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The enzymic degradation of ovalbumin and its glycopeptides.

ABSTRACT: 1. Ovalbumin glycopeptides, freed from all amino acids other than aspartic acid and a small proportion of leucine by repeated digestion with Pronase, were hydrolysed by 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (glycoaspartamidase) to the corresponding oligosaccharides. The glycoaspartamidase did not attack ovalbumin itself. 2. Ovalbumin, with mannose/hexosamine ratio 5:4, lost 1.5moles of N-acetylglucosamine and more than 2moles of mannose after incubation with alpha-mannosidase and beta-N-acetylglucosaminidase respectively. 3. In ovalbumin glycopeptides with approximate mannose/hexosamine ratios 5:3 and 5:4, one and two N-acetylglucosamine residues respectively were accessible to the action of beta-N-acetylglucosaminidase. 4. A mixture of alpha-mannosidase and beta-N-acetylglucosaminidase, acting on an ovalbumin glycopeptide with mannose/hexosamine ratio 5:3.7, removed nearly 4moles of mannose and 1.5moles of N-acetylglucosamine. 5. alpha-Mannosidase removed about 1.5moles of mannose from the ovalbumin oligosaccharide with mannose/hexosamine ratio approx. 5:3. The subsequent action of beta-N-acetylglucosaminidase liberated less than 1mole of N-acetylglucosamine and made at least 1mole further of mannose accessible to alpha-mannosidase action. 6. It is concluded that the carbohydrate moiety of ovalbumin is linked through a glycosyl group to asparagine. In a molecule with mannose/hexosamine ratio 5:4, there are two beta-N-acetylglucosamine residues linked together in a terminal position, followed by alpha-mannose. There is also present a side chain containing two alpha-mannose units.

SUBMITTER: Conchie J

PROVIDER: S-EPMC1185198 | biostudies-other | 1969 Dec

REPOSITORIES: biostudies-other

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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.

Project description:1. Cell-free extracts, prepared from a non-fluorescent Pseudomonas grown on m-cresol, oxidized gentisate and certain alkyl-substituted gentisates with the consumption of 1 mol of oxygen and the formation of 1 mol of pyruvate from 1 mol of substrate. 2. In addition to pyruvate, malate was formed from gentisate; citramalate was formed from 3-methylgentisate and 4-methylgentisate; 2,3-dimethylmalate was formed from 3,4-dimethylgentisate. 3. One enantiomer, d-(-)-citramalate, was formed enzymically from 3-methylgentisate, 4-methylgentisate and citraconate. l-(+)-Citramalate was formed from mesaconate by the same extracts. When examined as its dimethyl ester by gas-liquid chromatography, enzymically formed 2,3-dimethylmalate showed the same behaviour as one of the two racemates prepared from the synthetic compound. 4. Maleate, citraconate and 2,3-dimethylmaleate were rapidly hydrated by cell extracts, but ethylfumarate and 2,3-dimethylfumarate were not attacked. 5. Cell extracts oxidized 1,4-dihydroxy-2-naphthoate to give pyruvate and phthalate. 6. Alkylgentisates were oxidized by a gentisate oxygenase (EC 1.13.1.4) present in Pseudomonas 2,5. The ring-fission products were attacked by maleylpyruvase, but not by fumarylpyruvase, and their u.v.-absorption spectra were those expected for alkyl-substituted maleylpyruvates. 7. When supplemented with ATP, CoA, succinate and Mg(2+) ions, an enzyme system from cells grown with 2,5-xylenol formed pyruvate from d- but not from l-citramalate. Extracts from cells grown with dl-citramalate or with itaconate attacked both d- and l-citramalate; other alkylmalates were cleaved in similar fashion to give pyruvate or 2-oxobutyrate. 8. These results accord with a general sequence of reactions in which the benzene nucleus of an alkylgentisate is cleaved to give an alkyl-substituted maleylpyruvate. The ring-fission products are hydrolysed to give pyruvate, plus alkylmalic acids which then undergo aldol fissions, probably as their CoA esters. In Pseudomonas 2,5 several homologous sequences of this general type appear to be catalysed by a single battery of enzymes with broad substrate specificities, whereas the metabolic capabilities of the fluorescent Pseudomonas 3,5 are more restricted. 9. Intact cells of both organisms metabolize d-malic acid by reactions that have not been elucidated, but are different from those which degrade alkylmalates.

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The enzymic degradation of ovalbumin and its glycopeptides.

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