N-glycosylation of purified rat and rabbit hepatic UDP-glucuronosyltransferases

Five UDP-glucuronosyltransferases (UDPGTs) have been isolated to apparent homogeneity from rat and rabbit liver and have been characterized for their glycoprotein nature by reacting these proteins with commercially available endo- and exoglycosidases. The enzymes studied were rat hepatic p-nitrophenol, 17 beta-hydroxysteroid, and 3 alpha-hydroxysteroid UDPGTs and rabbit hepatic p-nitrophenol and estrone UDPGTs. Hydrolysis of oligosaccharide moieties was evidenced by an increase in the mobility (decreased apparent molecular weight) of the protein subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified rabbit hepatic estrone and p-nitrophenol UDPGTs were hydrolyzed by almond glycopeptidase A and endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus (endo H), but not by endo-beta-N-acetylglucosaminidase D from Diplococus pneumoniae (endo D) suggesting that these transferases are glycoproteins of the high mannose type and not of the complex type. Likewise, purified rat hepatic 3 alpha-hydroxysteroid and p-nitrophenol UDPGTs were substrates for glycopeptidase A and endo H but not for endo D. One enzyme, 17 beta-hydroxysteroid UDPGT, was not glycosylated since it was not hydrolyzed by any of the three endoglycosidases. All four glycosylated UDPGTs could serve as substrates for jack bean alpha-mannosidase, confirming the high mannose nature of the oligosaccharide. Deglycosylation of the purified UDPGTs by endo H did not have an effect on the catalytic activities of these proteins.     

Purification and characterization of neutral alpha-mannosidase that is activated by Co2+ from Japanese quail oviduct

An alpha-mannosidase was purified from the magnum section of Japanese quail oviduct by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, mannan-Sepharose 4B chromatography, and hydroxyapatite chromatography. The purified alpha-mannosidase (referred to as neutral alpha-mannosidase) showed a single band on polyacrylamide gel with or without sodium dodecyl sulfate. Its molecular weight was found to be 330,000 by gel chromatography. Neutral alpha-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannopyranoside and the pyridylamino derivative of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (Km value was 3 mM). Mannosyl alpha 1-2 linkages in the pyridylamino derivative of Man alpha 1-2 Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc were hardly hydrolyzed. Its optimum pH was found to be 7.0. The activity of the enzyme was activated by CO2+, and was potently inhibited by Cu2+, Hg2+, swainsonine, and 1-deoxymannojirimycin.     

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.     

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.     

Molecular cloning and heterologous expression of beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from maize

Maize is considered a promising alternative production system for pharmaceutically relevant proteins. However, like in all other plant species asparagine-linked oligosaccharides of maize glycoproteins are modified with beta1,2-xylose and core alpha1,3-fucose sugar residues, which are considered to be immunogenic in mammals. This altered N-glycosylation when compared to mammalian cells may reduce the potential of maize as a production system for heterologous glycoproteins. Here we report the cloning and characterization of the cDNA sequences coding for the maize enzymes beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT). The cloned XylT and FucT cDNAs were shown to encode enzymatically active proteins, which were independently able to convert a mammalian acceptor glycoprotein into an antigen binding anti-plant N-glycan antibodies. The complete sequence of the XylT gene was determined. Evidence for the presence of at least three XylT and FucT gene loci in the maize genome was obtained. The identification of the two enzymes and their genes will allow the targeted downregulation or even elimination of beta1,2-xylose and core alpha1,3-fucose addition to recombinant glycoproteins produced in maize.     

Regulation of glycosylation. The influence of protein structure on N-linked oligosaccharide processing

The Sindbis virus glycoproteins, E1 and E2, comprise a useful model system for evaluating the effects of local protein structure on the processing of N-linked oligosaccharides by Golgi enzymes. The conversion of oligomannose to N-acetyllactosamine (complex) oligosaccharides is hindered to different extents at the four glycosylation sites, so that the complex/oligomannose ratio decreases in the order E1-Asn139 greater than E2-Asn196 greater than E1-Asn245 greater than E2-Asn318. The processing steps most susceptible to interference were deduced from the oligosaccharide compositions at hindered sites in virus from baby hamster kidney cells (BHK), chick embryo fibroblasts (CEF), and normal and hamster sarcoma virus (HSV)-transformed hamster fibroblasts (Nil-8). Persistence of Man6-9GlcNAc2 was taken to indicate interference with alpha 2-mannosidase(s) I (alpha-mannosidase I), Man5GlcNAc2, with UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase I (GlcNAc transferase I), and unbisected hybrid glycans, with GlcNAc transferase I-dependent alpha 3(alpha 6)-mannosidase (alpha-mannosidase II). Taken together, the results indicate that all four sites acquire a precursor oligosaccharide with equally high efficiency, but alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are all impeded at E2-Asn318 and, to a lesser extent, at E1-Asn245. In contrast, sialic acid and galactose transfer to hybrid glycans (in BHK cells) is virtually quantitative even at E2-Asn318. E2-Asn318 carried no complex oligosaccharides, but the structures of those at E1-Asn245 indicate almost complete GlcNAc transfer by UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase II (GlcNAc transferase II), galactosylation, and sialylation. Because the E2-Asn318 and E1-Asn245 glycans have previously been shown to be less accessible to a steric probe than those at E2-Asn196 or E1-Asn139, a simple explanation for these results would be that alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are more susceptible to steric hindrance than are the later processing steps examined. Finally, in addition to these site-specific effects, the overall extent of viral oligosaccharide processing varied with host and cellular growth status. For example, alpha-mannosidase I processing is more complete in BHK cells compared to CEF, and in confluent Nil-8 cells compared to subconfluent or HSV-transformed Nil-8 cells.     

Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases

Endoplasmic reticulum (ER) class I alpha1,2-mannosidase (also known as ER alpha-mannosidase I) is a critical enzyme in the maturation of N-linked oligosaccharides and ER-associated degradation. Trimming of a single mannose residue acts as a signal to target misfolded glycoproteins for degradation by the proteasome. Crystal structures of the catalytic domain of human ER class I alpha1,2-mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin. Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2' and O-3' hydroxyls and stabilizing the six-membered rings of both inhibitors in a (1)C(4) conformation. This is the first direct evidence of the role of the calcium ion. The lack of major conformational changes upon inhibitor binding and structural comparisons with the yeast alpha1, 2-mannosidase enzyme-product complex suggest that this class of inverting enzymes has a novel catalytic mechanism. The structures also provide insight into the specificity of this class of enzymes and provide a blueprint for the future design of novel inhibitors that prevent degradation of misfolded proteins in genetic diseases.     

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor?manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor?manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K(cat)/K(m) (mM(-1) s(-1) = 0.41 for D-mannose-6-phosphate), but failed to show GDP-D-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor?manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.     

Molecular characterization of the Thermomonospora curvata aglA gene encoding a thermotolerant alpha-1,4-glucosidase

The cloning, sequencing and structural characterization of a gene encoding a thermostable alpha-1,4-glucosidase from Thermomonospora curvata is described. DNA sequence analysis revealed four open reading frames designated aglA, aglR, aglE and aglF. The aglA gene encodes a thermostable alpha-1,4-glucosidase from T. curvata and is situated between two genes, aglR and aglE. Genes aglA, aglE and aglF are transcribed in the same direction, while aglR is transcribed in the opposite direction. By comparing the amino acid sequence of the alpha-1,4-glucosidase from T. curvata with other alpha-glucanases, it appears that the enzyme is a member of the alpha-amylase family. The proteins of this family have an (alpha/beta)8 barrel super secondary structure. The topology of the alpha-1,4-glucosidase was predicted by computer-assisted analysis. The topology of the secondary structures of the alpha-1,4-glucosidase resembles the structure of barley alpha-amylase, but the primary structure resembles most closely the oligo-1,6-glucosidase from Bacillus cereus. Putative catalytic residues (D221, E281 and D343) and calcium binding residues (N116, E179, D191, H224 or G225) are proposed.     

Studies on the substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct by using sugar chains from glycoproteins.

The substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co2+ hydrolyzed the Man alpha 1-3 and Man alpha 1-6 bonds from the non-reducing termini of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (M5A), but hardly hydrolyzed the Man alpha 1-2 bonds of Man9GlcNAc2. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Man beta 1-4GlcNAc2. For the substrates with the GlcNAc structure at their reducing ends (Man5GlcNAc, Man6GlcNAc and Man9GlcNAc), the hydrolysis rate was remarkably increased: Man5GlcNAc was hydrolyzed 16 times faster than M5A, and Man2GlcNAc 40 times faster than Man9GlcNAc2. The enzyme did not hydrolyze Man alpha 1-2 residue(s) linked to Man alpha 1-3Man beta 1-4GlcNAc. The end products were as follows: [formula; see text] These results suggest that oligomannose-type sugar chains with the GlcNAc structure at their reducing ends seem to be native substrates for neutral alpha-mannosidase and the enzyme seems to hydrolyze endo-beta-N-acetylgucosaminidase digests of oligomannose-type sugar chains in the cytosol.     

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.     

Novel endo-alpha-N-acetylgalactosaminidases with broader substrate specificity

In an effort to identify novel endo-alpha-N-acetylgalactosaminidases (endo-alpha-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-alpha-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp. (EngAL). All enzymes were incubated with various synthetic substrates, and natural glycoproteins and the released sugars were detected by colorimetric assay and thin layer chromatography analysis. The Core 1 disaccharide Gal beta 1,3GalNAc alpha 1pNP was the most rapidly hydrolyzed substrate by all enzymes tested. EngEF exhibited the highest k(cat) for this substrate. EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAc beta 1,3GalNAc alpha 1pNP. This is the first report of endo-alpha-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans. Interestingly, there were no significant differences in transglycosylation activities when Gal beta 1,3GalNAc alpha 1pNP or GlcNAc beta 1,3GalNAc alpha 1pNP was incubated with various 1-alkanols in the presence of the endo-alpha-GalNAcases tested in this work.     

Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.     

Novel glycoproteins of the halophilic archaeon Haloferax volcanii

Archaea possess many eukaryote-like properties, including the ability to glycosylate proteins. Using oligosaccharide staining and lectin binding, this study revealed the existence of several glycosylated Haloferax volcanii membrane proteins, besides the previously reported surface layer (S-layer) glycoprotein. While the presence of glycoproteins in archaeal S-layers and flagella is well-documented, few archaeal glycoproteins that are not part of these structures have been reported. The glycosylated 150, 98, 58 and 54 kDa protein species detected were neither precursors nor breakdown products of the 190 kDa S-layer glycoprotein. Furthermore, these novel glycoproteins were outwardly oriented and intimately associated with the membrane.     

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.     

Effect of isomers of swainsonine on glycosidase activity and glycoprotein processing

The chemical synthesis of swainsonine [(1S,2R,8R,8 alpha R)-trihydroxyindolizidine] from trans-1,4-dichloro-2-butene was previously described [Adams, C. E., Walker, F. J., & Sharpless, K. B. (1985) J. Org. Chem. 50, 420-424]. A modification of that synthesis provided two other isomers, referred to here as "Glc-swainsonine" [(1S,2S,8R,8 alpha R)-trihydroxyindolizidine] and "Ido-swainsonine" [(1S,2S,8S,8 alpha R)-trihydroxyindolizidine]. To determine whether these new compounds had biological activity, they were compared to swainsonine as inhibitors of a number of commercially available glycosidases. While swainsonine is a potent inhibitor of jack bean alpha-mannosidase but does not inhibit other glycosidases, its two isomers were inactive on alpha-mannosidase but did inhibit other enzymes. Thus, Glc-swainsonine was an inhibitor of the fungal alpha-glucosidase amyloglucosidase, and this inhibition was of a competitive nature (Ki = 5 X 10(-5) M) with respect to the substrate p-nitrophenyl alpha-D-glucopyranoside. This alkaloid also inhibited beta-glucosidase, but much less effectively than alpha-glucosidase. On the other hand, Ido-swainsonine was more effective toward beta-glucosidase than toward alpha-glucosidase, and this inhibition was also of a competitive nature. None of these inhibitors were effective against beta-mannosidase or alpha- or beta-galactosidase. Glc-swainsonine was also tested against the glycoprotein processing glycosidases. Surprisingly, in this respect, the alkaloid was like swainsonine in that it inhibited mannosidase II but had no effect or only slight effect on glucosidase I, glucosidase II, and mannosidase I. Glc-swainsonine also inhibited glycoprotein processing in cell culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoesterase domains associated with DNA polymerases of diverse origins.

Computer analysis of DNA polymerase protein sequences revealed previously unidentified conserved domains that belong to two distinct superfamilies of phosphoesterases. The alpha subunits of bacterial DNA polymerase III and two distinct family X DNA polymerases are shown to contain an N-terminal domain that defines a novel enzymatic superfamily, designated PHP, after polymerase and histidinol phosphatase. The predicted catalytic site of the PHP superfamily consists of four motifs containing conserved histidine residues that are likely to be involved in metal-dependent catalysis of phosphoester bond hydrolysis. The PHP domain is highly conserved in all bacterial polymerase III alpha subunits, but in proteobacteria and mycoplasmas, the conserved motifs are distorted, suggesting a loss of the enzymatic activity. Another conserved domain, found in the small subunits of archaeal DNA polymerase II and eukaryotic DNA polymerases alpha and delta, is shown to belong to the superfamily of calcineurin-like phospho-esterases, which unites a variety of phosphatases and nucleases. The conserved motifs required for phospho-esterase activity are intact in the archaeal DNA polymerase subunits, but are disrupted in their eukaryotic orthologs. A hypothesis is proposed that bacterial and archaeal replicative DNA polymerases possess intrinsic phosphatase activity that hydrolyzes the pyrophosphate released during nucleotide polymerization. As proposed previously, pyrophosphate hydrolysis may be necessary to drive the polymerization reaction forward. The phosphoesterase domains with disrupted catalytic motifs may assume an allosteric, regulatory function and/or bind other subunits of DNA polymerase holoenzymes. In these cases, the pyrophosphate may be hydrolyzed by a stand-alone phosphatase, and candidates for such a role were identified among bacterial PHP superfamily members.     

Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea

N-linked glycosylation is recognized as an important post-translational modification across all three domains of life. However, the understanding of the genetic pathways for the assembly and attachment of N-linked glycans in eukaryotic and bacterial systems far outweighs the knowledge of comparable processes in Archaea. The recent characterization of a novel trisaccharide [beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc]N-linked to asparagine residues in Methanococcus voltae flagellin and S-layer proteins affords new opportunities to investigate N-linked glycosylation pathways in Archaea. In this contribution, the insertional inactivation of several candidate genes within the M. voltae genome and their resulting effects on flagellin and S-layer glycosylation are reported. Two of the candidate genes were shown to have effects on flagellin and S-layer protein molecular mass and N-linked glycan structure. Further examination revealed inactivation of either of these two genes also had effects on flagella assembly. These genes, designated agl (archaeal glycosylation) genes, include a glycosyl transferase (aglA) involved in the attachment of the terminal sugar to the glycan and an STT3 oligosaccharyl transferase homologue (aglB) involved in the transfer of the complete glycan to the flagellin and S-layer proteins. These findings document the first experimental evidence for genes involved in any glycosylation process within the domain Archaea.