Isolation and Characterization of a Bacillus subtilis Mutant with a Defective N-Glycosidase Activity for Uracil-Containing Deoxyribonucleic Acid

Crude cell extracts of Bacillus subtilis 168T exhibit enzyme activity capable of releasing free uracil from phage PBS1 deoxyribonucleic acid (DNA) in the presence of ethylenediaminetetraacetate. By measuring the enzyme activity in 300 clones that emanated from mutagenized cells, we obtained a mutant strain that did not show this N-glycosidase activity. The mutant strain, designated as TKJ6901 (urg-1) exhibited no physiological abnormalities. We observed the intracellular action of the enzyme by following the fate of uracil-containing DNA in cells from wild-type and mutant cultures. When infection with phage PBS1 was allowed in the presence of chloramphenicol, extensive degradation of phage DNA was observed only in the wild-type cells. When bromouracil residues were converted to uracil residues by ultraviolet light irradiation in the presence of cysteamine, the DNA was extensively fragmented in the wild-type cells. These single-strand breaks were rejoined upon postirradiation incubation. In contrast, such fragmentation of the DNA was not observed in the mutant cells, indicating that the uracil residues were not removed from the DNA. This demonstrated that the N-glycosidase activity was involved in the excision of uracil in DNA. A transformation assay with four types of recipient strains with combinations of N-glycosidase and DNA polymerase I deficiencies indicated that DNA polymerase I was involved in the later steps of this base excision repair pathway initiated by the action of the N-glycosidase.     

Excision repair of uracil incorporated in DNA as a result of a defect in dUTPase.

Mutants of Escherichia coli that are severely defective in the enzyme dUTPase (dut) accumulate short (4 to 5 S) Okazaki fragments following brief pulses with [³H]thymidine. The transient appearance of DNA fragments in these mutants is plausibly explained by the misincorporation of uracil in DNA as a result of an increase in available dUTP, followed by its rapid excision and repair. The evidence in support of this interpretation is the following: (1) accumulation of short DNA fragments can be partially suppressed by a mutation in dCTP deaminase, presumably by decreasing the intracellular level of dUTP relative to dTTP; (2) accumulation of the short DNA fragments can be almost completely suppressed by a mutation in uracil N-glycosidase, probably by preventing the introduction of nicks at the sites of uracil incorporation; (3) introduction of DNA polymerase I or DNA ligase mutations into dUTPase-defective strains results in the persistence of the 4 to 5 S fragments and rapid cessation of DNA synthesis. Uracil N-glycosidase, DNA polymerase I and DNA ligase must therefore be involved in the excision repair of uracil-containing DNA.     

Identification and Characterization of a Novel Prokaryotic Peptide: N-GLYCOSIDASE FROM ELIZABETHKINGIA MENINGOSEPTICA*

Peptide:N-glycosidase (PNGase) F, the first PNGase identified in prokaryotic cells, catalyzes the removal of intact asparagine-linked oligosaccharide chains from glycoproteins and/or glycopeptides. Since its discovery in 1984, PNGase F has remained as the sole prokaryotic PNGase. Recently, a novel gene encoding a protein with a predicted PNGase domain was identified from a clinical isolate of Elizabethkingia meningoseptica. In this study, the candidate protein was expressed in vitro and was subjected to biochemical and structural analyses. The results revealed that it possesses PNGase activity and has substrate specificity different from that of PNGase F. The crystal structure of the protein was determined at 1.9 Å resolution. Structural comparison with PNGase F revealed a relatively larger glycan-binding groove in the catalytic domain and an additional bowl-like domain with unknown function at the N terminus of the candidate protein. These structural and functional analyses indicated that the candidate protein is a novel prokaryotic N-glycosidase. The protein has been named PNGase F-II.     

Cuticle proteins of the boll weevil,Anthonomus grandis,abdomen: Structural similarities and glycosylation

Cuticle proteins are thought to be important in defining the structural and functional differences occurring in insect cuticle. In order to explain and better understand the structural similarities among the cuticle proteins of the cotton boll weevil, Anthonomus grandis Boheman, described in a previous study (Stiles and Leopold, 1990, Insect Biochem.20, 113–125) three series of monoclonal antibody producing hybridoma cell lines were produced. Larval, pupal or adult cuticle proteins were used as antigens. While some of the monoclonal antibodies were specific for one or two cuticle proteins from a single developmental stage, the majority showed multiple cuticle protein binding patterns on Western blots. To determine whether this cross-reaction was due to common oligosaccharide chains bound to the proteins, lectins were used to probe Western blots. Many of the cuticle proteins were found to be glycosylated. The majority of the Con A reactive carbohydrate could be removed from the protein by N-glycosidase F digestion (specific for N-asparagine linked carbohydrate). N-glycosidase F digestion did not reduce the multiple cross-reactions of the monoclonal antibodies, nor did periodate oxidation of the CP. The carbohydrate remaining after enzyme digestion is presumably O-linked to serine/threonine.     

Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies

Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.     

Plant N-glycan profiling of minute amounts of material

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring α(1,3)- or α(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.     

Expression,purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system

The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial O-glycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative.     

Detection of glycoproteins in Borrelia burgdorferi

The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two major outer surface exposed proteins of 31 kDa and 34 kDa showed to be glycosylated and gel filtration high pressure liquid chromatography (HPLC) of proteins of B. burgdorferi metabolically labeled with ¹⁴C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.Abbreviations N-glycosidase F peptide-N-glycosidase F (EC 3.5.1.52) - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WB Western blotting - HPLC high pressure liquid chromatography - SDS sodium dodecyl sulphate - mAb monoclonal antibody - MIAF mouse immune ascitic fluid - SS Schiff staining - Osp Outer surface protein     

A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the α-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease.     

High-throughput quantitative analysis of plant N-glycan using a DNA sequencer

High-throughput quantitative analytical method for plant N-glycan has been developed. All steps, including peptide N-glycosidase (PNGase) A treatment, glycan preparation, and exoglycosidase digestion, were optimized for high-throughput applications using 96-well format procedures and automatic analysis on a DNA sequencer. The glycans of horseradish peroxidase with plant-specific core α(1,3)-fucose can be distinguished by the comparison of the glycan profiles obtained via PNGase A and F treatments. The peaks of the glycans with (91%) and without (1.2%) α(1,3)-fucose could be readily quantified and shown to harbor bisecting β(1,2)-xylose via simultaneous treatment with α(1,3)-mannosidase and β(1,2)-xylosidase. This optimized method was successfully applied to analyze N-glycans of plant-expressed recombinant antibody, which was engineered to contain a minor amount of glycan harboring β(1,2)-xylose. These results indicate that our DNA sequencer-based method provides quantitative information for plant-specific N-glycan analysis in a high-throughput manner, which has not previously been achieved by glycan profiling based on mass spectrometry.     

Localization of Rabies Virus Glycoprotein into the Endoplasmic Reticulum Produces Immunoprotective Antigen

Rabies virus surface glycoprotein (rabies G-protein) with (G+RS) and without (G?RS) endoplasmic reticulum retrieval signal was expressed and characterized in tobacco plants. Transgenically expressed rabies G-protein was estimated at 0.015?C0.38?% of total leaf protein. The relative migration of the rabies G-protein on SDS-PAGE was at the position, as anticipated for the viral coat protein (~66?kDa). Immunolocalization by confocal microscopy established that immunoprotective G+RS expressed in tobacco was primarily confined to ER. G+RS showed binding to Con A lectin and was susceptible to N-glycosidase F activity similar to native rabies G-protein. However, the G?RS transgenically expressed in tobacco leaves was glycosylated differently and was resitant to N-glycosidase F. Immunological studies and Rapid Fluorescent Foci Inhibition Test (RFFIT) showed that G+RS was immunogenic and immunoprotective, whereas G?RS was moderately immunogenic but non-protective against live virus challenge. Hence, plants can express the antigenic component of rabies virus with suitable glycosylation, which is important to give protection against rabies virus infection.     

A new series of N2-substituted-5-(p-toluenesulfonylamino)phthalimide analogues as α-glucosidase inhibitors

Several members of a new family of non-sugar-type α-glycosidase inhibitors, bearing a 5-(p-toluenesulfonylamino)phthalimide moiety and various substituent at the N2 position, were synthesized and their activities were investigated. The newly synthesized compounds displayed different inhibition profile towards yeast α-glycosidase and rat intestinal α-glycosidase. Almost all the compounds had strong inhibitory activities against yeast α-glycosidase. Regarding rat intestinal α-glycosidase, only analogs with N2-aromatic substituents displayed varying degrees of inhibitory activities on rat intestinal maltase and lactase and nearly all compounds showed no inhibition against rat intestinal α-amylase. Structure–activity relationship studies indicated that 5-(p-toluenesulfonylamino)phthalimide moiety is a favorable scaffold to exert the α-glucosidase inhibitory activity and substituents at the N2 position have considerable influence on the efficacy of the inhibition activities.     

Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry

A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate–peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI–MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies.     

4-Aminopyrazolo[3,4-d]pyrimidine (4-APP) as a novel inhibitor of the RNA and DNA depurination induced by Shiga toxin 1

Shiga toxin 1 (Stx1) catalyses the removal of a unique and specific adenine from 28S RNA in ribosomes (RNA-N-glycosidase activity) and the release of multiple adenines from DNA (DNA glycosylase activity). Added adenine behaves as an uncompetitive inhibitor of the RNA-N-glycosidase reaction binding more tightly to the Stx1–ribosome complex than to the free enzyme. Several purine derivatives and analogues have now been assayed as inhibitors of Stx1. Most of the compounds showed only minor differences in the rank order of activity on the two enzymatic reactions catalysed by Stx1. The survey highlights the importance of the amino group in the 6-position of the pyrimidine ring of adenine. Shifting (2-aminopurine) or substituting (hypoxanthine, 6-mercaptopurine, 6-methylpurine) the group greatly decreases the inhibitory power. The presence of a second ring, besides the pyrimidine one, is strictly required. Substitution, by introducing an additional nitrogen, of the imidazole ring of adenine with triazole leads to loss of inhibitory power, while rearrangement of the nitrogen atoms of the ring from the imidazole to the pyrazole configuration greatly enhances the inhibitory power. Thus 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), the isomer of adenine with the five-membered ring in the pyrazole configuration, is by far the most potent inhibitor of both enzymatic reactions catalysed by Stx1. This finding opens perspectives on therapeutic strategies to protect endothelial renal cells once endocytosis of Stx1 has occurred (haemolytic uraemic syndrome). In the RNA-N-glycosidase reaction 4-APP binds, as adenine, predominantly to the Stx1–ribosome complex (uncompetitive inhibition), while inhibition of the DNA glycosylase activity by both inhibitors is of the mixed type.     

Novel 2-aminopyridine liganded Pd(II) N-heterocyclic carbene complexes: Synthesis,characterization, crystal structure and bioactivity properties

In this work, the synthesis, crystal structure, characterization, and enzyme inhibition effects of the novel a series of 2-aminopyridine liganded Pd(II) N-heterocyclic carbene (NHC) complexes were examined. These complexes of the Pd-based were synthesized from PEPPSI complexes and 2-aminopyridine. The novel complexes were characterized by using ¹³C NMR, ¹H NMR, elemental analysis, and FTIR spectroscopy techniques. Also, crystal structures of the two compounds were recorded by using single-crystal X-ray diffraction assay. Also, these complexes were tested toward some metabolic enzymes like α-glycosidase, aldose reductase, butyrylcholinesterase, acetylcholinesterase enzymes, and carbonic anhydrase I, and II isoforms. The novel 2-aminopyridine liganded (NHC)PdI₂(2-aminopyridine) complexes (1a-i) showed Ki values of in range of 5.78 ± 0.33–22.51 ± 8.59 nM against hCA I, 13.77 ± 2.21–30.81 ± 4.87 nM against hCA II, 0.44 ± 0.08–1.87 ± 0.11 nM against AChE and 3.25 ± 0.34–12.89 ± 4.77 nM against BChE. Additionally, we studied the inhibition effect of these derivatives on aldose reductase and α-glycosidase enzymes. For these compounds, compound 1d showed maximum inhibition effect against AR with a Ki value of 360.37 ± 55.82 nM. Finally, all compounds were tested for the inhibition of α-glycosidase enzyme, which recorded efficient inhibition profiles with Ki values in the range of 4.44 ± 0.65–12.67 ± 2.50 nM against α-glycosidase.     

Development and application of mass spectrometric methods for the analysis of progranulin N-glycosylation

PGRN is a modular protein with 7 1/2 repeats of the granulin domain separated by short spacer sequences. Elevated expression of PGRN is associated with cancer growth, while mutations of PGRN cause frontotemporal lobar degeneration (FTLD), an early onset form of dementia. PGRN is a glycoprotein, containing five N-glycosylation consensus sequons, three of which fall within granulin domains. A method tailored to enable detailed analysis of the PGRN oligosaccharides and glycopeptides has been developed. The approach involves in-gel deglycosylation using peptide-N-glycosidase F (PNGase F) followed by permethylation of the released oligosaccharides. Permethylation was applied for rapid sample clean-up and to improve sensitivity of MS detection and mass spectrometric fragmentation. Reversed-phase monolithic LC–ESI–MS/MS was used for analysis of permethylated oligosaccharides, enabling structural characterization of released N-linked glycans in one chromatographic run. In-gel tryptic digestion was further applied to the gel pieces containing deglycosylated protein, for N-glycosylation site determination. In addition, glycopeptides were produced using in-solution pronase digestion to identify species of N-glycan attached at particular sites. The method developed was applied to progranulin (PGRN) to characterize the structures of the released glycans and to identify the sites of glycosylation. Glycosylation of four out of five potential PGRN N-glycosylation consensus sites was demonstrated (the final one remains undetermined), with one of the four observed to be partially occupied. Two of the observed glycosylation sites occur within granulin domains, which may have important implications for understanding the structural basis of PGRN action.     

Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.Revisions requested 30 September 2004; Revisions received 22 October 2004     

Dolichylphosphate-dependent Glycosyl Transfer Reactions in the Endoplasmic Reticulum of Castor Bean Endosperm

In the endosperm of Ricinus communis (castor bean) a number of glycosyl transferases were found to be present during germination. They catalyze the incorporation of mannose from guanosine diphosphate mannose and of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine into a glycolipid fraction, which had all of the properties of dolichylphosphate and pyrophosphate sugars, respectively. The sugar moiety of dolichylphosphate mannose is transferred to a lipid-oligosaccharide, containing more than 6 hexose units. When the membranes are preincubated with nonradioactive guanosine diphosphate mannose and uridine diphosphate N-acetylglucosamine, radioactivity from dolichylphosphate [¹⁴C]mannose is also transferred to a glycopolymer. In addition, the formation of radioactive glycoproteins from guanosine diphosphate [¹⁴C]mannose has been demonstrated using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography.     

Mixed function oxidases from germinating castor bean endosperm

Homogenates from germinating castor bean endosperm were fractionated by sucrose density gradient centrifugation and examined for mixed function oxidase activity. Activity of cinnamic acid 4-hydroxylase and p-chloro-N-methylaniline N-demethylase was highest in the endoplasmic reticulum fraction. Activity of both enzymes is dependent on NADPH and on molecular oxygen; both activities are inhibited by carbon monoxide. When challenged with a number of potential inhibitors the enzymes responded in ways fairly typical of mixed function oxidases from other plants and animals. The N-demethylase appears to be specific for N-methylarylamines. In the absence of NADPH, cumene hydroperoxide is able to support N-demethylation. The mechanistic significance of this activity is discussed.     

Altered N-glycan profile of IgG-depleted serum proteins in Hashimoto's thyroiditis

BackgroundHashimoto's thyroiditis (HT) is an autoimmune disease characterized by chronic inflammation of thyroid gland. Although HT is the most common cause of hypothyroidism, the pathogenesis of this disease is not fully understood. Glycosylation of serum proteins was examined in HT only to a limited extent. The study was designed to determine the glycosylation pattern of IgG-depleted sera from HT patients.MethodsSerum N-glycans released by N-glycosidase F (PNGase F) digestion were analyzed by normal-phase high-performance liquid chromatography (NP-HPLC). N-glycan structures in each collected HPLC fraction were determined by liquid chromatography-mass spectrometry (LC-MS) and exoglycosidase digestion. Fucosylation and sialylation was also analyzed by lectin blotting.ResultsThe results showed an increase of monosialylated tri-antennary structure (A3G3S1) and disialylated diantennary N-glycan with antennary fucose (FA2G2S2). Subsequently, we analyzed the serum N-glycan profile by lectin blotting using lectins specific for fucose and sialic acid. We found a significant decrease of Lens culinaris agglutinin (LCA) staining in HT samples, which resulted from the reduction of α1,6-linked core fucose in HT serum. We also observed an increase of Maackia amurensis II lectin (MAL-II) reaction in HT due to the elevated level of α2,3-sialylation in HT sera.ConclusionsThe detected alterations of serum protein sialylation might be caused by chronic inflammation in HT. The obtained results complete our previous IgG N-glycosylation analysis in autoimmune thyroid patients and show that the altered N-glycosylation of serum proteins is characteristic for autoimmunity process in HT.General SignificanceThyroid autoimmunity is accompanied by changes of serum protein sialylation.