hydrolysis and exoglycosidase digestion. The structures of the N-glycans were of the highmannose types with xylose and fucose; however, among them two new N-glycans, Manalpha1-6(Manalpha1-3)Manalpha1-6(Xylbeta1-2)+ ++Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-3)GlcNAc and Manalpha1-3Manalpha1-6(Xylbeta1-2)Manbeta1-4G lcNAcbeta1-4(Fucalpha1-3 )GlcNAc, were found. Judging from these structures together with Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc reported previously [Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) J. Biochem. 125, 560- 565], a processing pathway for N-glycans in C. papaya is inferred in which the activity of Golgi alpha-mannosidase II is incomplete.     

Asparaginyl glycopeptides with a low mannose content are hydrolyzed by endo-beta-N-acetylglucosaminidase H

Substrates susceptible to endo-beta-N-acetylglucosaminidase H were reduced in size through alpha-mannosidase treatment and periodate oxidation to yield the following compounds: (Man)4(GlcNAc)2Asn, [Manalpha 1 leads to 6Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNACAsn]; (Man)3(GlcNAc)2Asn, [Manalpha 1 leads to 3Man-alpha 1 leads to 6Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn]; (Man)2(GlcNAc)2Asn, [Manalpha 1 leads to 6Manbeta1 leads to 4GlcNAcbeta 1 leads to 4BlcNAcAsm]. Comparison of the relative rates of hydrolysis of these compounds with (Man)5(GlcNAc)2-Asn, the most active substrate to date for the endoglycosidase, revealed (Man)4(GlcNAc)2Asn to be hydrolyzed faster than (Man)5(GlcNAc)2Asn and (Man)3-(GlcNAc)2Asn to be equal to or slightly better than (Man)5(GlcNAc)2Asn as a substrate. (Man)2(GlcNAc)2-Asn was completely hydrolyzed but at a rate that was about 10(4) slower than (Man)5(GlcNAc)2Asn, which is comparable to that for (Man)3(GlcNAc)2Asn(aa)x [Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn(aa)x], obtained from immunoglobulin M. (Man)1(GlcNAc)2Asn, [Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn] was hydrolyzed at a 100-fold slower rate than the latter glycopeptide. The effective range of endo-beta-N-acetylglucosaminidase H has thus been extended to compounds containing as few as 2 mannosyl residues.     

Urinary oligosaccharides of fucosidosis. Evidence of the occurrence of X-antigenic determinant in serum-type sugar chains of glycoproteins.

Urine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucose-rich glycopeptides. Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucalpha1 leads to 6GlcNAc, Fucalpha1 leads to 2Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 2Manalpha1 leads to 3Manbeta1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to3)GlcNAcbeta1 leads to 2Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc, and Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 6Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc. In additon, the structure of a minor decasaccharide was found to be Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to [Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to]Manbeta1 leads to 4GlcNAc.     

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.     

Accumulation of free complex-type N-glycans in MKN7 and MKN45 stomach cancer cells

During the N-glycosylation reaction, it has been shown that 'free' N-glycans are generated either from lipid-linked oligosaccharides or from misfolded glycoproteins. In both cases, occurrence of high mannose-type free glycans is well-documented, and the molecular mechanism for their catabolism in the cytosol has been studied. On the other hand, little, if anything, is known with regard to the accumulation of more processed, complex-type free oligosaccharides in the cytosol of mammalian cells. During the course of comprehensive analysis of N-glycans in cancer cell membrane fractions [Naka et al. (2006) J. Proteome Res. 5, 88-97], we found that a significant amount of unusual, complex-type free N-glycans were accumulated in the stomach cancer-derived cell lines, MKN7 and MKN45. The most abundant and characteristic glycan found in these cells was determined to be NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1-3Manbeta1-4GlcNAc. Biochemical analyses indicated that those glycans found were cytosolic glycans derived from lysosomes due to low integrity of the lysosomal membrane. Since the accumulation of these free N-glycans was specific to only two cell lines among the various cancer cell lines examined, these cytosolic N-glycans may serve as a specific biomarker for diagnosis of specific tumours. A cytosolic sialidase, Neu2, was shown to be involved in the degradation of these sialoglycans, indicating that the cytosol of mammalian cells might be equipped for metabolism of complex-type glycans.     

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.     

Developmental regulation of asparagine-linked oligosaccharide synthesis in Dictyostelium discoideum

In the preceding report we demonstrated that the expression of two developmentally regulated alpha-mannosidase activities is induced in Dictyostelium discoideum during its differentiation from single-cell amoebae to multicellular organism (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18477-18484). These activities, designated membrane alpha-mannosidase I (MI) and membrane alpha-mannosidase II (MII), were shown to have several properties in common with rat liver Golgi alpha-mannosidases I and II, respectively, suggesting that MI and MII may play a role in the processing of asparagine-linked oligosaccharides in developing D. discoideum. In this study we analyzed the structures of the asparagine-linked oligosaccharides synthesized by D. discoideum at various stages of development to determine the timing and extent of asparagine-linked oligosaccharide processing. Cells were labeled with [2-3H] mannose, and then total cellular glycoproteins were digested with Pronase to generate glycopeptides that were fractionated on concanavalin A-Sepharose. Glycopeptides from each fraction were digested with endoglycosidase H, both before and after desulfation by solvolysis, and the released, neutral oligosaccharides were sized by high pressure liquid chromatography. At early stages of development, D. discoideum contain predominantly large high mannose-type oligosaccharides (Man9GlcNAc and Man8GlcNAc). Some of these are modified by GlcNAc residues attached beta 1-4 to the mannose-linked alpha 1-6 to the beta-linked core mannose (the "intersecting" position), as well as by fucose, sulfate, and phosphate. In contrast, the oligosaccharides found at late stages of development (18-24 h) have an array of sizes from Man9GlcNAc to Man3GlcNAc. These are still modified by GlcNAc, fucose, sulfate, and phosphate, but the percent of larger high mannose oligosaccharides that are modified with GlcNAc in the intersecting position decreases after 6 h of development, in parallel with the decrease in the intersecting GlcNAc transferase activity. Similarly, the changes in the size of asparagine-linked oligosaccharides synthesized during development correlate well with the appearance of MI and MII activities and suggest that these developmentally regulated alpha-mannosidase activities function in the processing of these oligosaccharides. This is supported further by the observation that oligosaccharide processing was inhibited in late stage cells labeled in the presence of either deoxymannojirimycin, an inhibitor of MI, or swainsonine, an inhibitor of MII.     

Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver

An alpha-mannosidase activity has been identified in a preparation of rat liver endoplasmic reticulum and shown to be distinct from the previously described Golgi alpha-mannosidases I and II and the lysosomal alpha-mannosidase. The enzyme was solubilized with deoxycholate and separated from other alpha-mannosidases by passage over concanavalin A-Sepharose to which it does not bind. The endoplasmic reticulum alpha-mannosidase cleaves alpha-1,2-linked mannoses from high mannose oligosaccharides and, unlike Golgi alpha-mannosidase I, is active against p-nitrophenyl-alpha-D-mannoside (Km = 0.17 mM). It has no activity toward GlcNAc-Man5GlcNAc2 peptide, the specific substrate of the Golgi alpha-mannosidase II. The endoplasmic reticulum alpha-mannosidase activity toward p-nitrophenyl-alpha-D-mannoside is relatively insensitive to swainsonine, an inhibitor of both the lysosomal alpha-mannosidase and Golgi alpha-mannosidase II. We propose that the endoplasmic reticulum alpha-mannosidase is responsible for the removal of mannose residues from asparagine-linked high mannose type oligosaccharides prior to their entry into the Golgi.     

First evidence for occurrence of Galbeta1-3GlcNAcbeta1-4Man unit in N-glycans of insect glycoprotein: beta1-3Gal and beta1-4GlcNAc transferases are involved in N-glycan processing of royal jelly glycoproteins

On a way of structural analysis of total N-glycans linked to glycoproteins in royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985-1989 (2002)), we found that some complex type N-glycans containing a beta1-3galactose residue occur on the insect glycoproteins. Up to date, it has been considered that naturally occurring insect glycoproteins do not bear the galactose-containing N-glycans, therefore, in this report we describe the structural analysis of the complex type N-glycans of royal jelly glycoproteins.By a combination of endo- and exo-glycosidase digestions, IS-MS analysis, and 1H-NMR spectroscopy, the structures of the beta1-3 galactose-containing N-glycan were identified as the following; GlcNAcbeta1-2Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, Manalpha1-3Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this is the first report showing that the Galbeta1-3GlcNAcbeta1-4Man unit occurs in N-glycans of insect glycoproteins, indicating a beta1-3 galactosyl transferase and beta1-4GlcNAc transferase (GNT-IV) are expressed in the honeybee cells.     

Characterization and subcellular localization of human neutral class II alpha-mannosidase [corrected

A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral alpha-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe(2+), Co(2+), and Mn(2+), and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe(2+) than Co(2+). Without activating cations the main reaction product was Man(8)GlcNAc, and Cu(2+) completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.     

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.     

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.     

Human IgG1 expression in silkworm larval hemolymph using BmNPV bacmids and its N-linked glycan structure

A Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expressing heavy and light chains of human 29IJ6 IgG was constructed and used to secrete recombinant antibody into silkworm larval hemolymph. Fifth instar silkworm larvae were reared and injected into the dorsum of the larvae with recombinant cysteine protease- and chitinase-deficient BmNPV (BmNPV-CP(-)-Chi(-)) bacmid/29IJ6 IgG and harvested after approximately 6 days. The total yield of recombinant 29IJ6 IgG was 36 microg/larvae, which is equivalent to 8 mg/kg of larvae. The recombinant antibody was purified to homogeneity using a HiTrap rProtein A FF column with a purification yield of 83.1%. The purified protein was identified by Western blot and ELISA experiments. The N-linked glycan structure of the purified protein was determined by the HPLC mapping method. The N-glycans of the 29IJ6 IgG glycoprotein produced in, and secreted by the silkworm larvae were composed exclusively of two kinds of paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc and Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc.     

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).     

Substrate specificity and molecular cloning of the lily endo-beta-mannosidase acting on N-glycan

Endo-beta-mannosidase, which hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans, was recently purified to homogeneity from lily (Lilium longiflorum) flowers as a heterotrimer [Ishimizu, T., Sasaki, A., Okutani, S., Maeda, M., Yamagishi, M., and Hase, S. (2004) J. Biol. Chem. 279, 38555-38562]. Here, we describe the substrate specificity of the enzyme and cloning of its cDNA. The purified enzyme hydrolyzed pyridylaminated (PA-) Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc (n = 0-2) to Man(n)Manalpha1-6Man and GlcNAcbeta1-4GlcNAc-PA. It did not hydrolyze PA-sugar chains containing Manalpha1-3Manbeta and/or Xylbeta1-2Manbeta. The best substrate among the PA-sugar chains tested was Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA with a K(m) value of 1.2 mM. However, the enzyme displayed a marked preference for the corresponding glycopeptide, Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-peptide (K(m) value 75 microM). These results indicate that the substrate recognition by the enzyme involves the peptide portion attached to the N-glycan. Sequence information on the purified enzyme was used to clone the corresponding cDNA. The monocotyledonous lily enzyme (952 amino acids) displays 68% identity to its dicotyledonous (Arabidopsis thaliana) homologue. Our results show that the heterotrimeric enzyme is encoded by a single gene that gives rise to three polypeptides following posttranslational proteolysis. The enzyme is ubiquitously expressed, suggesting that it has a general function such as processing or degrading N-glycans.