Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thirteen high mannose isomers have been structurally characterized within three glycomers, Man(5)GlcNAc(2), Man(7)GlcNAc(2), and Man(8)GlcNAc(2) released from bovine ribonuclease B, six previously unreported. The study was carried out with a single ion trap instrument involving no chromatography. Three previously characterized isomers from Man(7) and Man(8) (three each) have been identified plus one unreported Man(7) isomer. Incomplete alpha-glucosidase activity on the Man(6) and Man(7) glycoproteins appears to account for two additional isomeric structures. The preeminence of ion traps for detail analysis was further demonstrated by resolving three new isomers within the Man(5) glycomer summing to the six previously unreported structures in this glycoprotein. All reported structures represent a distribution of Golgi processing remnants that fall within the Man(9)GlcNAc(2) footprint. Topologies were defined by ion compositions along a disassembly pathway while linkage and branching were aided by spectral identity in a small oligomer fragment library. Isomers from this glycoprotein appear to represent a distribution of Golgi processing remnants, and an alphanumeric classification scheme has been devised to identify all products. Although numerous analytical strategies have been introduced to identify selected components of structure, it has been the continued focus of this and previous reports to only build upon protocols that can be integrated into a high throughput strategy consistent with automation. Duplication of these and results from comparable standards could bring an important analytical focus to carbohydrate sequencing that is greatly lacking.
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PMID:The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap MS. 1918 40

Homogeneous N-glycoproteins carrying defined natural N-glycans are essential for detailed structural and functional studies. The transglycosylation activity of the endo-beta-N-acetylglucosaminidases from Arthrobacter protophormiae (Endo-A) and Mucor hiemalis (Endo-M) holds great potential for glycoprotein synthesis, but the wild-type enzymes are not practical for making glycoproteins carrying native N-glycans because of their predominant activity for product hydrolysis. In this article, we report studies of two endoglycosidase-based glycosynthases, EndoM-N175A and EndoA-N171A, and their usefulness in constructing homogeneous N-glycoproteins carrying natural N-glycans. The oligosaccharide oxazoline corresponding to the biantennary complex-type N-glycan was synthesized and tested with the two glycosynthases. The EndoM-N175A mutant was able to efficiently transfer the complex-type glycan oxazoline to a GlcNAc peptide and GlcNAc-containing ribonuclease to form the corresponding homogeneous glycopeptide/glycoprotein. The EndoA-N171A mutant did not recognize the complex-type N-glycan oxazoline but could efficiently use the high-mannose-type glycan oxazoline for transglycosylation. These mutants possess the transglycosylation activity but lack the hydrolytic activity toward the product. Kinetic studies revealed that the dramatically enhanced synthetic efficiency of the EndoA-N171A mutant was due to the significantly reduced hydrolytic activity toward both the Man(9)GlcNAc oxazoline and the product as well as to its enhanced activity for transglycosylation. Thus, the two mutants described here represent the first endoglycosidase-based glycosynthases enabling a highly efficient synthesis of homogeneous natural N-glycoproteins.
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PMID:Glycosynthases enable a highly efficient chemoenzymatic synthesis of N-glycoproteins carrying intact natural N-glycans. 1919 9

A simple, sensitive, and rapid quantitative LC-MS/MS assay was designed for the simultaneous quantification of free and glycoprotein bound monosaccharides using a multiple reaction monitoring (MRM) approach. This study represents the first example of using LC-MS/MS methods to simultaneously quantify all common glycoprotein monosaccharides, including neutral and acidic monosaccharides. Sialic acids and reduced forms of neutral monosaccharides are efficiently separated using a porous graphitized carbon column. Neutral monosaccharide molecules are detected as their alditol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids are detected as deprotonated ions [M - H](-). The new method exhibits very high sensitivity to carbohydrates with limits of detection as low as 1 pg for glucose, galactose, and mannose, and below 10 pg for other monosaccharides. The linearity of the described approach spans over three orders of magnitudes (pg to ng). The method effectively quantified monosaccharides originating from as little as 1 microg of fetuin, ribonuclease B, peroxidase, and alpha(1)-acid glycoprotein human (AGP) with results consistent with literature values and with independent CE-LIF measurements. The method is robust, rapid, and highly sensitive. It does not require derivatization or postcolumn addition of reagents.
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PMID:Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins. 1931 80

Glycoanalysis is important in the manufacture and quality control of protein therapeutics. An emerging method for glycoanalysis is the use of lectin arrays. Critical to the performance of these arrays is the immobilization of lectin molecules. Polydopamine has recently been shown to adsorb to a wide variety of surfaces. In this study, polydopamine (pDA) was used to modify gold, indium, and iridium surfaces and promote the adhesion of the alpha-mannose-specific lectin concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the alpha-mannose-presenting glycoprotein ribonuclease B (RNase B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selective affinity of RNase B for Con A. Surface-MALDI-TOF MS experiments revealed that the affinity of polydopamine-immobilized Con A for the glycoforms of RNase B is significantly affected by slight variations in oligosaccharide structure and composition. Specifically, surface-bound Con A binds certain Man7, Man8, and Man9 RNase B glycoforms more strongly than Man5 and Man6 glycoforms.
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PMID:Selective binding of RNase B glycoforms by polydopamine-immobilized concanavalin A. 1951 1

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.
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PMID:Identification and quantification of glycoproteins using ion-pairing normal-phase liquid chromatography and mass spectrometry. 1952 81

Lectins, a class of proteins that reversibly and non-enzymatically bind specific sugars, have been purified from different kinds of legumes. In this study, a 48-kDa lectin (KBL) was purified from Korean large black soybeans using liquid chromatography. The specific hemagglutinating activity of the KBL was 4096 titer/mg. EDTA-induced loss of hemagglutinating activity of KBL could be recovered by addition of Fe(3+) ions and some divalent cations as Ca(2+), Mn(2+), Fe(2+), Cu(2+), Zn(2+), and Pb(2+). Sugars such as D-(+)-galactose, D-(+)-raffinose, L-(+)-arabinose, alpha-D-(+)-melibiose, and alpha-lactose could inhibit the hemagglutinating activity of the lectin. Furthermore, the protein showed high thermal stability as well as stability over a wide range of pH values. KBL inhibited HIV-1 reverse transcriptase activity with an IC(50) of 1.38 microM. However, it was destitute of cytokine releasing, mitogenic, ribonuclease and antifungal activities. In addition, inhibitory activity toward nasopharyngeal cell lines was undetectable in KBL at concentrations up to 20 microM.
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PMID:Biochemical and functional properties of a lectin purified from korean large black soybeans--a cultivar of glycine max. 1971 33

Phospholipids are used as an additive in capillary electrophoresis to enhance the separation of glycans derived from alpha1-acid glycoprotein, fetuin, and ribonuclease B. The properties of phospholipid preparations are dependent upon composition, hydration, and temperature. Separation performance is evaluated as a function of these variables. A preparation of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), with [DMPC]/[DHPC] = 2.5, in 10% lipid/aqueous buffer at 25 degrees C provides the best separation efficiency at an electric field strength of 400 V/cm. Resolution is enhanced with the additive. Concanavalin A, a lectin selective for high mannose and mannose branching glycans, and alpha1-2,3 mannosidase, an enzyme that cleaves 1-2 and 1-3 mannopyranosyl residues, are incorporated in the separation to provide additional selectivity and to expand the application of phospholipid additives for glycan separation.
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PMID:Transformable capillary electrophoresis for oligosaccharide separations using phospholipid additives. 2007 30

BMY-28864 is a derivative of carbohydrate-binding antibiotic pradimicin A. This study aimed to examine the carbohydrate-binding specificity of BMY-28864 by a direct binding assay using neoglycolipids synthesized by conjugation of various oligosaccharides with dipalmitoylphosphatidylethanolamine (DPPE). Neoglycolipids were chromatographed on a thin layer chromatography plate and then subjected to BMY-28864 binding analysis. Binding of BMY-28864 to neoglycolipids such as Manalpha1-3Man-DPPE, Manalpha1-6Man-DPPE, Man3-DPPE, and Man5-DPPE and those bearing oligosaccharides derived from ribonuclease B, all of which bear mannose residues at the non-reducing ends, was detected. This study showed that pradimicin A derivative BMY-28864 selectively bound to mannose residues at the non-reducing ends and that neoglycolipids bearing various carbohydrate structures will be helpful as carbohydrate probes to detect carbohydrate-binding low molecular weight compounds.
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PMID:Binding of pradimicin A derivative BMY-28864 to neoglycolipids bearing mannose residues at the non-reducing ends. 2010 11

Regulatory agencies' expectations for biotherapeutic approval are becoming more stringent with regard to product characterization, where minor species as low as 0.1% of a given profile are typically identified. The mission of this manuscript is to demonstrate a multi-method approach toward de novo glycan characterization and quantitation, including minor species at or approaching the 0.1% benchmark. Recently, unexpected isomers of the Man(5)GlcNAc(2) (M(5)) were reported (Prien JM, Ashline DJ, Lapadula AJ, Zhang H, Reinhold VN. 2009. The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap mass spectrometry (MS). J Am Soc Mass Spectrom. 20:539-556). In the current study, quantitative analysis of these isomers found in commercial M(5) standard demonstrated that they are in low abundance (<1% of the total) and therefore an exemplary "litmus test" for minor species characterization. A simple workflow devised around three core well-established analytical procedures: (1) fluorescence derivatization; (2) online rapid resolution reversed-phase separation coupled with negative-mode sequential mass spectrometry (RRRP-(-)-MS(n)); and (3) permethylation derivatization with nanospray sequential mass spectrometry (NSI-MS(n)) provides comprehensive glycan structural determination. All methods have limitations; however, a multi-method workflow is an at-line stopgap/solution which mitigates each method's individual shortcoming(s) providing greater opportunity for more comprehensive characterization. This manuscript is the first to demonstrate quantitative chromatographic separation of the M(5) isomers and the use of a commercially available stable isotope variant of 2-aminobenzoic acid to detect and chromatographically resolve multiple M(5) isomers in bovine ribonuclease B. With this multi-method approach, we have the capabilities to comprehensively characterize a biotherapeutic's glycan array in a de novo manner, including structural isomers at >/=0.1% of the total chromatographic peak area.
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PMID:A multi-method approach toward de novo glycan characterization: a Man-5 case study. 2011 Feb 46

Many plant phytochemicals constitute binary enzyme-glucoside systems and function in plant defence. In brassicas, the enzyme myrosinase is confined to specific myrosin cells that separate the enzyme from its substrate; the glucosinolates. The myrosinase-catalysed release of toxic and bioactive compounds such as isothiocyanates, upon activation or tissue damage, has been termed 'the mustard oil bomb' and characterized as a 'toxic mine' in plant defence. The removal of myrosin cells and the enzyme that triggers the release of phytochemicals have been investigated by genetically modifying Brassica napus plants to remove myrosinase-storing idioblasts. A construct with the seed myrosin cell-specific Myr1.Bn1 promoter was used to express a ribonuclease, barnase. Transgenic plants ectopically expressing barnase were embryo lethal. Co-expressing barnase under the control of the Myr1.Bn1 promoter with the barnase inhibitor, barstar, under the control of the cauliflower mosaic virus 35S promoter enabled a selective and controlled death of myrosin cells without affecting plant viability. Ablation of myrosin cells was confirmed with light and electron microscopy, with immunohistological analysis and immunogold-electron microscopy analysis showing empty holes where myrosin cells normally are localized. Further evidence for a successful myrosin cell ablation comes from immunoblots showing absence of myrosinase and negligible myrosinase activity, and autolysis experiments showing negligible production of glucosinolate hydrolysis products. The plants where the myrosin defence cells have been ablated and named 'MINELESS plants'. The epithiospecifier protein profile and glucosinolate levels were changed in MINELESS plants, pointing to localization of myrosinases and a 35 kDa epithiospecifier protein in myrosin cells and a reduced turnover of glucosinolates in MINELESS plants.
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PMID:Removing the mustard oil bomb from seeds: transgenic ablation of myrosin cells in oilseed rape (Brassica napus) produces MINELESS seeds. 2021 77


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