Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.
Animals ; Aspartic Acid Endopeptidases ; Biology ; Cathepsin D ; Cathepsins ; Clonorchiasis ; Clonorchis sinensis ; Eggs ; Helminths ; Humans ; Intestines ; Metacercariae ; Ovum ; Parasites ; Peptide Hydrolases ; Rats ; Reproduction

In this paper,metabolomics and network pharmacology were used to investigate the bioactive components of Harrisonia perforata and their possible mechanisms of action. Metabolites in the flowers,fruits,branches,leaves and stalks of H. perforata were analyzed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Meanwhile,multiple statistical analysis methods including principal component analysis( PCA) and orthogonal partial least squares discriminant analysis( OPLS-DA)were applied to screen and identify differential compounds. With metabolomics method,9 differential compounds were preliminarily identified from leaves and other non-traditional medicinal parts. Subsequently,these compounds were explored by using network pharmacology. With gastrointestinal absorption and drug-likeness as limiting conditions,they were imported into the Swiss ADME,from which 7 compounds with potential medicinal activity were obtained. Then,their targets were predicted by PharmMapper,with Human Protein Targets Only and Normalized Fit Score>0. 9 set as limiting conditions,and 60 standardized potential targets were identified with Uniprot. KEGG( Kyoto encyclopedia of genes and genomes) pathway data was obtained using metascape and the " potential active ingredients-target-pathway" network was constructed with Cytoscape 3. 7. 2. The enrichment analysis of KEGG demonstrated that the 60 targets were enriched in 78 signaling pathways( min overlap: 3,P value cutoff: 0. 01,min enrichment: 1. 5),many of which are related to anti-bacteria,anti-inflammation and anti-virus,such as IL-17 signaling pathway,RIG-I-like receptor signaling pathway and NOD-like receptor signaling pathway. Finally,depending on the clinical activity of H. perforata,the relevant signaling pathways were analyzed through experimental data and literature. Dehydroconiferyl alcohol was reported to have the anti-inflammatory effect and perforamone D to possess the antimycobacterial activity. The KEGG pathway enrichment analysis showed that dehydroconiferyl alcohol could act on the Alzheimer's disease( AD) signaling pathway by targeting CDK5 R1 and BACE1. ACh E inhibitor is the most promising drug to treat AD,while dehydroconiferyl alcohol has been proved to inhibit ACh E according to literature. The experimental results revealed that the extract of leaves of H. perforata can effectively inhibit the growth of Staphylococcus aureus. These are consistent with the enrichment analysis results of KEGG. This study explored the bioactive components and pharmacodynamics of the leaves of the H. perforata,laying a theoretical foundation for its in-depth development and rational application.
Amyloid Precursor Protein Secretases ; Aspartic Acid Endopeptidases ; Drugs, Chinese Herbal/pharmacology* ; Humans ; Metabolomics ; Simaroubaceae

Glutinous rice wine is a traditional food in south of China and it can coagulate milk. It has been proved that its function of coagulating milk is because of the presence of milk-clotting enzyme produced by microorganisms in glutinous rice wine. The aim of this work is to isolate milk-clotting enzyme producing strain from glutinous rice wine and study the fermentation condition. We screened out four bacteria and fungus by gradient dilution. It was proved that mold played the most important role in the production of milk-clotting enzyme. This is further confirmed by casein plate method. The optimization of fermentation conditions revealed that two times concentrated potato medium supplemented with 5% glucose without additional nitrogen was better for production of the enzyme. The enzyme activity was increased 144% under the conditions established.
Aspartic Acid Endopeptidases ; biosynthesis ; Bacteria ; isolation & purification ; metabolism ; Culture Media ; Fermentation ; Oryza ; metabolism ; Wine ; microbiology ; Yeasts ; cytology ; isolation & purification ; metabolism

Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.
Aspartic Acid/chemistry* ; Bacterial Outer Membrane Proteins/metabolism ; Blotting, Western ; Escherichia coli/enzymology* ; Escherichia coli/chemistry ; Micrococcal Nuclease/metabolism ; Mutagenesis, Site-Directed ; Oligonucleotides ; Protein Precursors/metabolism ; Serine Endopeptidases/metabolism* ; Serine Endopeptidases/genetics ; Serine Endopeptidases/chemistry* ; Structure-Activity Relationship

To generate active recombinant human beta-secreatase (BACE1) for studying its interaction with its inhibitors, we constructed two recombinant plasmids, pPIC9K-MetBACE22 (bearing pro-bace1 gene) and pPIC9K-MetBACE46 (bearing bace1 gene). These two plasmids were then transformed into Pichia pastoris GS115 by electroporation to obtain the recombinant strains 9k-B22 and 9k-B46. After induction in buffered methanol complex medium, we found the supernatant activity of 9k-B22 significantly higher than that of 9k-B46. The culture filtrate of 9k-B22 was concentrated, and then purified by HisTrap affinity column. The purified proteins, showing good BACE1 protease activity, were found to be a mixture of glycoproteins because they can be stained by periodic acid-Schiff reagent. After this mixture was treated with Endo H(f) (a recombinant protein of endoglycosidase H), we found two new adjacent bands around 50 kDa on SDS-PAGE. These two bands were cut and subjected to peptide mass fingerprint analysis, and identified as proBACE1 and BACE1 proteins. Enzyme assays revealed that the activities of both BACE1 proteins in glycosylated and deglycosylated form were lower than that of commercial BACE1 (expressed in HEK-293), inferring glycosylation and the type of glycosylation are crucial to the activity. However, we found no apparent difference in the inhibition of those all above three enzyme forms by one known BACE1 inhibitor. This observation demonstrated that the glycosylation of BACE1 by Pichia pastoris does not affect its interaction with this inhibitor. After optimization of culture conditions, the production of BACE1 in Pichia pastoris was enhanced to about 1 mg/L. This work enables us to further investigate the interaction of BACE1 and its inhibitors, and assists in discovering and optimizing BACE1 inhibitors as anti-Alzheimer's disease agents.
Amyloid Precursor Protein Secretases ; biosynthesis ; genetics ; Aspartic Acid Endopeptidases ; biosynthesis ; genetics ; Electroporation ; Humans ; Pichia ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichiaexpressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.
Adolescent ; Adult ; Aged ; Amino Acid Sequence ; Animals ; Antibodies ; Aspartic Endopeptidases/chemistry/genetics/*immunology ; Child ; Cockroaches/immunology ; Epitopes ; Gene Expression Regulation ; Humans ; Immunoglobulin E/*immunology ; Middle Aged ; Young Adult

OBJECTIVETo explore the effect of Panax notoginseng saponin (PNS) on the activity and content of beta-secretase in the brain of senescence accelerated mouse-prone 8 (SAMP8) mice with Alzheimer's disease.

METHODSTotally 32 SAMP8 mice were randomly divided into the normal control group, the high dose PNS group (200 mg/kg), the low dose group (100 mg/kg), and the huperzine A group (0.3 mg/kg), 8 in each group. Equal volume of double distilled water was given to those in the normal control group. All medication was given by gastrogavage, once daily for two successive months. The activity of BACE1 was assayed by direct immunofluorescent method (DIF). The content of BACE1 protein was detected by Western blot.

RESULTSThe relative fluorescence units (RFU/microg) was 2.008 +/- 0.031 in the high dose PNS group, 2.221 +/- 0.029 in the low dose PNS group, and 2.267 +/- 0.076 in the huperzine A group, all lower than that in the normal control group (2.403 +/- 0.058; all P < 0.01). The content of BACE1 protein was 0.900 +/- 0.028 in the high dose PNS group, 1.000 +/- 0.032 in the low dose PNS group, and 0.837 +/- 0.080 in the huperzine A group, all lower than that in the normal control group (2.210 +/- 0.074, all P < 0.01).

CONCLUSIONPNS higher than 100 mg/kg could decrease the activity of BACE1 and down-regulate the content of BACE1 protein in the brain of SAMP8 mice.

Aging ; Alzheimer Disease ; metabolism ; Amyloid Precursor Protein Secretases ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; metabolism ; Brain ; metabolism ; Disease Models, Animal ; Male ; Mice ; Panax notoginseng ; RNA, Messenger ; genetics ; Saponins ; pharmacology

The bglS gene encoding endo-l,3-1,4-beta-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone alpha-factor (MFalpha1(S)), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-betaG) was preliminarily screened by the clearing hydrolysis zone formed after the barley beta-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-beta-glucanase assay methods showed that the recombinant strain SC-betaG had high endo-l,3-1,4-beta-glucanase expression level with the maximum of 69.3 U/(h.ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-beta-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.
Amino Acid Sequence ; Aspartic Acid Endopeptidases ; genetics ; Base Sequence ; Glycoside Hydrolases ; genetics ; Molecular Sequence Data ; Mutagenesis, Insertional ; Polymerase Chain Reaction ; Recombination, Genetic ; Saccharomyces cerevisiae ; genetics ; Saccharomyces cerevisiae Proteins ; genetics

OBJECTIVETo investigate the effects of exogenous hydrogen sulfide (H2S) on β-site APP cleaving enzyme 1 (BACE-1) and β-amyloid peptide (Aβ) metabolism in primary culture of neurons under high-glucose condition.

METHODSThe cortical neurons in primary culture under normal and high glucose (60 mmol/L) conditions for 24 h were exposed to 25, 50 and 100 µmol/L NaHS. Aβ1-42 concentration in the cell culture was measured by ELISA, and BACE-1 mRNA and protein levels were detected by fluorescent quantitative real-time PCR and Western blotting, respectively.

RESULTSCompared with the neurons cultured in normal glucose, the neurons exposed to high glucose showed significantly increased Aβ1-42 concentration and BACE-1 mRNA and protein expressions (P<0.05). Exposure to 25, 50 and 100 µmol/L NaHS significantly decreased Aβ1-42 concentration and BACE-1 mRNA and protein expressions in the high-glucose cell culture (P<0.05).

CONCLUSIONNeurons exposed to high glucose exhibit increased Aβ1-42 levels and BACE-1 mRNA and protein expressions, which can be concentration-dependently decreased by NaHS.

Amyloid Precursor Protein Secretases ; metabolism ; Amyloid beta-Peptides ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; metabolism ; Cells, Cultured ; Culture Media ; chemistry ; Glucose ; chemistry ; Hydrogen Sulfide ; pharmacology ; Neurons ; drug effects ; metabolism ; Peptide Fragments ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the diagnostic value of HNF-1β and Napsin A for ovarian clear cell carcinomas, serous carcinomas, endometrioid adenocarcinomas and metastatic Krukenberg tumors.

METHODSImmunohistochemical EnVision method was used to detect the expression of HNF-1β and Napsin A in 38 cases of ovarian clear cell carcinoma, 30 cases of high-grade serous carcinoma, 22 cases of endometrioid adenocarcinoma and 16 cases of metastatic Krukenberg tumor. Expression of HNF-1β and Napsin A were compared, and sensitivity and specificity of clear cell carcinoma of the ovary were analysed.

RESULTSThe positive rate of HNF-1β in the ovarian clear cell carcinoma was 100%(38/38), higher than those in high-grade serous carcinoma and endometrioid adenocarcinoma (P<0.05), although significant difference was not observed from that of metastatic Krukenberg tumor (P>0.05). Napsin A expressed in 97.4% (37/38) of ovarian clear cell carcinoma, 6.7% (2/30) of high-grade serous carcinoma, 22.7% (5/22) of endometrioid adenocarcinoma. Napsin A expression in clear cell carcinoma was higher than those in high-grade serous carcinoma and endometrioid adenocarcinoma (P<0.01), and no expression of Napsin A was seen in metastatic Krukenberg tumor (P>0.05). The sensitivity and specificity of HNF-1β in the diagnosis of ovarian clear cell carcinoma were 100% and 52.9%, those of Napsin A were 97.4% and 91.2%, those of both HNF-1β and Napsin A were 97.4% and 91.2%, respectively. The sensitivity and specificity of HNF-1β or Napsin A in the diagnosis of ovarian clear cell carcinoma were 100% and 52.9%, respectively.

CONCLUSIONSHNF-1β is a more sensitive marker for the diagnosis of ovarian clear cell carcinoma, whereas Napsin A is a more specific marker. The combined detection of HNF-1β and Napsin A may be helpful for the diagnosis of clear cell carcinoma of the ovary.

Adenocarcinoma, Clear Cell ; diagnosis ; Aspartic Acid Endopeptidases ; genetics ; Biomarkers, Tumor ; genetics ; Carcinoma, Endometrioid ; diagnosis ; Cystadenocarcinoma, Serous ; diagnosis ; Female ; Hepatocyte Nuclear Factor 1-alpha ; genetics ; Humans ; Immunohistochemistry ; Krukenberg Tumor ; diagnosis ; Ovarian Neoplasms ; diagnosis ; Sensitivity and Specificity