Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei

Abstract Thelohanellus kitauei , is a group of obligate parasitic Myxozoans, which causes intestinal giant-cystic disease of common carp ( Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris . Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G 19 G 20 , Q 73 VVAG 77 , and C-terminal L 102 P 103 . Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (-13 kal/mol) with cathepsin L whereas a higher binding energy (-8.6 kal/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli . Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-TK-cystatin vaccine in carp culture in the future.

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Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei

10.21203/rs.3.rs-26747/v1 ◽

2020 ◽

Author(s):

Fengli Zhang ◽

Yalin Yang ◽

Chenchen Gao ◽

Yuanyuan Yao ◽

Rui Xia ◽

...

Keyword(s):

Binding Energy ◽

Inhibitory Activity ◽

Cathepsin B ◽

Cathepsin L ◽

Cysteine Proteases ◽

Economic Losses ◽

Amino Acid Residues ◽

Gene Encoding ◽

Multiple Sequence ◽

Cystatin Gene

Abstract Thelohanellus kitauei, is a group of obligate parasitic Myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G19G20, Q73VVAG77, and C-terminal L102P103. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (-13 kal/mol) with cathepsin L whereas a higher binding energy (-8.6 kal/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-TK-cystatin vaccine in carp culture in the future.

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Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens

Parasite ◽

10.1051/parasite/2021005 ◽

2021 ◽

Vol 28 ◽

pp. 9

Author(s):

Shiro Murata ◽

Ayaka Taniguchi ◽

Masayoshi Isezaki ◽

Sotaro Fujisawa ◽

Eishi Sakai ◽

...

Keyword(s):

Cysteine Protease ◽

Cathepsin L ◽

Cysteine Proteases ◽

Mite Species ◽

Economic Losses ◽

Dermanyssus Gallinae ◽

Physiological Processes ◽

Poultry Houses ◽

Poultry Red Mites ◽

Potential Use

Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.

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Cysteine Proteases and Cell Differentiation: Excystment of the Ciliated Protist Sterkiella histriomuscorum

Eukaryotic Cell ◽

10.1128/ec.2.6.1234-1245.2003 ◽

2003 ◽

Vol 2 (6) ◽

pp. 1234-1245 ◽

Cited By ~ 21

Author(s):

Eduardo Villalobo ◽

Clara Moch ◽

Ghislaine Fryd-Versavel ◽

Anne Fleury-Aubusson ◽

Loïc Morin

Keyword(s):

Cyst Wall ◽

Protein Sequence ◽

Calcium Binding ◽

Cathepsin B ◽

Cathepsin L ◽

Signal Transduction Pathway ◽

Cysteine Proteases ◽

Reverse Transcription Pcr ◽

Phylogenetic Cluster ◽

Extracellular Matrix Components

ABSTRACT The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.

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Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination

PLANT PHYSIOLOGY ◽

10.1104/pp.109.146019 ◽

2009 ◽

Vol 151 (3) ◽

pp. 1531-1545 ◽

Cited By ~ 103

Author(s):

Manuel Martinez ◽

Ines Cambra ◽

Laura Carrillo ◽

Mercedes Diaz-Mendoza ◽

Isabel Diaz

Keyword(s):

Seed Germination ◽

Gene Family ◽

Cathepsin L ◽

Cysteine Proteases ◽

Cystatin Gene

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Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

Biochemical Journal ◽

10.1042/bj3400113 ◽

1999 ◽

Vol 340 (1) ◽

pp. 113-117 ◽

Cited By ~ 14

Author(s):

Victor J. CHAN ◽

Paul M. SELZER ◽

James H. McKERROW ◽

Judy A. SAKANARI

Keyword(s):

Cathepsin B ◽

Leishmania Major ◽

Cathepsin L ◽

Expression System ◽

Specific Inhibitor ◽

Structural Similarity ◽

Cysteine Proteases ◽

Recombinant Enzyme ◽

Yeast Expression System ◽

S2 Subsite

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

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Cathepsin Cs Are Key for the Intracellular Survival of the Protozoan Parasite, Toxoplasma gondii

Journal of Biological Chemistry ◽

10.1074/jbc.m606764200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4994-5003 ◽

Cited By ~ 38

Author(s):

Xuchu Que ◽

Juan C. Engel ◽

David Ferguson ◽

Annette Wunderlich ◽

Stanislas Tomavo ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Cathepsin B ◽

Parasitophorous Vacuole ◽

Cathepsin L ◽

Protozoan Parasite ◽

Cysteine Proteases ◽

Intracellular Survival ◽

Chemotherapeutic Agents ◽

Cathepsin C

Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli β-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.

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Action of human liver cathepsin B on the oxidized insulin B chain

Biochemical Journal ◽

10.1042/bj2130467 ◽

1983 ◽

Vol 213 (2) ◽

pp. 467-471 ◽

Cited By ~ 34

Author(s):

M J McKay ◽

M K Offermann ◽

A J Barrett ◽

J S Bond

Keyword(s):

Amino Acid ◽

Cathepsin B ◽

Human Liver ◽

Degradation Products ◽

Cysteine Proteinase ◽

Cathepsin L ◽

Amino Acid Content ◽

Peptide Bond ◽

Cleavage Sites ◽

Amino Acid Residues

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.

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Selectivity of propeptide-enzyme interaction in cathepsin L-like cysteine proteases

Biological Chemistry ◽

10.1515/bc.2009.023 ◽

2009 ◽

Vol 390 (2) ◽

pp. 167-174 ◽

Cited By ~ 15

Author(s):

Klaus Schilling ◽

Alexandra Körner ◽

Saskia Sehmisch ◽

Annett Kreusch ◽

Ramona Kleint ◽

...

Keyword(s):

Inhibitory Activity ◽

Tertiary Structure ◽

Cathepsin L ◽

Cysteine Proteases ◽

Systematic Investigation ◽

Cross Reactivity ◽

X Ray ◽

Chaperone Function ◽

Inhibitory Function ◽

Enzyme Unit

Abstract Cathepsin L-like endopeptidases of the papain family are synthesized as proenzymes. N-terminal proregions are essential for folding and latency of the enzyme unit. While selectivity has been reported for the inhibitory function of papain-family propeptides, there is no systematic investigation of the selectivity of their chaperone-like function to date. The chaperone-like cross-reactivity between the cathepsins S, K, and L were thoroughly quantified in trans-experiments, i.e., with isolated propeptides and mature enzymes, and compared to the inhibitory cross-reactivity. The three endopeptidases have been chosen due to only minimal evolutionary distance and nearly identical X-ray structures of their zymogenes. The intramolecular chaperone function of the proregion was found to be more selective than the inhibitory activity and significant differences were found between the selectivity profiles, underlining the assumption that the inhibitory and the chaperone-like propeptide functions are autonomous. Considering the data published on the intramolecular chaperone-like propeptide function within other protease classes as well, our data suggest that intrinsically structured propeptides are more selective than intrinsically unstructured propeptides, i.e., those adopting tertiary structure elements only in complex with their maternal enzyme.

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Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives

Molecules ◽

10.3390/molecules24132348 ◽

2019 ◽

Vol 24 (13) ◽

pp. 2348 ◽

Cited By ~ 2

Author(s):

Florencia Ferraro ◽

Alicia Merlino ◽

Jorge Gil ◽

Hugo Cerecetto ◽

Ileana Corvo ◽

...

Keyword(s):

Cathepsin L ◽

Md Simulations ◽

Cysteine Proteases ◽

Current Treatment ◽

Economic Losses ◽

New Chemical Entities ◽

Ligand Interactions ◽

Bovine Spermatozoa ◽

Low Cytotoxicity

Infections caused by Fasciola species are widely distributed in cattle and sheep causing significant economic losses, and are emerging as human zoonosis with increasing reports of human cases, especially in children in endemic areas. The current treatment is chemotherapeutic, triclabendazole being the drug of preference since it is active against all parasite stages. Due to the emergence of resistance in several countries, the discovery of new chemical entities with fasciolicidal activity is urgently needed. In our continuous search for new fasciolicide compounds, we identified and characterized six quinoxaline 1,4-di-N-oxide derivatives from our in-house library. We selected them from a screening of novel inhibitors against FhCL1 and FhCL3 proteases, two essential enzymes secreted by juvenile and adult flukes. We report compounds C7, C17, C18, C19, C23, and C24 with an IC50 of less than 10 µM in at least one cathepsin. We studied their binding kinetics in vitro and their enzyme-ligand interactions in silico by molecular docking and molecular dynamic (MD) simulations. These compounds readily kill newly excysted juveniles in vitro and have low cytotoxicity in a Hep-G2 cell line and bovine spermatozoa. Our findings are valuable for the development of new chemotherapeutic approaches against fascioliasis, and other pathologies involving cysteine proteases.

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SCREENING OF α-GLUCOSIDASE INHIBITORS FROM TERMINALIA CATAPPA L. FRUITS USING MOLECULAR DOCKING METHOD AND IN VITRO TEST

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2016v8i12.14800 ◽

2016 ◽

Vol 8 (12) ◽

pp. 184 ◽

Cited By ~ 1

Author(s):

Bina Lohita Sari ◽

Abdul Mun’im ◽

Arry Yanuar ◽

Rezi Riadhi

Keyword(s):

Molecular Docking ◽

Amino Acid ◽

Binding Energy ◽

Ethyl Acetate ◽

Active Compound ◽

Inhibitory Activity ◽

Ethyl Acetate Extract ◽

Amino Acid Residues ◽

Terminalia Catappa

Objective: Terminalia catappa L. (T. catappa L.) fruit has inhibitory activity on α-glucosidase, therefore, can be a potential natural source for the treatment of type II diabetes mellitus. Inhibitory activity of ethanol fruit extract with IC50 3.02 µg/ml was the strongest inhibition when compared with 54 medicinal plants used as an antidiabetic agent in Indonesia. This project was aimed to find the active compound from T. catappa L. fruit using molecular docking, identification ethyl acetate subfraction using TLC and GC-MC, determine in vitro test on α-glucosidase inhibitory activity from ethyl acetate extract and subfraction.Methods: Molecular docking using AutoDock 4.2 was performed to predict the binding modes of α-glucosidase enzyme from Saccharomyces cereviciae with 13 chemical constituents of T. catappa. α-Glucosidase enzyme was obtained from Protein Data Bank (PDB code: 3A4A). Acarbose, voglibose and miglitol were used as standards. Docking result determines the highest binding energy (ΔG) and inhibition constants (Ki) as an active compound. Visualization of amino acid residues around the active compound was identified with PyMOL and LigPlot. Screening of active compound was carried out by T. catappa L. fruit remaceration extraction use hexane and ethyl acetate. Ethyl acetate extract was separated on silica gel column chromatography using n-hexane, ethyl acetate and methanol sequentially based on polarity of each solvent. Identification of an active compound from ethyl acetate sub fractions using TLC and GC-MS method. The inhibitory activity of the active compound of α-glucosidase was determined with in vitro test using α-glucosidase enzyme.Results: The highest binding energy and inhibition constant is β–sitosterol with ΔG-10.61 kcal/mol and Ki 0.02 µM. The ligand was situated around of 18 amino acid residues. Ethyl acetate subfractions A, B and C showed that subfraction B contains similar spot characteristic and Rf value (0.42) with β-Sitosterol standard. Identification with GC-MS gave β–sitosterol acetate and sitostenone. Redocking process of β–sitosterol acetate and sitostenone showed ΔG-11.14 kcal/mol and-9.79 kcal/mol with Ki 0.01 μM and 0.07 μM respectively. In vitro test of acarbose, ethyl acetate extract and subfraction B gave IC50 17.52; 192.51 and 296.28 µg/ml.Conclusion: Three steroids that are β-sitosterol, β-sitosterol acetate and sitostenone were the active compounds responsible for α-glucosidase inhibitory activity of T. catappa L. fruit. According to the in vitro test, ethyl acetate extract has stronger α-glucosidase inhibitory activity than ethyl acetate subfraction B.

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