STUDIES ON A GLYCOPEPTIDE FROM OVALBUMIN.
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ABSTRACT:
SUBMITTER: CLAMP JR
PROVIDER: S-EPMC1206535 | biostudies-other | 1965 Feb
REPOSITORIES: biostudies-other
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Project description:1. The denaturation of ovalbumin by the reagents sodium dodecyl sulphate and guanidinium chloride was investigated, by following the changes in sedimentation velocity, optical rotatory dispersion and viscosity as a function of denaturant concentration. 2. With sodium dodecyl sulphate both the optical-rotatory-dispersion parameters a(0) and b(0) become more negative, the sedimentation coefficient decreases and the viscosity increases; significant differences in the denaturation profiles are observed. The change in each parameter is indicative of only limited denaturation. 3. With guanidinium chloride the transition occurs over the concentration range 1-4m: more extensive changes occur in all the physical parameters than with sodium dodecyl sulphate. The values of a(0) and b(0) are indicative of complete denaturation. Reduction by mercaptoethanol produces only minor further changes. 4. Renaturation was attempted from both denaturants, the removal of reagent being accomplished reversibly by controlled slow dialysis. Partial renaturation was observed, but aggregated or insoluble material was produced in both cases at relatively low concentrations of denaturant. Similar behaviour was observed with fully reduced protein in guanidinium chloride-mercaptoethanol; complete renaturation could not be brought about even at very low protein concentrations.
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Project description:Aberrant glycosylation of ?-dystroglycan (?-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of ?-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from ?-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid-phase glycopeptide synthesis using hyper-acid-sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide, and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme POMGnT1, which attaches ?(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-glycan processing on ?-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated ?-DG.
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Project description:The mechanism of high-level resistance to vancomycin in enterococci consists of the synthesis of peptidoglycan terminating in D-alanyl-D-lactate instead of the usual D-alanyl-D-alanine. This alternate cell wall biosynthesis pathway is ensured by the collective actions of three enzymes: VanH, VanA, and VanX. The origin of this resistance mechanism is unknown. We have cloned three genes encoding homologs of VanH, VanA, and VanX from two organisms which produce glycopeptide antibiotics: the A47934 producer Streptomyces toyocaensis NRRL 15009 and the vancomycin producer Amycolatopsis orientalis C329.2. The predicted amino acid sequences are highly similar to those found in VRE: 54 to 61% identity for VanH, 59 to 63% identity for VanA, and 61 to 64% identity for VanX. Furthermore, the orientations of the genes, vanH, vanA, and vanX, are identical to the orientations found in vancomycin-resistant enterococci. Southern analysis of total DNA from other glycopeptide-producing organisms, A. orientalis 18098 (chloro-eremomycin producer), A. orientalis subsp. lurida (ristocetin producer), and Amycolatopsis coloradensis subsp. labeda (teicoplanin and avoparcin producer), with a probe derived from the vanH, vanA, and vanX cluster from A. orientalis C329.2 revealed cross-hybridizing DNA in all strains. In addition, the vanH, vanA, vanX cluster was amplified from all glycopeptide-producing organisms by PCR with degenerate primers complementary to conserved regions in VanH and VanX. Thus, this gene sequence is common to all glycopeptide producers tested. These results suggest that glycopeptide-producing organisms may have been the source of resistance genes in vancomycin-resistant enterococci.
| S-EPMC105782 | biostudies-literature
Project description:Allergy is a major public health concern, the main treatment for which is symptomatic relief with anti-inflammatory drugs. A key clinical challenge is to induce specific tolerance in order to control allergen-specific memory B and T cells, and specifically block effector cell responses. Our lab recently developed antigen-specific regulatory T-cell (Treg) therapies as a treatment for adverse responses. Recently, we created a chimeric antigen receptor (CAR) approach in which we engineered a target protein antigen, ovalbumin (OVA), linked with the transmembrane and signal transduction domains, CD28-CD3? to directly target B cells and sensitized mast cells in an allergy model. We named this receptor "BAR" for B-cell Antibody Receptor. Murine or human Tregs, transduced with a BAR containing OVA or control Tregs expressing an unrelated antigen, were successfully expanded in vitro and tested in the murine OVA-alum allergy model with measurable titers of anti-OVA IgE. Because BAR Tregs express the target antigen and could interact with specific IgE on sensitized mast cells, we first demonstrated that intravenously injected OVA-BAR Tregs did not directly lead to a drop in temperature or release of mediators in plasma indicative of anaphylaxis. Forty-eight hours later, mice were challenged intraperitoneally with 200??g OVA to induce an anaphylactic reaction, and temperature immediately measured for 30?min. We found that OVA-BAR Tregs protected mice from hypothermia, whereas mice given control BARs (expressing an unrelated antigen) or PBS showed substantial temperature drops indicative of anaphylaxis when systemically challenged with OVA. Importantly, this effect was also demonstrated in a passive anaphylaxis model in which mice that received anti-OVA IgE antibody were protected from hypothermia when treated with OVA-BAR Tregs prior to systemic OVA challenge. These results provide proof of principle that engineered allergen-specific T-regulatory cells can provide clinical protection against severe allergic reactions in individuals already IgE-sensitized to an allergen.
| S-EPMC6742773 | biostudies-literature
Project description:Glycopeptide antibiotics (GPAs) are a key weapon in the fight against drug resistant bacteria, with vancomycin still a mainstream therapy against serious Gram-positive infections more than 50 years after it was first introduced. New, more potent semisynthetic derivatives that have entered the clinic, such as dalbavancin and oritavancin, have superior pharmacokinetic and target engagement profiles that enable successful treatment of vancomycin-resistant infections. In the face of resistance development, with multidrug resistant (MDR) S. pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) together causing 20-fold more infections than all MDR Gram-negative infections combined, further improvements are desirable to ensure the Gram-positive armamentarium is adequately maintained for future generations. A range of modified glycopeptides has been generated in the past decade via total syntheses, semisynthetic modifications of natural products, or biological engineering. Several of these have undergone extensive characterization with demonstrated in vivo efficacy, good PK/PD profiles, and no reported preclinical toxicity; some may be suitable for formal preclinical development. The natural product monobactam, cephalosporin, and β-lactam antibiotics all spawned multiple generations of commercially and clinically successful semisynthetic derivatives. Similarly, next-generation glycopeptides are now technically well positioned to advance to the clinic, if sufficient funding and market support returns to antibiotic development.
| S-EPMC5952257 | biostudies-literature
Project description:To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over‑expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper‑mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. Our proteome and transcriptome analyses have been performed during stationary‑phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets. Keywords: Molecular markers, antibiotic resistance, glycopeptides, growth-phase
2007-03-09 | GSE5188 | GEO
Project description:One hundred thirty-two glycopeptide-resistant Enterococcus faecium (GREF) isolates from different hospitals and pig and poultry farms in Belgium were compared on the basis of (i) their antibiotic susceptibilities, (ii) their SmaI pulsed-field gel electrophoresis (PFGE) patterns, and (iii) the organization of their Tn1546 or related elements in order to detect possible phenotypic and genotypic relationships among both groups of isolates. Human and animal vanA-positive GREF isolates were found to have similar susceptibility patterns; they remained susceptible to gentamicin and were, in general, susceptible to ampicillin. PFGE demonstrated a very high degree of genomic heterogeneity in both groups of isolates. However, indistinguishable isolates were found within different farms or hospitals, and in two instances, epidemiologically unrelated pig and human isolates showed indistinguishable PFGE patterns. In total, eight different transposon types were identified, and all were related to the prototype transposon Tn1546. The two predominant types, Tn1546 and type 2 transposons, which differed at three band positions, were present in both human and animal isolates. Type 2 transposons were significantly associated with pig isolates. The other types were seldom detected. These data suggest a possible exchange of glycopeptide resistance markers between animals and humans.
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Project description:The incidence of antibiotic resistance among pathogenic microorganisms is increasing at an alarming rate. Resistance against front-line therapeutics such as the glycopeptide antibiotic vancomycin has emerged and has spread to highly virulent pathogens, including Staphylococcus aureus. Glycopeptide antibiotics are natural products from the Actinomycetes that have a characteristic heptapeptide core. The chemical diversity of the class is achieved through glycosylation, halogenation, methylation, and acylation of the core, modifications that are implicated in improved solubility, stability, or activity of the molecule. Sulfation is yet another modification observed infrequently in glycopeptides, but its role is not known. Although glycopeptide sulfotransferases are found in the environmental metagenome and must therefore serve an evolutionary purpose, all previous studies have reported decreased antibiotic activity with sulfation. We report that sulfation of glycopeptides has little effect on the compound's ability to bind its target, the d-Ala-d-Ala peptidoglycan precursors of the bacterial cell wall. However, sulfation does impact glycopeptide dimerization, and importantly, sulfated glycopeptides are significantly less potent inducers of the resistance gene cluster vanHAX in actinomycetes. Our results begin to unravel the mystery of the biological role of glycopeptide sulfation and offer a potential new strategy for the development of new antibiotics that avoid resistance.
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Project description:1. The isolation, characterization and properties of two by-products in the preparation of 2-acetamido-3,4,6-tri-O- acetyl-2-deoxy-beta-d-glucopyranosylamine are described. They are bis(2-acetamido-2-deoxy-d-glucopyranosyl)amines. 2. An independent synthesis of the bis-glycopyranosylamines is reported and conditions are given for their preparation in high yield. 3. Further improvements are given for the synthesis of 2-acetamido-1-N-(beta-l- aspartyl)-2-deoxy-beta-d-glucopyranosylamine and the alpha-l-aspartyl isomer. 4. The synthesis of 2-acetamido-1-N-acetyl-2-deoxy-beta-d-glucopyranosylamine is described.
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Project description:Historically, researchers have put considerable effort into developing automation systems to prepare natural biopolymers such as peptides and oligonucleotides. The availability of such mature systems has significantly advanced the development of natural science. Over the past twenty years, breakthroughs in automated synthesis of oligosaccharides have also been achieved. A machine-driven platform for glycopeptide synthesis by a reconstructed peptide synthesizer is described. The designed platform is based on the use of an amine-functionalized silica resin to facilitate the chemical synthesis of peptides in organic solvent as well as the enzymatic synthesis of glycan epitopes in the aqueous phase in a single reaction vessel. Both syntheses were performed by a peptide synthesizer in a semiautomated manner.
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