Presence Of Dityrosine Research Articles

Formation of di-Tyrosine in pasteurized milk during shelf storage

Formation of the protein crosslink di-Tyrosine was studied in PET-bottled pasteurized milk exposed to fluorescent light in a commercial display cabinet. An HPLC method with fluorescence detection was developed and intra-laboratory validated using pure di-Tyrosine synthesized on purpose. Di-Tyrosine was detected after 1-day lightening and increased up to 7 days, reaching around 250 and 320 µg/g protein in whole and partly skimmed milk, respectively. Afterward, a progressive decrease occurred. By transmission electron microscopy with specific immune gold labelling, presence of di-Tyrosine was observed for the first time on the surface of casein micelles of lightened milk. The crosslink formation, however, did not bring to protein aggregation phenomena detectable by laser light scattering measurements. Exposure to light also induced degradation of riboflavin and decrease of yellowness index. Di-Tyrosine proved to be a suitable indicator to evaluate the progress of protein oxidation in pasteurized milk during storage on the market.

ROLE OF PEROXIDASE AND H2O2 IN CROSS-LINKING OF GLUTEN PROTEINS

ABSTRACT The extent of protein cross-linking in wheat dough influences the viscoelastic properties of dough. We have studied peroxidase-mediated cross-linking of proteins in in vitro system using isolated wheat glutenin protein subunits. Isolated glutenin subunits were treated with peroxidase/H2O2 system as well as with H2O2 alone. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of treated subunits under reducing and nonreducing conditions revealed that peroxidase/H2O2 was able to cross-link glutenin protein subunits by disulfide bonds and nondisulfide covalent cross-links. The fluorescence spectrum of cross-linked proteins indicated the presence of dityrosine. On the other hand, treatment of glutenin protein subunits with H2O2 alone formed disulfide cross-links only. Peroxidase purified from wheat bran showed thiol oxidase activity. By virtue of thiol oxidizing and peroxidatic activity, wheat bran peroxidase may be able to form disulfide and dityrosine cross-links in glutenin subunits. Based on the results, we have proposed a mechanism for the role of peroxidase and H2O2 in cross-linking gluten proteins. PRACTICAL APPLICATIONS Wheat milling industry generates a large amount of bran and it is a waste by-product. Peroxidase isolated from wheat bran cross-linked the glutenin protein subunits. Therefore, wheat bran peroxidase can be used as a dough improver to achieve required dough properties. The knowledge of role of peroxidase in dough formation could be used in wheat breeding programs to develop new wheat varieties. Gluten is a potential source for the manufacture of biodegradable films, and peroxidase could be a tool to control cross-linking gluten properties to obtain desired biofilm characteristics.

Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima

Apicomplexan parasites such as Eimeria maxima possess a resilient oocyst wall that protects them upon excretion in host faeces and in the outside world, allowing them to survive between hosts. The wall is formed from the contents of specialised organelles – wall-forming bodies – found in macrogametes of the parasites. The presence of dityrosine in the oocyst wall suggests that peroxidase-catalysed dityrosine cross-linking of tyrosine-rich proteins from wall-forming bodies forms a matrix that is a crucial component of oocyst walls. Bioinformatic analyses showed that one of these tyrosine-rich proteins, EmGAM56, is an intrinsically unstructured protein, dominated by random coil (52–70%), with some α-helix (28–43%) but a relatively low percentage of β-sheet (1–11%); this was confirmed by nuclear magnetic resonance and circular dichroism. Furthermore, the structural integrity of EmGAM56 under extreme temperatures and pH indicated its disordered nature. The intrinsic lack of structure in EmGAM56 could facilitate its incorporation into the oocyst wall in two ways: first, intrinsically unstructured proteins are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGAM56; and, second, its flexibility could facilitate cross-linking between these tyrosine-rich derivatives. An in vitro cross-linking assay was developed using a recombinant 42 kDa truncation of EmGAM56. Peroxides, in combination with plant or fungal peroxidases, catalysed the rapid formation of dityrosine cross-linked polymers of the truncated EmGAM56, as determined by western blotting and HPLC, confirming this protein’s propensity to form dityrosine bonds.

CYP56 (Dit2p) in Candida albicans : Characterization and Investigation of Its Role in Growth and Antifungal Drug Susceptibility

The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

Site-Specific Cross-Linking of Proteins through Tyrosine Hexahistidine Tags

The genetic addition of hexahistidine (H(6)) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H(6) tags enabled proteins to be covalently cross-linked under mild conditions in a manner similar to the natural, site-specific cross-linking of tyrosines into dityrosine. A series of seven hexahistidine tags with tyrosines placed in various positions (H(6)Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H(6)Y-tagged I28 dimerized in the presence of excess Ni(2+) with a K(D) of 200 microM. Treatment of Ni(2+)-dimerized H(6)Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni(2+)-catalyzed cross-linking of the Y residues engineered into the H(6) tag. The protein oligomerization was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The presence of dityrosine in the cross-linked proteins was confirmed by fluorescence emission at 410 nm. Proteins lacking the Y residue in the H(6) tag treated with the same oxidative conditions did not cross-link or exhibit dityrosine fluorescence, despite the presence of an endogenous Y residue. The method may have potential uses in other protein conjugation applications such as protein labeling and interfacial immobilization of proteins on artificial surfaces.

Phytopathogenic filamentous (Ashbya, Eremothecium) and dimorphic fungi (Holleya, Nematospora) with needle-shaped ascospores as new members within the Saccharomycetaceae.

Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives of the Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenic fungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region). Based on sequence information from both ITS regions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in a single genus as suggested by Kurtzman (1995; J Industr. Microbiol. 14, 523-530). In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowia remains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequences support the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremothecium or dimorphic genera like Holleya and Nematospora with needle-shaped ascospores within the family Saccharomycetaceae. After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromyces appears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus Saccharomyces. The data of Cai et al. (1996; Int. J. Syst. Bacteriol. 46, 542-549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K. marxianius, K. dobzhanskii, K. wickerhamii and K. aestuarii, which again can be included in the family Saccharomycetaceae. The phylogenetic age of the Metschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution.

The Content of Dityrosine in Chick and Rabbit Aorta Proteins

The possible presence of dityrosine in elastin derived by two different methods and in structural glycoproteins from aortas of 1 day old chicks, adult rabbits and fetal rabbits was determined by a sensitive spectrofluorimetric procedure. Only chick tissues were found to contain dityrosine, 0.3 residues/100,000 total amino acid residues in aortic elastin and 12-15 residues/100,000 residues in the structural glycoproteins. No dityrosine could be detected in any of the fetal or mature rabbit tissues. However, related fluorescent compounds with different excitation-emission maxima and different elution times were obtained by ion exchange chromatography of structural glycoproteins partially hydrolyzed under alkaline conditions.

Chemical nature of monogenean sclerites. I. Stabilization of clamp-protein by formation of dityrosine.

The clamp sclerites ofPricea multaeand other higher monogeneans was fuchsin-positive in Mallory's and Masson's triple stain, and the staining was not affected by detanning agents, suggesting some form of stabilization in the protein. The sclerites were positive in Millon's, Morner's and Xanthoproteic tests for aromatic amino acids, but negative in tests for sulphydryl and disulphide groups. Quinone, phenol and phenol oxidase were absent. They stained with basic dyes such as toluidine blue and methylene blue in the pH range of 3·5–9·5, and also exhibited bluish fluorescence in u.v. light which was not affected by lipid solvents, indicating the presence of dityrosine and recalling the protein present in the ligament cuticle of insects. These features indicate that the clampprotein is stabilized by the formation of dityrosine. It is suggested that this protein may be responsible for the biological properties of the clamp sclerites. This mode of stabilization by tyrosyl residues of the protein, resulting in the formation of dityrosine, is the first record of its kind to be reported in platyhelminths.