Proteolysis Characteristics Research Articles

Endopeptidase O promotes Streptococcus suis immune evasion by cleaving the host- defence peptide cathelicidins

ABSTRACT Streptococcus suis is a zoonotic Gram-positive bacterium that causes invasive infections such as sepsis and meningitis, threatening public health worldwide. For successful establishment of infection, the bacterium should subvert the innate effectors of immune defence, including the cathelicidin family of host-defence peptides that combat pathogenic bacteria by directly disrupting cell membranes and coordinating immune responses. Here, our study shows that an extracellular endopeptidase O (PepO) of S. suis contributes to assisting the bacterium to resist cathelicidin-mediated killing, as the deletion of the pepO gene makes S. suis more sensitive to the human cathelicidin LL-37, as well as its mouse equivalent, mCRAMP. This protease targets and cleaves both LL-37 and mCRAMP, degrading them into shorter peptides with only a few amino acids, thereby abrogating their ability to kill S. suis. By cleaving LL-37 and mCRAMP, PepO impairs their chemotactic properties for neutrophil migration and undermines their anti-apoptosis activity, which is required for prolonging neutrophil lifespan. Also, PepO inhibits the ability of LL-37 and mCRAMP to promote lysosome development in macrophages. Moreover, the loss of PepO attenuates organ injury and decreases bacterial burdens in a murine model of S. suis bacteraemia. Taken together, these data provide novel insights into the role of the intrinsic proteolytic characteristics of PepO in S. suis-host interaction. Our findings demonstrate that S. suis utilizes the PepO protease to cleave cathelicidins, which is an immunosuppressive strategy adopted by this bacterium to facilitate pathogenesis.

Revealing the proteolytic characteristics of lactobacillus, lacticaseibacillus, and lactiplantibacillus isolates by in vitro and in situ perspectives

This study examined in vitro and in situ proteolytic characteristics of 8 lactobacilli strains isolated from Turkish Tulum cheese, consisting of L. plantarum, L. helveticus, L. rhamnosus, and L. paracasei, whose beneficial properties and safety concerns previously evaluated. The studied isolates presented versatile proteolytic activity with acidic, neutral, and alkaline proteases. The proteolytic activity of isolates against azocasein substrate was determined between 10.30 and 19.90 U/mg, and L. plantarum isolates had the highest proteolytic activity at varying pH values. Isolates 15, 449, 480 and 675 harboured proteinase genes in the catalytic region of prtR and prtP, and in the prtP/prtM intergenic region. All isolates were also evaluated in the production of fermented milk. As a result of SDS-PAGE analysis of fermented milk samples, L. plantarum isolates 449, 480 and 675 showed more proteolytic activity on milk caseins. Besides, isolate 480 was also effective in hydrolysis of beta-lactoglobulin. L. plantarum isolates resulted in an increase in amino acid (AA) concentrations especially in arginine, alanine, histidine, and proline during the 24-h fermentation of milk. AA production abilities varied depending on the strain. Generally, isolate 675 offered superior AA production ability. The proteolytic properties of cultures are effective on both human health and technological properties of the product in terms of reducing the allergenicity of milk proteins, forming functional peptides, and improving the sensory properties of the product. Based on the results of this study, L. plantarum isolates 449, 480 and 675 could potentially be used as starter cultures or co-cultures for these purposes.

Proteolysis characteristics and aroma development during ripening of functional IranianLighvancheese

Probiotic Lighvan cheese was studied during 60 days of ripening to determine microbial analysis, proteolysis characteristics, and aroma development. Viability of Bifidobacterium lactis subsp. animalis, water-soluble nitrogen (WSN), total nitrogen (TN), ripening index (RI), trichloroacetic acid-soluble nitrogen (TCA-SN), α-and β-galactosidase activities, aroma compounds, and organoleptic assessments were analyzed. After 60 days of ripening, the survival of B. lactis subsp. animalis was 6.84 log cfu/g. Values for RI and TCA-SN increased continuously throughout ripening. α-and β-galactosidase activities were markedly more pronounced in the present of B. lactis. The final concentration of volatile compounds in probiotic Lighvan cheeses was higher than traditional Lighvan cheese. Among volatile compounds, acetic acid, ethanol, and acetoin were present in the greatest quantities. B. lactis subsp. animalis did not influence sensory characteristics of cheese samples. Novelty impact statement Bifidobacterium lactis affected the proteolysis characteristics of the traditional Lighvan cheese samples; it did not alter the sensory properties of the probiotic samples. At the end of ripening, the cheeses to which bifidobacteria had been added contained significantly (p < 0.05) higher α-and β-galactosidase activity than traditionally produced Lighvan cheese. Most of the volatile compounds identified were present in all cheese samples throughout ripening, with their amounts changing significantly (p < 0.05) as ripening progressed; the final concentration of volatile compounds in most of the probiotic Lighvan cheese samples was higher than in the traditional Lighvan cheese. ​

Influence of mixed starters on the degradation of proteins and the formation of peptides with antioxidant activities in dry fermented sausages

This study aimed to investigate the influence of mixed starters (Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201) with a high protease activity on the degradation of proteins and the formation of peptides with antioxidant activities during the fermentation and ripening of dry fermented sausages. The starters mainly affected the proteolytic characteristics during the fermentation stage, and their beneficial effects persisted until the end of ripening. Electrophoresis analysis indicated that the starters could more rapidly and severely hydrolyze proteins on days 1 and 4 than on other days. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric ion reducing antioxidant power (FRAP) value of the peptides extracted from the starter-inoculated group significantly increased (P < 0.05) compared with those from the control group. After 23 days of ripening, the free amino acid (FAA) content also increased significantly (P < 0.05) because of the addition of starters. Thus, mixed starters could help delay oxidative changes in dry fermented sausages.

Proteolysis of Reduced-Salt Cheddar Cheese as Affected by Casein to Fat Ratio, Rennet, and pH at Drainage

Effects of change in pH at drainage (6.2, 5.9, and 5.6), rennet concentration (0.1 and 0.3 mL rennet/L of milk) and casein to fat ratio (0.6, 0.7, and 0.8) on proteolytic characteristics of salt-reduced Cheddar cheese were investigated. Chemical composition, lactic acid bacteria and proteolysis were measured. At the same drainage pH, cheeses with C/F ratio 0.6 had significantly higher moisture and ash content compared other ratios. The pH decreased significantly from day 0 (right after pressing) to 120 day and then remained constant. Total LAB growth in cheeses made with 0.3 ml rennet /L was higher at the same C/F ratio and pH at drainage of 6.2. Total plate count at day 180 was significantly higher compared with day 0 (right after pressing). At the same drainage pH, C/F ratio and storage time, water soluble nitrogen contents in cheeses made with 0.3 ml/L rennet were higher compared with 0.1 ml/L. Total free amino acids and trichloroacetic acid-soluble nitrogen contents increased significantly during storage. More hydrophobic peaks appeared as the rennet concentration was increased from 0.1 to 0.3 ml/L. Extra-hydrophobic peptides % in cheese at day 0 and pH at drainage of 5.6, were higher compared to cheese where pH at drainage was 6.2. Increase in C/F ratio and rennet concentration, especially at low pH, showed similar proteolysis as cheese containing 2.5-3% salt.

Biocleaning of historical documents: The use and characterization of bacterial enzymatic resources

Living microorganisms have been used for biocleaning of cultural heritage items, but the application of only extracellular enzyme mixtures (EEMs) from isolated bacteria has not previously been considered for biorestoration work. In this investigation, the proteolytic characteristics of the EEMS from three bacterial isolates (Paracoccus sp., Bacillus flexus and Exiguobacterium undae) were determined and their extent of inhibition in the presence of the proteolytic inhibitors 1,10-phenanthroline monohydrate, EDTA, leupeptin, bestatin and various metal ions was examined. These EEMs were used to remove the animal glue present on the spine of bound newspapers. FT-IR analysis showed that animal glue was effectively removed by a solution of tylose and EEM. The EEM of E. undae gave the best results. Our study describes a simple strategy for the production and characterization of enzymatic mixtures targeted to the degradation of a specific substrate and their subsequent use for biocleaning historical objects.

Effect of cold and frozen temperatures on artisanal goat cheese containing probiotic lactic acid bacteria isolates (Lactobacillus plantarum TW14 and Lactobacillus rhamnosus TW2).

Aim:The research was conducted to determine the effect of temperature and storage duration on the physicochemical, lipolytic, microbiological, and proteolytic characteristics of goat cheese made using Lactobacillus plantarum TW14 and Lactobacillus rhamnosus TW2 bacteria.Materials and Methods:The cheese was stored at 4°C and −20°C for 0, 15, 30, 45, and 60 days. Observations were made on its physicochemical, lipolysis, and microbiological characteristics. The proteolysis pattern was measured with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Results:The protein, fat, ash and total solids levels of cold-stored cheese were higher than the frozen-stored one. The frozen-stored cheese’s free fatty acids (FFA) and acid degree value (ADV) levels are lower than those of the cold-stored cheese as indicated by the partial lipolysis event. The total yeast in the frozen-stored cheese is lower than that in the frozen-stored cheese. Finally, the electrophoresis profile indicates that proteolysis of the frozen-stored cheese is formed since there have been detected αs1-casein, αs2-casein, β-casein, and κ-casein in the casein breakdown during the 60-day storage.Conclusion:The physicochemical characteristics of cold-stored cheese are better than the cheese stored at frozen temperature. However, frozen-stored cheese produces lower FFA and ADV than cold-stored cheese and lipolysis occurs only partially.

In vitro potency determination of botulinum neurotoxin serotype A based on its receptor-binding and proteolytic characteristics

Botulinum neurotoxins (BoNTs) inhibit the release of the neurotransmitter acetylcholine from motor neurons, resulting in highly effective muscle relaxation. In clinical and aesthetic medicine, serotype BoNT/A, which is most potent for humans, is widely used to treat a continuously increasing spectrum of disorders associated with muscle overactivity. Because of the high toxicity associated with BoNTs, it is mandatory to precisely determine the potency of every batch produced for pharmaceutical purposes. Here we report a new quantitative functional in vitro assay for BoNT/A. In this binding and cleavage (BINACLE) assay, the toxin is first bound to specific receptor molecules. Then a chemical reduction is performed, thereby releasing the light chain of BoNT/A and activating its proteolytic domain. The activated light chain is finally exposed to its substrate protein SNAP-25, and the fragment resulting from the proteolytic cleavage of this protein is quantified in an antibody-mediated reaction. The BoNT/A BINACLE assay offers high specificity and sensitivity with a detection limit below 0.5 mouse lethal dose (LD50)/ml. In conclusion, this new in vitro assay for determining BoNT/A toxicity represents an alternative to the LD50 test in mice, which is the “gold standard” method for the potency testing of BoNT/A products.

Recyclable trypsin immobilized magnetic nanoparticles based on hydrophilic polyethylenimine modification and their proteolytic characteristics

In this work, recyclable trypsin immobilized magnetic nanoparticles based on hydrophilic branched polyethylenimine (PEI) modification were synthesized under mild conditions.

In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics

Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD50 test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1mouseLD50/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.

Neuronal ADAM10 Promotes Outgrowth of Small-Caliber Myelinated Axons in the Peripheral Nervous System.

The regulation of myelination and axonal outgrowth in the peripheral nervous system is controlled by a complex signaling network involving various signaling pathways. Members of the A Disintegrin And Metalloproteinase (ADAM) family are membrane-anchored proteinases with both proteolytic and disintegrin characteristics that modulate the function of signaling molecules. One family member, ADAM17, is known to influence myelination by cleaving and thus regulating one of the key signals, neuregulin-1, which controls peripheral nervous system myelination. A similar function for ADAM10 had been suggested by previous in vitro studies. Here, we assessed whether ADAM10 exerts a similar function in vivo and deleted ADAM10 in a cell type-specific manner in either neurons or Schwann cells. We found that ADAM10 is not required in either Schwann cells or neurons for normal myelination during development or for remyelination after injury. Instead, ADAM10 is required specifically in neurons for the outgrowth of myelinated small-fiber axons in vitro and after injury in vivo. Thus, we report for the first time a neuron-intrinsic function of ADAM10 in axonal regeneration that is distinct from that of the related protein family member ADAM17 and that may have implications for targeting ADAM function in nervous system diseases.

High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development.

This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.

Investigation of bi-enzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics

The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic–inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24h to 50s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight.

"BINACLE" assay for in vitro detection of active tetanus neurotoxin in toxoids.

Tetanus neurotoxin (TeNT) consists of two protein chains connected by a disulfide linkage: The heavy chain mediates the toxin binding and uptake by neurons, whereas the light chain cleaves synaptobrevin and thus blocks neurotransmitter release.Chemically inactivated TeNT (tetanus toxoid) is utilized for the production of tetanus vaccines. For safety reasons, each toxoid bulk has to be tested for the "absence of toxin and irreversibility of toxoid". To date, these mandatory tests are performed as toxicity tests in guinea pigs. A replacement by an animal-free method for the detection of TeNT would be desirable. The BINACLE (BINding And CLEavage) assay takes into account the receptor-binding as well as the proteolytic characteristics of TeNT: The toxin is bound to immobilized receptor molecules, the light chains are then released by reduction and transferred to a microplate containing synaptobrevin, and the fragment resulting from TeNT-induced cleavage is finally detected. This assay offers a higher specificity for discriminating between toxic TeNT and inactivated toxoid molecules than other published assays. Validation studies have shown that the BINACLE assay allows the sensitive and robust detection of TeNT in toxoids, and thus may indeed represent a suitable alternative to the prescribed animal safety tests for toxoids from several European vaccine manufacturers. Product-specific validations (and possibly adaptations) of the assay protocol will be required. A European collaborative study is currently being initiated to further examine the applicability of the method for toxoid testing. The final aim is the inclusion of the method into the European Pharmacopoeia.

Influence of pig rennet on proteolysis, organic acids content and microbiota of Pecorino di Farindola, a traditional Italian ewe’s raw milk cheese

The use of pig rennet is very ancient and in Italy is only applied in the manufacture of Pecorino di Farindola cheese. In order to evaluate the key role of this rennet in the establishment of peculiar features of Pecorino di Farindola, cheeses made from raw ewes’ milk using calf (A) and kid (B) rennets were compared to those produced with pig (C) rennet. The use of pig rennet for Pecorino di Farindola cheese making confers physico-chemical and proteolytic characteristics that differentiate it from cheeses produced with other coagulants. However, no microbiological differences were observed. Chesses made with pig and kid rennets were characterised by higher proteolysis after 7days of ripening. The content of isovaleric and propionic acids at the end of ripening was correlated with the presence of propionibacteria.

Substrate Cleavage Profiling Suggests a Distinct Function of Bacteroides fragilis Metalloproteinases (Fragilysin and Metalloproteinase II) at the Microbiome-Inflammation-Cancer Interface

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

오징어 먹즙 첨가가 저 식염 오징어 젓갈의 단백질분해 특성에 미치는 영향

오징어 먹즙 첨가가 저 식염 오징어 젓갈의 단백질분해 특성에 미치는 영향

Proteolytic Characteristics of Cathepsin D Related to the Recognition and Cleavage of Its Target Proteins

Cathepsin D (CD) plays an important role in both biological and pathological processes, although the cleavage characteristics and substrate selection of CD have yet to be fully explored. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the CD cleavage sites in bovine serum albumin (BSA). We found that the hydrophobic residues at P1 were not only a preferential factor for CD cleavage but that the hydrophobicity at P1’ also contributed to CD recognition. The concept of hydrophobic scores of neighbors (HSN) was proposed to describe the hydrophobic microenvironment of CD recognition sites. The survey of CD cleavage characteristics in several proteins suggested that the HSN was a sensitive indicator for judging the favorable sites in peptides for CD cleavage, with HSN values of 0.5–1.0 representing a likely threshold. Ovalbumin (OVA), a protein resistant to CD cleavage in its native state, was easily cleaved by CD after denaturation, and the features of the cleaved peptides were quite similar to those found in BSA, where a higher HSN value indicated greater cleavability. We further conducted two-dimensional gel electrophoresis (2DE) to find more proteins that were insensitive to CD cleavage in CD-knockdown cells. Based on an analysis of secondary and three-dimensional structures, we postulated that intact proteins with a structure consisting of all α-helices would be relatively accessible to CD cleavage.

Proteolysis characteristics of sarcoplasmic, myofibrillar, and stromal proteins separated from grass carp and antioxidant properties of their hydrolysates

Proteolysis of grass carp sarcoplasmic, myofibrillar, and stromal proteins by 5 commercial proteases were studied. Sarcoplasmic and myofibrillar protein could be well hydrolyzed by Alcalase 2.4 L to reach high protein recoveries (PR) (71.86±2.46 and 80.77±3.05%, respectively), while the maximum PR for stromal protein was only 42.83±2.84%. However, stromal hydrolysates, containing mostly 6–10 kDa fraction, exhibited higher ·OH scavenging activities due to its high content of antioxidant-assisting amino acids. Alcalase 2.4 L and pancreatin 6.0, which produced hydrolysates with relative high degree of hydrolysis (DH), were used for further hydrolysis of whole grass carp protein with the assistance of response surface methodology (RSM). The results showed that serine proteases (Alcalase 2.4 L and pancreatin 6.0) could produce sarcoplasmic, myofibrillar, or stromal hydrolysates with relatively high PR, DH, and strong ·OH scavenging activity, which may be used to prepare antioxidant hydrolysates from grass carp.

Assessment of proteolysis and lipolysis in cheeses made using artisanal lamb rennet paste

AbstractRennet paste contains chymosin and pepsin as major enzymatic coagulant, and lipolytic enzymes such as pre-gastric lipase (PGL) and gastric lipase (GL), able to release free fatty acids during cheese ripening. Main factors influencing rennet paste features are diet (milk feeding, weaning) and slaughter age. Little is known about the effects of probiotic addition to milk substitute on the microbial and enzyme feature of rennet, and thus on the characteristics of cheese. In the present study the effects of different rennet pastes on the Pecorino foggiano cheese in terms of proteolytic and lipolytic characteristics were investigated. Twenty-four lambs were divided into six groups of four according to a 3x2 factorial design with lambs being subjected to three different feeding systems: mother suckling (MS), artificial rearing (AR), and artificial rearing with Lactobacillus acidophilus (Strain DSM 20079; DSMZ, Braunshweig-Germany; cells were suspended in milk replacer at the concentration of 7log 10 cfu...