Identification of antioxidant peptides in enzymatic hydrolysates of carp (Cyprinus carpio) skin gelatin

The protein by-products from carp (Cyprinus carpio) are normally discarded as industrial waste during fish processing. The objective of this study was to identify and characterise the peptides with a potential antioxidant activity that are released from carp skin proteins during hydrolysis by the Protamex enzyme mixture. This study shows that a hydrolysate of carp skin gelatin and its reversed-phase chromatography fractions have strong in vitro antioxidant properties. Among these fractions, the alanine-tyrosine (Ala-Tyr) dipeptide was identified as the major compound with high antioxidant potential. The peptide has good stability during in vitro enzymatic digestion assay and can inhibit the angiotensin-converting enzyme (ACE). In conclusion, our study proves that both the unfractionated hydrolysate of carp skin gelatin and the above-mentioned Ala-Tyr dipeptide represents attractive novel compounds for the formulation of antioxidant foods

Antibacterial peptides of the moth Galleria mellonella

The work describes purification and biochemical characterization of two inducible antimicrobial peptides from the hemolymph of Galleria mellonella. The peptides were isolated by a sequence of reversed-phase chromatography steps from the hemolymph of larvae immunized with viable bacteria. The first peptide is a member of the cecropin family while the second one is rich in proline residues and has a unique sequence

Collagen type II modification by hypochlorite

Oxidation of proteins is a common phenomenon in the inflammatory process mediated by highly reactive agents such as hypochlorite (HOCl/OCl-) produced by activated neutrophils. For instance, in rheumatoid arthritis hypochlorite plays an important role in joint destruction. One of the major targets for HOCl/OCl- is collagen type II (CII) - the primary cartilage protein. In our study, HOCl/OCl- mediated collagen II modifications were tested using various methods: circular dichroism (CD), HPLC, ELISA, dynamic light scattering (DLS), fluorimetry and spectrophotometry. It was shown that hypochlorite action causes deamination with consecutive carbonyl group formation and transformation of tyrosine residues to dichlorotyrosine. Moreover, it was shown that ammonium chloramine (NH2Cl) formed in the reaction mixture reacts with CII. However, in this case the yield of carbonyl groups and dichlorotyrosine is lower than that observed for HOCl/OCl- by 50%. CD data revealed that collagen II exists as a random coil in the samples and that chlorination is followed by CII fragmentation. In the range of low HOCl/OCl- concentrations (up to 1 mM) 10-90 kDa peptides are predominant whereas massive production of shorter peptides was observed for high (5 mM) hypochlorite concentration. DLS measurements showed that chlorination with HOCl/OCl- decreases the radius of collagen II aggregates from 30 to 6.8 nm. Taking into account the fact that chlorinated collagen is partially degraded, the DLS results suggest that smaller micelles are formed of the 10-90 kDa peptide fraction. Moreover, collagen chlorination results in epitope modification which affects CII recognition by anti-CII antibodies. Finally, since in the synovial fluid the plausible hypochlorite concentration is smaller than that used in the model the change of size of molecular aggregates seems to be the best marker of hypochlorite-mediated collagen oxidation

Properties of chemically oxidized kininogens

Kininogens are multifunctional proteins involved in a variety of regulatory processes including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N-chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation suggests involvement of other structural effects detrimental for kinin production. Indeed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation

Isolation and characterization of acetylated derivative of recombinant insulin lispro produced in Escherichia coli

PURPOSE: Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis. METHODS: For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed. RESULTS: Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro—one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine. CONCLUSIONS: The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro

The effect of Galleria mellonella hemolymph polypeptides on Legionella gormanii

Among Legionella species, which are recognized to be pathogenic for humans, L. gormanii is the second prevalent causative agent of community-acquired pneumonia after L. pneumophila. Anti-L. gormanii activity of Galleria mellonella hemolymph extract and apolipophorin III (apoLp-III) was examined. The extract and apoLp-III at the concentration 0.025 mg/ml caused 75% and 10% decrease of the bacteria survival rate, respectively. The apoLp-III-induced changes of the bacteria cell surface were analyzed for the first time by atomic force microscopy. Our studies demonstrated the powerful anti-Legionella effects of the insect defence polypeptides, which could be exploited in drugs design against these pathogens

Anti-Legionella dumoffii activity of Galleria mellonella defensin and apolipophorin III

The gram-negative bacterium Legionella dumoffii is, beside Legionella pneumophila, an etiological agent of Legionnaires’ disease, an atypical form of pneumonia. The aim of this study was to determine the antimicrobial activity of Galleria mellonella defense polypeptides against L. dumoffii. The extract of immune hemolymph, containing a mixture of defense peptides and proteins, exhibited a dose-dependent bactericidal effect on L. dumoffii. The bacterium appeared sensitive to a main component of the hemolymph extract, apolipophorin III, as well as to a defense peptide, Galleria defensin, used at the concentrations 0.4 mg/mL and 40 μg/mL, respectively. L. dumoffii cells cultured in the presence of choline were more susceptible to both defense factors analyzed. A transmission electron microscopy study of bacterial cells demonstrated that Galleria defensin and apolipophorin III induced irreversible cell wall damage and strong intracellular alterations, i.e., increased vacuolization, cytoplasm condensation and the appearance of electron-white spaces in electron micrographs. Our findings suggest that insects, such as G. mellonella, with their great diversity of antimicrobial factors, can serve as a rich source of compounds for the testing of Legionella susceptibility to defense-related peptides and proteins

Defence response of Galleria mellonella larvae to oral and intrahemocelic infection with Pseudomonas entomophila

We report differences in the course of infection of G. mellonella larvae with P. entomophila via intrahemocelic and oral routes. Survival curves, larval morphology, histology, and induction of defence response were investigated. Larvae injected with 10 and 50 cells of P. entomophila activated a dose-dependent immune response, which was manifested by induction of immune-related genes and dose-dependent defence activity in larval hemolymph. In contrast, after the oral application of the pathogen, antimicrobial activity was detected in whole hemolymph of larvae infected with the $10^{3}$ but not $10^{5}$ dose in spite of the induction of immune response manifested as immune-relevant gene expression and defence activity of electrophoretically separated low-molecular hemolymph components. Among known proteins induced after the P. entomophila infection, we identified proline-rich peptide 1 and 2, cecropin D-like peptide, galiomycin, lysozyme, anionic peptide 1, defensin-like peptide, and a 27 kDa hemolymph protein. The expression of the lysozyme gene and the amount of protein in the hemolymph were correlated with inactivity of hemolymph in insects orally infected with a higher dose of P. entomophila, pointing to its role in the host-pathogen interaction

Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91

Staphylococcus aureus strain CH-91, isolated from a broiler chicken with atopic dermatitis, has a highly proteolytic phenotype that is correlated with the disease. We describe the isolation and biochemical and molecular characterization of the AI-type lantibiotic BacCH91 from S. aureus CH-91 culture medium. The bacteriocin was purified using a three-stage procedure comprising precipitation with ammonium sulfate, extraction with organic solvents, and reversed-phase HPLC. The BacCH91 peptide is thermostable and highly resistant to cleavage by both prokaryotic and eukaryotic peptidases. The MIC for the Gram-positive bacteria ranged from 2.5 nM for Microococcus luteus through 1.3–6.0 μM for staphylococcal strains up to more than 100 μM for Lactococcus lactis. BacCH91 was ineffective against the Gram-negative strains tested at the maximal concentration (100 μM). The amino acid sequence of BacCH91 is similar to that of epidermin and gallidermin. The encoding gene (bacCH91) occurred in two allelic variants distinguishable in the restriction fragment length polymorphism assay. Variant I, identified in S. aureus CH-91, dominated in S. aureus strains of poultry origin, although strains with variant II were also identified in this group. S. aureus strains of human origin were characterized exclusively by variant II