Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Isolation and characterization of collagen type I crosslink from skin: high-resolution NMR reveals diastereomers of hydroxylysinonorleucine crosslink.

Skin is made up of mainly collagen type I and its structure is stabilised by the formation of covalent immature and mature crosslinks. In this study, collagen immature crosslink hydroxylysinonorleucine (HLNL) was isolated from bovine skin in high purity using two sequential purification steps. These consisted of preparative fibrous cellulose and size exclusion chromatography. The purified crosslink was then analysed using tandem mass spectrometry and high-resolution nuclear magnetic resonance (NMR) spectroscopy. The mass of singly and doubly charged ions of HLNL was 292.1865 and 146.5970 m/z and their optimised fragmentation energy was 17 keV and 5 keV, respectively. The 13C NMR of HLNL showed a doubled-up peak at 67.84 and 67.91 ppm which corroborated a diastereomeric form of collagen immature crosslink HLNL and both are chiroptically indistinguishable. The chemical structure was fully resolved using 1H, 13C and DEPT-135 high-resolution NMR spectroscopy and compared with other previous studies. We also obtained for the first time the 2D NMR spectra COSY and HSQC of HLNL. We therefore suggested that collagen organization into specific fibrils' orientation may be affected by the different configuration of these diastereomers of HLNL.

Design and Synthesis of Broad Spectrum Trypanosomatid Selective Inhibitors.

Neglected tropical diseases caused by parasitic infections are an ongoing and increasing concern that have a devastating effect on the developing world due to their burden on human and animal health. In this work, we detail the preparation of a focused library of substituted-tetrahydropyran derivatives and their evaluation as selective chemical tools for trypanosomatid inhibition and the follow-on development of photoaffinity probes capable of labeling target protein(s) in vitro. Several of these functionalized compounds maintain low micromolar activity against Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, and Leishmania donovani. In addition, we demonstrate the utility of the photoaffinity probes for target identification through preliminary cellular localization studies.

Unique fluorescence and high-molecular weight characteristics of protein isolates from manuka honey (Leptospermum scoparium).

This study compared the fluorescence properties (λex/em=350/450nm) and molecular size of proteins from manuka and non-manuka honey. The fluorescence characteristics of non-manuka and manuka proteins differ markedly, whereby manuka honey protein fluorescence increases with increasing methylglyoxal (MGO) content of the honey. It was concluded that manuka honey proteins are modified due to MGO-derived glycation and crosslinking reactions, thus resulting in fluorescent structures. The molecular size of honey proteins was studied using size exclusion chromatography. Manuka honey proteins contain a significantly higher amount of high molecular weight (HMW) fraction compared to non-manuka honey proteins. Moreover, HMW fraction of manuka honey proteins was stable against reducing agents such as dithiothreitol, whereas HMW fraction of non-manuka honey proteins was significantly decreased. Thus, the chemical nature of manuka honey HMW fraction is probably covalent MGO crosslinking, whereas non-manuka HMW fraction is caused by disulfide bonds. Storage of a non-manuka honey, which was artificially spiked with MGO and DHA, did not induce above mentioned fluorescence properties of proteins during 84days of storage. Hence, MGO-derived fluorescence and crosslinking of honey proteins can be useful parameters to characterize manuka honey.

Selecting a DNA-Encoded Chemical Library against Non-immobilized Proteins Using a "Ligate-Cross-Link-Purify" Strategy.

DNA-encoded chemical libraries (DELs) have recently emerged and become an important technology platform in biomedical research and drug discovery. DELs containing large numbers of compounds can be prepared and selected against biological targets to discover novel ligands and inhibitors. In practice, DELs are usually selected against purified and immobilized proteins using the typical "bind-wash-elute" protocol; however, selection methods compatible with non-immobilized proteins would be able to greatly expand the target scope of DELs beyond purified proteins to more-complex and biologically relevant targets. Using a novel "ligate-cross-link-purify" strategy, we report here a method capable of selecting DELs against unmodified and non-immobilized protein targets. In addition, this method has shown excellent capability in identifying binders with moderate and weak affinities.

A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa.

Galactosylglycerolipids (GGLs) and chlorophyll are characteristic components of chloroplast in photosynthetic organisms. Although chlorophyll is anchored to the thylakoid membrane by phytol (tetramethylhexadecenol), this isoprenoid alcohol has never been found as a constituent of GGLs. We here described a novel GGL, in which phytol was linked to the glycerol backbone via an ether linkage. This unique GGL was identified as an Alkaline-resistant and Endogalactosylceramidase (EGALC)-sensitive GlycoLipid (AEGL) in the marine green alga, Ulva pertusa. EGALC is an enzyme that is specific to the R-Galα/ß1-6Galß1-structure of galactolipids. The structure of U. pertusa AEGL was determined following its purification to 1-O-phytyl-3-O-Galα1-6Galß1-sn-glycerol by mass spectrometric and nuclear magnetic resonance analyses. AEGLs were ubiquitously distributed in not only green, but also red and brown marine algae; however, they were rarely detected in terrestrial plants, eukaryotic phytoplankton, or cyanobacteria.

Tea derived galloylated polyphenols cross-link purified gastrointestinal mucins.

Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm) heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria.

Multipart copolyelectrolyte adhesive of the sandcastle worm, Phragmatopoma californica (Fewkes): catechol oxidase catalyzed curing through peptidyl-DOPA.

Tube-building sabellariid polychaetes have major impacts on the geology and ecology of shorelines worldwide. Sandcastle worms, Phragmatopoma californica (Fewkes), live along the western coast of North America. Individual sabellariid worms build tubular shells by gluing together mineral particles with a multipart polyelectrolytic adhesive. Distinct sets of oppositely charged components are packaged and stored in concentrated granules in separate cell types. Homogeneous granules contain sulfated macromolecules as counter-polyanion to polycationic Pc2 and Pc5 proteins, which become major components of the fully cured glue. Heterogeneous granules contain polyphosphoproteins, Pc3A/B, paired with divalent cations and polycationic Pc1 and Pc4 proteins. Both types of granules contain catechol oxidase that catalyzes oxidative cross-linking of L-DOPA. Co-secretion of catechol oxidase guarantees rapid and spatially homogeneous curing with limited mixing of the preassembled adhesive packets. Catechol oxidase remains active long after the glue is fully cured, perhaps providing an active cue for conspecific larval settlement.

Optimizing the enrichment of cross-linked products for mass spectrometric protein analysis.

RATIONALE: Chemical cross-linking in combination with a mass spectrometric analysis of the created cross-linked products is an area of growing interest for deriving low-resolution structural information of proteins and protein complexes. One of the greatest challenges is the complexity of the created cross-linking mixtures, which can be met by a charge-based enrichment of cross-linked peptides after proteolytic digestion using strong cation-exchange (SCX) chromatography. METHODS: SCX chromatography was used for the enrichment of cross-linked peptides with the N-hydroxysuccinimide ester bis(sulfosuccinimidyl)succinate (BS(3)) prior to a mass spectrometric analysis by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS. Bovine serum albumin (BSA) and glutathione S-transferase (GST) were employed as model proteins. RESULTS: Conditions for SCX enrichment were optimized for obtaining as many interpeptide cross-linked peptides as possible in order to maximize the amount of structural information from a single experiment. With an SCX-based enrichment step of cross-linked products within BSA using the cross-linker BS(3), 154 interpeptidal cross-linking products were identified during nano-HPLC/nano-ESI-MS/MS analyses, whereas analyses without a prior SCX enrichment allowed the identification of merely 20 cross-linked products. The application of the SCX enrichment strategy for the analysis of cross-linked products of GST with BS(3) allowed the identification of 26 interpeptidal cross-linked products compared with 16 without SCX enrichment. CONCLUSIONS: For both proteins investigated herein, BSA and GST, the introduction of an SCX-based enrichment step prior to nano-HPLC/nano-ESI-MS/MS of cross-linked products led to a considerable gain in structural information.

Cross-linking of dithiols by mitomycin C.

Upon reduction, the antitumor drug mitomycin C undergoes a cascade of reactions to give a bis-electrophile that alkylates cellular nucleophiles. We recently reported that dithiols activate mitomycin C by reduction, and we report here that dithiols, after executing the reductive activation of mitomycin C, are bis-alkylated by the activated drug to form S,S'-cross-links as the predominant end products. The diastereomeric pair of adducts formed by 1,3-propanedithiol has been fully characterized by UV, HRMS, CD, and NMR experiments. Racemic dithiol (+/-)-dithiothreitol gave four diastereomeric cross-links, and (+/-)-dihydrolipoic acid gave eight cross-links (two regioisomers with four diastereomers each) that were partially characterized by UV and MS. The observed dependence of cross-link formation on dithiol concentration indicated the requirement of a second reduction step by dithiol, prior to the alkylation of the second arm of the dithiol. The existence of unidentified reaction pathways was manifested by the formation of unexpected intermediates during the course of the reaction of mitomycin C with dithiols and by the formation of unsoluble mitosene derivatives in the reaction between equimolar amounts of dithiol and mitomycin C. Mechanistic details of the reaction are addressed in light of these results. Finally, we discuss the potential relevance of our findings for the interaction of mitomycin C with dithiol-containing proteins.

Cross-linked nucleic acids: isolation, structure, and biological role.

This review includes literature data on main types of reagents inducing covalent cross-links of nucleic acids. Reactivity of cross-linking agents, preferable sites for their binding, and methods for determination of location of cross-links in duplex are discussed. Biological responses of cells to cross-linking in nucleic acids, i.e. replication and transcription blocking, onset of repair processes, and apoptotic cell death are considered, as well as application of cross-linking reagents as medicinal drugs for solving molecular-biological problems.

The crosslinking and antimicrobial properties of tunichrome.

Tunichromes are small peptides containing one or more dehydrodopa derived units that have been identified in the blood cells of at least eleven species of tunicates. Incubation of tunichromes isolated from Ascidia nigra hemocytes (or model dopa-containing compounds) under oxidative conditions with either lysozyme, cytochrome c or ovalbumin resulted in a time-dependent polymerization of these test proteins to dimers, trimers, tetramers and potentially to other oligomers. These results indicate that the oxidation products of tunichromes possess inherent crosslinking properties. Hence it is possible that tunichromes participate in tunic production by forming adducts and crosslinks with structural proteins and/or carbohydrate polymers, similar to the well-understood process of insect cuticle hardening. Since such crosslinking potentials could also be beneficial for defense reactions against invading microorganisms, antibacterial activity of tunichromes was tested using both a radial diffusion assay and the Microtox(R) test. Tunichromes exhibited antimicrobial activity against gram-negative bacteria Escherichia coli and Photobacterium phosphorium. However, they did not show any antimicrobial activity against the gram-positive bacteria Staphylococcus aureus at the concentrations tested. We propose that the crosslinking and antimicrobial functions are both based on the reactivity of dehydrodopa units present in the tunichromes, and their subsequent ability to form highly reactive quinone methides.

K2P--a novel cross-link from human lens protein.

We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by LC-MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure as being 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl-amino)-3-hydroxy-2, 3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues and a five-carbon atom ring. We assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of water-soluble [WS] protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross-link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.

Specificity of metal ion cross-linking in marine mussel adhesives.

In an effort to understand the formation of marine bioadhesives, mussel protein extracts were cured with various reagents and the enhanced cross-linking ability of Fe3+ was found.

Model studies on the modification of proteins by lipoxidation-derived 2-hydroxyaldehydes.

2-hydroxyaldehydes have been previously identified as products of lipid peroxidation, and although they represent the simplest reducing sugars, their potential for modification of proteins under physiological conditions has not been investigated. Here, 2-hydroxyaldehydes were found to condense with amines in two ways, implicating potential pathways for modification of lysine residues. A fluorescent 4,5-dialkyl-3-hydroxypyridinium with ex/em 327/390 nm and a nonfluorescent 4-alkylimidazolium cross-linking product were isolated and characterized by (1)H NMR, (13)C NMR, high-resolution mass spectrometry, and, in the former case, through independent synthesis. Both reactions appear to proceed through Amadori rearrangement of the initial Schiff base. On the basis of the UV absorbance of the 3-hydroxypyridinium, the latter was estimated to represent modification of 1.5% of the lysines of RNase incubated with 0.5 mM 2-hydroxyheptanal for 10 days at 25 degrees C. The 4-alkylimidazolium is proposed to contribute to the protein cross-linking observed by gel electrophoresis in the incubation of RNase with higher concentrations of 2-hydroxyheptanal.

Feasibility study using a natural compound (reuterin) produced by Lactobacillus reuteri in sterilizing and crosslinking biological tissues.

Bioprostheses derived from biological tissues have to be fixed and subsequently sterilized before they can be implanted in humans. Currently available crosslinking agents and sterilants used in the fixation or sterilization of biological tissues such as glutaraldehyde and formaldehyde are all highly cytotoxic, which may impair the biocompatibility of bioprostheses. Therefore, it is desirable to provide an agent suitable for use in biomedical applications that is of low cytotoxicity and may form sterile and biocompatible crosslinked products. To achieve this goal, a natural compound (reuterin), produced by Lactobacillus reuteri in the presence of glycerol, was used by our group. It is known that reuterin has antibacterial, antimycotic, and antiprotozoal activities. Additionally, as in the case with formaldehyde, reuterin may react with the free amino groups in biological tissues by using its aldehyde functional group. Therefore, it was speculated that reuterin could be used as a crosslinking agent and a sterilant for biological tissues in the same way as glutaraldehyde and formaldehyde. In the study, the production of reuterin, produced by Lactobacillus reuteri under control conditions, was reported. Preparative chromatography was used to purify reuterin. Also, the minimal inhibitory concentration and minimal bactericidal concentration of reuterin and its antimicrobial activity on a contaminated tissue were investigated. In addition, the cytotoxicity of reuterin was evaluated. Glutaraldehyde, the most commonly used sterilant in the sterilization of biological tissues, was employed as a control. Furthermore, the feasibility of using reuterin as a crosslinking agent in fixing biological tissues was studied. Fresh and the glutaraldehyde-fixed tissues were used as controls. The results obtained in the minimal inhibitory concentration and minimal bactericidal concentration studies and in the sterilization study of a contaminated tissue indicated that the antimicrobial activity of reuterin is significantly superior to its glutaraldehyde counterpart. In addition, the results obtained in the 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide assay showed that reuterin is significantly less cytotoxic than glutaraldehyde. Additionally, it was found that reuterin is an effective crosslinking agent for biological tissue fixation. The reuterin-fixed tissue had comparable free amino group content, denaturation temperature, and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue. In conclusion, the results obtained in this study indicate that reuterin is an effective agent in the sterilization and fixation of biological tissues.

Cyclopentenosine, major trifunctional crosslinking amino acid isolated from acid hydrolysate of elastin.

A trifunctional crosslinking amino acid named cyclopentenosine (CP) was isolated from the hydrolysate of bovine nuchal ligament elastin. CP and its derivatives were identified by spectroscopic methods. CP was found to consist of a 2-cyclopenten-1-one structure and its imine-enamine tautomers with enantiomers in H(2)O. A model reaction for the formation of the CP crosslink using model compounds for allysine (propanal) and lysine (n-butylamine) demonstrated that CP is composed of 2-cyclopenten-1-one and alpha, beta, gamma, delta-unsaturated aldehyde derived from three allysine residues. An ion-paired high-performance liquid chromatographic method for the determination of CP was developed. Among various bovine tissues the nuchal ligament had the highest concentration of CP. The age-related changes in the concentration of CP were examined in the aorta from rat (short-lived species) and bovine (long-lived species). The CP content was very low in the newborn rat but increased markedly with growth. After maturity, the CP content remained nearly the same or slightly decreased. In bovine aorta, the CP content scarcely changed from 7 months to 16 years.

A cross-linked monoclonal antibody fragment for improved tumor targeting.

Cross-linked F(ab')2 fragments derived from PR1A3, a murine monoclonal antibody used in radioimmunoscintigraphy of colorectal tumors, were produced using the bifunctional reagent bismaleimidohexane (BMH) as follows: Digestion of PR1A3 with pepsin gave F(ab')2 fragments which were purified by ion-exchange chromatography. Fab' was produced by reduction of F(ab')2 with cysteine. Following reaction with BMH, cross-linked F(ab')2 fragments, XL-F(ab')2, were isolated by preparative size-exclusion HPLC. Analysis by HPLC and SDS-PAGE demonstrated the presence of a molecule of approximately 100 kDa containing a nonreducible 50,000 MWt chain. Competitive and direct radioligand binding assays demonstrated that the XL-F(ab')2 had a capacity to bind to antigen similar to that of unmodified F(ab')2. The biodistribution of 125I-labeled XL-F(ab')2 and unmodified F(ab')2 was compared in a nude mouse human tumor xenograft model at 4, 24, and 48 h after injection. Differences between the two preparations were most significant after 24 or 48 h. Tumor uptake of the XL-F(ab')2 was greater and normal tissue retention less than with the unmodified fragment. Tumor to normal tissue ratios at 48 h ranged from 6.2 to 35.2 for XL-F(ab')2 while for the normal F(ab')2 they ranged from 1.5 to 14.2. These results suggest that cross-linked antibody fragments may produce better tumor targeting in clinical application.

Enzymatic oxidation of the bifunctional wheat inhibitor of subtilisin and endogenous alpha-amylase.

Oxidation of the bifunctional wheat inhibitor of subtilisin and endogenous alpha-amylase catalyzed by horseradish peroxidase results in the loss of the inhibitory activity against both enzymes. The enzymatic oxidation is accompanied by modification of one methionine and two tryptophan residues in the protein. The results obtained, together with data on chemical modification and limited proteolysis, allow us to conclude that Met34-Ala35 is the reactive site of the inhibitor responsible for the interaction with subtilisin. It is supposed that the reactive site of the inhibitor responsible for the interaction with alpha-amylase contains one or two tryptophan residues.

Characterization of a saporin mitotoxin specifically cytotoxic to cells bearing the granulocyte-macrophage colony-stimulating factor receptor.

When granulocyte-macrophage colony-stimulating factor (GM-CSF) is chemically conjugated to the ribosome-inactivating protein saporin, the resulting protein conjugate is highly toxic for cells expressing the GM-CSF receptor. Structural and Western blot analyses of the purified conjugate establish that it contains equimolar amounts of the starting materials and is free of any contamination by the non-conjugated components. The resulting bifunctional reagent is specifically cytotoxic to cells expressing the GM-CSF receptor, but is ineffective to cells that do not express the receptor. The cytotoxic activity is inhibited in a dose-dependent manner by GM-CSF, but not by any one of five other peptide growth factors. This is the first report of a mitotoxin for cells that express the GM-CSF receptor and which promises to be a valuable tool to study the expression of the GM-CSF receptor in normal and pathological states.