Cathepsin G Research Articles

Exogenous serpin B1 restricts immune complex-mediated NET formation via inhibition of a chymotrypsin-like protease and enhances microbial phagocytosis

Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation; however, its mechanism(s) of action is currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA, or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion, we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA, and A2138-mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis, and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysosomes.

KLK7 expression in human tumors: a tissue microarray study on 13,447 tumors

BackgroundKallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine protease which is essential for the desquamation of corneocytes and thus plays a pivotal role in maintaining skin homeostasis. In cancer, KLK7 overexpression was suggested to represent a route for metastasis through cleavage of cell junction and extracellular matrix proteins of cancer cells.MethodsTo comprehensively determine KLK7 protein expression in normal and neoplastic tissues, a tissue microarray containing 13,447 samples from 147 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry.ResultsKLK7 positivity was found in 64 of 147 tumor categories, including 17 tumor categories with at least one strongly positive case. The highest rate of KLK7 positivity was found in squamous cell carcinomas from various sites of origin (positive in 18.1%-63.8%), ovarian and endometrium cancers (4.8%-56.2%), salivary gland tumors (4.8%-13.7%), bilio-pancreatic adenocarcinomas (20.0%-40.4%), and adenocarcinomas of the upper gastrointestinal tract (3.3%-12.5%). KLK7 positivity was linked to nodal metastasis (p = 0.0005), blood vessel infiltration (p = 0.0037), and lymph vessel infiltration (p < 0.0001) in colorectal adenocarcinoma, nodal metastasis in hepatocellular carcinoma (p = 0.0382), advanced pathological tumor stage in papillary thyroid cancer (p = 0.0132), and low grade of malignancy in a cohort of 719 squamous cell carcinomas from 11 different sites of origin (p < 0.0001).ConclusionsThese data provide a comprehensive overview on KLK7 expression in normal and neoplastic human tissues. The prognostic relevance of KLK7 expression and the possible role of KLK7 as a drug target need to be further investigated.

Effect of enzymatic processing on the physicochemical properties of wheat gluten protein enzymatic products

AbstractBackground and ObjectivesWheat gluten is a high‐quality, low‐priced, and nutritious plant protein. Applications of wheat gluten protein could be expanded by improving its solubility through enzymatic modifications. Herein, we investigated the effect of proteolytic processing on the physicochemical properties of wheat gluten protein.FindingsThe degree of hydrolysis, trichloroacetic acid‐soluble nitrogen (TCA‐SN), and peptidyl nitrogen content of the enzymatic digestion products increased, and the TCA‐SN content exceeded 60% in all cases. The ζ‐potentials of all products after enzymatic digestion were significantly decreased and were negative. Three enzymatic solutions increased the stability of the solution system and the surface hydrophobicity of the enzymatic products to different degrees. The products after enzymatic digestion by alkaline protease had better antioxidant capacity.ConclusionsThis study provides theoretical support for expanding the applications for wheat protein to achieve the added value of wheat products and enhance the utilization rate of these readily available raw materials.Significance and NoveltyThis study investigates the dual enzyme stepwise enzymatic hydrolysis of wheat gluten protein by alkaline protease and neutral protease, which increases the water‐soluble protein content of wheat gluten protein. The resulting hydrolysate has a high peptide nitrogen content.

Aspartic protease supplementation enhancing the performance, carcass characteristics, nutrient digestibility and economic viability, without changing blood parameters and salivary cortisol of pigs

Aiming to study the performance, carcass characteristics, nutrient digestibility, blood parameters, salivary cortisol levels, and economic viability of pigs administered aspartic protease, a total of 135 pigs were housed in pens in a randomized block design, divided into five treatments with nine replications. The experimental diets were positive control (PC), basic diet with a 5.0% reduction in protein and amino acid requirements; negative control (NC) with a 7.5% reduction in protein and amino acid requirements; NC + 100 g/mT of aspartic protease (NC100); NC + 150 g/mT of aspartic protease (NC150); and NC + 200 g/mT of neutral serine protease (NC200). The inclusion of protease, independently of the source and amount, increased the average daily weight gain (P < 0.05) of animals compared with the control treatments (PC and NC), improved feed conversion (P < 0.05) in early stages, and improved diet digestibility (P < 0.05) compared with the PC. Treatment with NC150 and NC200 resulted in greater carcass weights (P < 0.05) than treatment with the PC. NC100 led to a greater carcass yield than PC (P < 0.05), and NC150 resulted in a greater loin eye area than PC (P < 0.05). No differences (P > 0.05) in the blood parameters or salivary cortisol levels were found. Regarding economic viability, proteases increased the profitability, with NC150 leading to the best results. Thus, the use of aspartic proteases is recommended to improve performance and further facilitate pork production.

Recombinant Chymotrypsin-like Peptidase from Tenebrio molitor with a Non-Canonical Substrate-Binding Site

Recombinant Chymotrypsin-like Peptidase from Tenebrio molitor with a Non-Canonical Substrate-Binding Site

Neutrophil extracellular traps promote invasion and metastasis via NLRP3-mediated oral squamous cell carcinoma pyroptosis inhibition

Neutrophil extracellular traps (NETs) are reticular structures composed of neutrophil elastase (NE), cathepsin G (CG) and DNA-histone enzyme complexes. Accumulating evidence has revealed that NETs play important roles in tumor progression, metastasis, and thrombosis. However, our understanding of its clinical value and mechanism of action in oral squamous cell carcinoma (OSCC) is limited and has not yet been systematically described. Here, we aimed to investigate the clinical significance of NETs in OSCC and the mechanisms by which they affect its invasive and metastatic capacity. Our results demonstrated that high enrichment of NETs is associated with poor prognosis in OSCC, and mechanistic studies have shown that NE in NETs promotes invasion and metastasis via NLRP3-mediated inhibition of pyroptosis in OSCC. These findings may provide a new therapeutic approach for OSCC.

Investigation of Genetic Association Between a High Activity Variant of Cathepsin G and Risk for Basal Cell Carcinoma.

Cathepsin G (CTSG) has been identified as an inhibitor of breast, bladder, and colorectal cancers. The G allele of the N125S (A/G, rs45567233) functional polymorphism of the CTSG gene confers increased serum CTSG activity and has been associated with cardiovascular and neurovascular diseases. This study examined the possible correlation between the pathogenesis of basal cell carcinoma (BCC) and the functional polymorphism CTSG N125S. A total of 197 DNA samples were examined, comprising 98 BCC patients and 99 control samples of Greek origin. The CTSG N125S polymorphism was molecularly genotyped using PCR amplification, followed by enzyme digestion, and agarose gel electrophoresis of the amplified DNA fragments. There was no statistically significant difference in the genotypic and allelic frequencies between the patient and the control groups. There is no association between the CTSG N125S polymorphism and pathogenesis of BCC.

A novel immobilized magnetic neutral protease used in extraction of protein from streptomycin residue: Preparation, characterization and application

Antibiotic residue is a hazardous waste which contains much protein; the extraction of protein from antibiotic residue using neutral protease is an effective utilization path of antibiotic residue. In this work, an immobilized magnetic neutral protease (IMNP) was successfully prepared by immobilizing free neutral protease on the surface of magnetic carrier, and characterized by a series of analysis methods (SEM, BET, FTIR, TGA, XRD, and VSM). IMNP can retain > 90 % enzyme activity of free neutral protease and extract ∼36 % protein from streptomycin residue within 4 h at the optimal extraction condition (water content in antibiotic residue = 97.5 %, IMNP loading = 5 %, pH = 7.0, and extraction temperature = 50 ℃). The loss of neutral protease from the surface of IMNP and the possible poisoning of IMNP caused by the toxic substances in the streptomycin residue significantly affected the relative enzyme activity of IMNP (29.4 % enzyme activity of IMNP lost after 6th use). IMNP showed better storage performance, pH and thermal tolerance performance than those of free neutral protease.

Airborne indoor allergen serine proteases and their contribution to sensitisation and activation of innate immunity in allergic airway disease.

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.

Effect of Neutral Protease on Freshness Quality of Shucked Pacific Oysters at Different Storage Conditions.

The aim of this study was to investigate the effect of neutral protease treatment on the biochemical properties of various parts of Pacific oysters (Crassostrea gigas) under different storage conditions. The mechanism of quality degradation in the mantle, adductor muscle, gill, and trunk of treated oysters stored at -1.5 °C (superchilling) or 4 °C (refrigeration) for several days using different storage methods was studied. The results showed that the oyster treated with the enzyme exhibited higher glycogen content, flavor nucleotide content, and sensory scores compared to the control group. Superchilling at -1.5 °C was observed to slow the increase in total volatile basic nitrogen (TVB-N), total viable count (TVC), and pH, while maintaining sensory scores better than refrigeration at 4 °C. Both wet superchilling (WS) and dry exposed superchilling (DeS) methods effectively preserved freshness and quality at -1.5 °C. The freshness of the oysters' body trunk changed most significantly. K value, K' value, and AEC value, as the evaluation indexes of oyster freshness, were affected by the storage medium. Therefore, neutral protease enhances the flavor of oysters in a short time, and oysters stored in wet superchilling or dry exposed superchilling conditions have an extended shelf life.

Optimization of MgCl2-assisted neutral protease system for sustainable beamhouse processing

The beamhouse plays a pivotal role in leather manufacturing. However, the conventional lime-sulfide system (LSS) used in the beamhouse causes significant environmental pollution due to the extensive use of chemical agents. In recent years, most research has focused on biological treatments, with enzymes emerging as a promising environmentally friendly alternative. In this study, we employed the salt-enzyme system (SES) to utilize MgCl2-assisted neutral protease to streamline processes and reduce pollution in the beamhouse. Additionally, response surface methodology (RSM) was utilized to optimize the experimental conditions for enhancing unhairing, fiber opening, and bating efficiency. In terms of environmental benefits, compared to LSS, SES exhibits a significant decrease in COD, NH3-N, and TS by 9.59 %, 26.27 %, and 76.94 %, respectively, highlighting its efficacy as an environmentally sustainable alternative. The environmental impacts of the beamhouse stage(LCA) approach by comparing two scenarios. The results showed that all the environmental bnificantly lower than those linked to LSS. The utilization of MgCl2-assisted neutral protease in a one-step beamhouse aligns with the trend of environmentally friendly and green production for the leather industry.

Impact of thermal ultrasound on enzyme inactivation and flavor improvement of sea cucumber hydrolysates

In this study, the effects of the thermal ultrasonic enzyme inactivation process on flavor enhancement in sea cucumber hydrolysates (SCHs) and its impact on the inactivation of neutral proteases (NPs) were investigated. The body wall of the sea cucumber was enzymatically hydrolyzed with NPs. On the one hand, the structure of NPs subjected to different enzyme inactivation methods was analyzed using ζ-potential, particle size, and Fourier transform infrared (FT-IR) spectroscopy. On the other hand, the microstructure and flavor changes of SCHs were examined through scanning electron microscopy, E-nose, and gas chromatography-ion mobility spectrometry (GC-IMS). The results indicated that thermal ultrasound treatment at 60 °C could greatly affect the structure of NPs, thereby achieving enzyme inactivation. Furthermore, this treatment generated more pleasant flavor compounds, such as pentanal and (E)-2-nonenal. Hence, thermal ultrasound treatment could serve as an alternative process to traditional heat inactivation of enzymes for improving the flavor of SCHs.

Development of a novel dual-enzyme modification method for the functionality improvement of egg yolk powder

Spray dried egg yolk powder (EYP) is a kind of food ingredient with high nutritional value. However, its low solubility and insufficient emulsifying properties restrict its applications in the food industry. In this study, a new method of dual enzymatic hydrolysis (phospholipase and protease) was proposed to improve the functional properties of EYP including emulsifying properties, solubility, rehydration time and so on. The optimal conditions were determined to be 50 U/g (phospholipase A1 (PLA1)) and 250 U/g (neutral protease). Under these optimal conditions, the solubility of HYP (hydrolyzed EYP) treated by dual enzyme (61.30%) was significantly higher than that of unhydrolyzed EYP (16.49%) or HYP treated by single PLA1 (23.97%). Meanwhile, the emulsifying stability index (ESI) of HYP (hydrolyzed EYP) treated by dual enzymes was about 16/1.4 times larger than unmodified EYP/single PLA1 modified HYP. The increasing electronegativity and decreasing surface hydrophobicity/surface tension of HYP could be responsible for the above results. Scanning electron microscopy (SEM) revealed that dual-enzyme hydrolysis could promote the formation of fine HYP with pore structure, enabling it to rehydrate more effectively. In addition, the usage of protease could improve the radical scavenging activity of dual enzyme modified HYP by releasing antioxidant peptides. This study could provide new insights to promote the techno-functionality of EYP, thus optimizing its utilization as a high-performance emulsifier in the food industry.

Prediction of viral protease inhibitors using proteochemometrics approach

Being widely accepted tools in computational drug search, the (Q)SAR methods have limitations related to data incompleteness. The proteochemometrics (PCM) approach expands the applicability area by using description for both protein and ligand structures. The PCM algorithms are urgently required for the development of new antiviral agents. We suggest the PCM method using the TLMNA descriptors, combining the MNA descriptors of ligands and protein sequence N-grams. Our method was validated on the viral chymotrypsin-like proteases and their ligands. We have developed an original protocol allowing us to collect a comprehensive set of 15 protein sequences and more than 9000 ligands from the ChEMBL database. The N-grams were derived from the 3D-based alignment, accurately superposing ligand-binding regions. In testing the ligand set in SAR mode with MNA descriptors, an accuracy above 0.95 was determined that shows the perspective of the antiviral drug search in virtual chemical libraries. The effective PCM models were built with the TLMNA descriptor. The strong validation procedure with pair exclusion simulated the prediction of interactions between the new ligands and new targets, resulting in accuracy estimation up to 0.89. The PCM approach shows slightly lower accuracy caused by more uncertainty compared with SAR, but it overcomes the problem of data incompleteness.

Abstract 1236: Characterization of CBX-250, a first-in-class TCR-mimetic-based T-cell engager targeting a cathepsin G peptide-HLA complex for the treatment of myeloid leukemia

Abstract While T-cell engaging therapies have demonstrated clear clinical benefit in the treatment of B-cell malignancies, their application to myeloid malignancies remains a challenge due to the limited repertoire of suitable tumor specific surface antigens. Targeting HLA-restricted peptides (pHLA) derived from intracellular cancer antigens provides an opportunity to explore the entire cancer proteome. CG1 (FLLPTGAEA) has been validated as an HLA-A*02:01 restricted leader peptide from Cathepsin G (CTSG) and is abundantly presented by leukemic versus normal myeloid cells. TCR-mimetic (TCRm) antibodies are ideally suited to target pHLA with high potency and specificity and can be engineered and manufactured using standard antibody technologies. Here we report the preclinical characterization of CBX-250, a novel CG1/HLA-A2 TCRm-CD3 bispecific T-Cell Engager (TCE). CBX-250 induced potent T-cell activation and T-cell mediated killing of leukemia cell lines with varying levels of target antigen density with sub-nM EC50 in vitro. In vivo, a closely related precursor of CBX-250 demonstrated potent tumor control in various AML and CML CDX models at doses as low as 0.01mpk. Although CTSG is a serine protease stored in neutrophil azurophilic granules, the CG1 peptide is preferentially presented by leukemia cells, as validated by mass spectrometry. Moreover, when co-culturing PBMCs with HLA-A2 neutrophils, we did not observe any CBX-250-mediated T-cell activation or IFNγ production. In silico predictions identified HLA-A2-restricted human peptides that share sequence or structural similarity to CG1. We determined the cross-reactivity risk of CBX-250 to be low, based on T-cell activation and T-cell mediated cytotoxicity assays against cells pulsed with supra-physiological concentrations of these peptides. Moreover, we observed no activity against target-negative cancer cells, including a CTSG-KO cell line. To further assess the specificity of CBX-250, we screened a panel of normal cells and found no meaningful T-cell activation or IFNγ production at CBX-250 concentrations exceeding its EC90. Finally, the CBX-250 precursor molecule demonstrated favorable safety in a double transgenic mouse model expressing human HLA-A2 and CG1. Following sequence optimization, CBX-250 demonstrated robust serum and pH stress stability and a favorable melting temperature, while retaining excellent potency and specificity. A full suite of analytical and biophysical assessments supports CBX-250’s favorable developability profile. In summary, these data provide strong preclinical evidence of the potency, specificity, safety, and developability of CBX-250, a novel, first-in-class, off-the-shelf, TCRm-based TCE for the treatment of myeloid malignancies. Citation Format: Geraldine Paulus, Benjamin Lee, Preethi Sankaran, Tanzila Rahman, Jessica Jimenez, Delainey O'Connor, Simon Yue, Yu Huang, Melissa Bikowitz, Sarah Jaffe, Shawn O'Malley, Bhupal Ban, Galina Gabriely, Tao Wang, Amanda Mak, Michael Princiotta, Chunhua Shi, Helen He, Ningping Feng, Jun Yan, Timothy Heffernan, Gheath Alatrash, Jeffrey Molldrem, Dmitri Wiederschain. Characterization of CBX-250, a first-in-class TCR-mimetic-based T-cell engager targeting a cathepsin G peptide-HLA complex for the treatment of myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1236.

The In Vitro Effects of Carprofen on Lipopolysaccharide-Induced Neutrophil Extracellular Trap Formation in Dairy Cows.

The objective of this study was to develop an in vitro model that mimics inflammatory reactions and neutrophil extracellular traps (NETs) formation by polymorphonuclear leukocytes (PMNs) in dairy cows. This model was used to examine the effect of carprofen (CA) on lipopolysaccharide (LPS)-induced NETs formation and expression of inflammatory factors. Peripheral blood samples were collected from 24 Holstein cows (3-11 days postpartum) and PMNs were isolated. In three replicates, PMNs were exposed to various treatments to establish an appropriate in vitro model, including 80 μg/mL of LPS for 2 h, followed by co-incubation for 1 h with 60 μmol/L CA and 80 μg/mL LPS. The effects of these treatments were evaluated by assessing NETs formation by extracellular DNA release, gene expression of pro-inflammatory cytokines, reactive oxygen species (ROS) production, and the expression of NETs-related proteins, including histone3 (H3), citrullinated histone (Cit-H3), cathepsin G (CG), and peptidyl arginine deiminase 4 (PAD4). The assessment of these parameters would elucidate the specific mechanism by which CA inhibits the formation of NETs through the PAD4 pathway instead of modulating the Nox2 pathway. This highlights CA's effect on chromatin decondensation during NETs formation. Statistical analyses were performed utilizing one-way ANOVA with Bonferroni correction. The results demonstrated that LPS led to an elevated formation of NETs, while CA mitigated most of these effects, concurrent the PAD4 protein level increased with LPS stimulating and decreased after CA administration. Nevertheless, the intracellular levels of ROS did not change under the presence of LPS. LPS supplementation resulted in an upregulation of H3 and Cit-H3 protein expression levels. Conversely, the CA administration inhibited their expression. Additionally, there was no change in the expression of CG with either LPS or LPS + CA co-stimulation. The gene expression of pro-inflammatory cytokines (tumor necrosis factor -α, interleukin (IL)-18, IL-1β, and IL-6) upregulated with LPS stimulation, while the treatment with CA inhibited this phenomenon. In conclusion, CA demonstrated a pronounced inhibitory effect on both LPS-induced NETs formation as well as the associated inflammatory response.

Insight into the solubilization mechanism of wheat gluten by protease modification from conformational change and molecular interaction perspective

The low solubility limits the utilization of other functional characteristics of wheat gluten (WG). This study effectively improved the solubility of WG through protease modification and explored the potential mechanism of protease modification to enhance the solubility of WG, further stimulating the potential application of WG in the food industry. Solubility of WG modified with alkaline protease, complex protease, and neutral protease was enhanced by 98.99%, 54.59%, and 51.68%, respectively. Notably, the content of β-sheet was reduced while the combined effect of hydrogen bond and ionic bond were increased after protease modification. Meanwhile, the reduced molecular size and viscoelasticity as well as the elevated surface hydrophobicity, thermostability, water absorption capacity, and crystallinity were observed in modified WG. Moreover, molecular docking indicated that protease was specifically bound to the amino acid residues of WG through hydrogen bonding, hydrophobic interaction, and salt bridge.

Statistical versus neural network-embedded swarm intelligence optimization of a metallo-neutral-protease production: activity kinetics and food industry applications

An integrated approach involving response surface methodology (RSM) and artificial neural network-ant-colony hybrid optimization (ANN-ACO) was adopted to develop a bioprocess medium to increase the yield of Bacillus cereus neutral protease under submerged fermentation conditions. The ANN-ACO model was comparatively superior (predicted r 2 = 98.5%, mean squared error [MSE] = 0.0353) to RSM model (predicted r 2 = 86.4%, MSE = 23.85) in predictive capability arising from its low performance error. The hybrid model recommended a medium containing (gL−1) molasses 45.00, urea 9.81, casein 25.45, Ca2+ 1.23, Zn2+ 0.021, Mn2+ 0.020, and 4.45% (vv−1) inoculum, for a 6.75-fold increase in protease activity from a baseline of 76.63 UmL−1. Yield was further increased in a 5-L bioreactor to a final volumetric productivity of 3.472 mg(Lh)−1. The 10.0-fold purified 46.6-kDa-enzyme had maximum activity at pH 6.5, 45–55 °C, with Km of 6.92 mM, Vmax of 769.23 µmolmL−1 min−1, kcat of 28.49 s−1, and kcat/Km of 4.117 × 103 M−1 s−1, at 45 °C, pH 6.5. The enzyme was stabilized by Ca2+, activated by Zn2+ but inhibited by EDTA suggesting that it was a metallo-protease. The biomolecule significantly clarified orange and pineapple juices indicating its food industry application.

Unveiling hidden interactions: Microorganisms, enzymes, and mangroves at different stages of succession in the Shankou Mangrove Nature Reserve, China

Understanding the interactions between microorganisms, soil extracellular enzymes, and mangroves is crucial for conserving and restoring mangrove ecosystems. However, the unique environments associated with mangroves have resulted in a lack of pertinent data regarding the interactions between these components. Root, stem, leaf, and soil samples were collected at three distinct stages of mangrove succession. Stoichiometry was employed to analyze the carbon, nitrogen, and phosphorus contents of these samples and to quantify extracellular enzyme activities, microbial biomass, and various physicochemical factors in the soil. The results showed that the trends of C, N, and P in the mangrove plants were consistent. Microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and microbial biomass phosphorus (MBP) were the highest in the Kandelia obovate community. Catalase (CAT) and β-D-G showed the highest content in K. obovate and Bruguiera gymnorrhiza, whereas cellulase showed the opposite trend. Urease was least abundant in the K. obovate community, whereas neutral protease (NPR) and acid phosphatase (ACP) were most abundant. The overall soil environment in mangroves exhibited a state of N limitation, with varying degrees of limitation observed across different succession stages. The demand for P became more intense in the later stages of succession, particularly in the K. obovate and B. gymnorrhiza communities. In conjunction with correlation analysis, it indicated that the input of mangrove plant litter had a significant regulatory influence on the C, N, and P contents in the soil. There was a significant positive correlation between MBC, MBN, and MBP, indicating synergistic effects of C, N, and P on soil microorganisms. Therefore, evaluating the nutrient ratios and sufficiency of mangroves allowed us to comprehensively understand the present environmental conditions. This study aims to develop sustainable management strategies for the conservation and restoration of mangroves.

Egg Yolk Selenopeptides: Preparation, Characterization, and Immunomodulatory Activity.

In this study, egg yolk selenium peptides (Se-EYP) were prepared using double-enzyme hydrolysis combined with a shearing pretreatment. The properties of the selenopeptides formed were then characterized, including their yield, composition, molecular weight distribution, antioxidant activity, in vitro digestion, and immunomodulatory activity. The peptide yield obtained after enzymatic hydrolysis using a combination of alkaline protease and neutral protease was 74.5%, of which 82.6% had a molecular weight <1000 Da. The selenium content of the lyophilized solid product was 4.01 μg/g. Chromatography-mass spectrometry analysis showed that 88.6% of selenium in Se-EYP was in the organic form, of which SeMet accounted for 60.3%, SeCys2 for 21.8%, and MeSeCys for 17.9%. After being exposed to in vitro simulated digestion, Se-EYP still had 65.1% of oligopeptides present, and the in vitro antioxidant activity was enhanced. Moreover, Se-EYP exhibited superior immune detection indices, including immune organ index, level of immune factors in the serum, histopathological changes in the spleen, and selenium content in the liver. Our results suggest that Se-EYP may be used as selenium-enriched ingredients in functional food products.