{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T03:20:46Z","timestamp":1773631246143,"version":"3.50.1"},"reference-count":47,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2022,6,23]],"date-time":"2022-06-23T00:00:00Z","timestamp":1655942400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Russian Foundation for Basic Research","award":["20-54-56044"],"award-info":[{"award-number":["20-54-56044"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 \u00b0C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.<\/jats:p>","DOI":"10.3390\/ijms23137001","type":"journal-article","created":{"date-parts":[[2022,6,23]],"date-time":"2022-06-23T21:25:00Z","timestamp":1656019500000},"page":"7001","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides"],"prefix":"10.3390","volume":"23","author":[{"given":"Elena A.","family":"Dvoryakova","sequence":"first","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria A.","family":"Klimova","sequence":"additional","affiliation":[{"name":"Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9291-877X","authenticated-orcid":false,"given":"Tatiana R.","family":"Simonyan","sequence":"additional","affiliation":[{"name":"Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ivan A.","family":"Dombrovsky","sequence":"additional","affiliation":[{"name":"Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marina V.","family":"Serebryakova","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Valeriia F.","family":"Tereshchenkova","sequence":"additional","affiliation":[{"name":"Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yakov E.","family":"Dunaevsky","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mikhail A.","family":"Belozersky","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Irina Y.","family":"Filippova","sequence":"additional","affiliation":[{"name":"Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4988-4842","authenticated-orcid":false,"given":"Elena N.","family":"Elpidina","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1743","DOI":"10.1016\/B978-0-12-382219-2.00404-X","article-title":"Introduction: The Clans and Families of Cysteine Peptidases","volume":"Volume 2","author":"Rawlings","year":"2013","journal-title":"Handbook of Proteolytic Enzymes"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/j.bbapap.2011.10.002","article-title":"Cysteine Cathepsins: From Structure, Function and Regulation to New Frontiers","volume":"1824","author":"Turk","year":"2012","journal-title":"Biochim. Biophys. Acta Proteins Proteom."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"387","DOI":"10.2174\/138161207780162962","article-title":"Emerging Roles of Cysteine Cathepsins in Disease and Their Potential as Drug Targets","volume":"13","author":"Vasiljeva","year":"2007","journal-title":"Curr. Pharm. Des."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.cbpb.2006.05.005","article-title":"Diversity of Digestive Proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) Larvae","volume":"145","author":"Vinokurov","year":"2006","journal-title":"Comp. Biochem. Physiol. B Biochem. Mol. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.cbpb.2006.05.004","article-title":"Fractionation of Digestive Proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) Larvae and Role in Protein Digestion","volume":"145","author":"Vinokurov","year":"2006","journal-title":"Comp. Biochem. Physiol. B Biochem. Mol. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Tribolium Genome Sequencing Consortium (2008). The Genome of the Model Beetle and Pest Tribolium castaneum. Nature, 452, 949\u2013955.","DOI":"10.1038\/nature06784"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1002\/arch.20299","article-title":"Digestive Proteolysis Organization in Two Closely Related Tenebrionid Beetles: Red Flour Beetle (Tribolium castaneum) and Confused Flour Beetle (Tribolium confusum)","volume":"70","author":"Vinokurov","year":"2009","journal-title":"Arch. Insect Biochem. Physiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1016\/j.ibmb.2012.04.010","article-title":"The 3D Structure and Function of Digestive Cathepsin L-like Proteinases of Tenebrio molitor Larval Midgut","volume":"42","author":"Beton","year":"2012","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/j.ibmb.2011.10.008","article-title":"Recombinant Expression, Localization and in Vitro Inhibition of Midgut Cysteine Peptidase (Sl-CathL) from Sugarcane Weevil, Sphenophorus levis","volume":"42","author":"Fonseca","year":"2012","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1931","DOI":"10.1038\/s41598-018-20154-1","article-title":"A Model Species for Agricultural Pest Genomics: The Genome of the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)","volume":"8","author":"Schoville","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"103679","DOI":"10.1016\/j.ibmb.2021.103679","article-title":"Primary Digestive Cathepsins L of Tribolium castaneum Larvae: Proteomic Identification, Properties, Comparison with Human Lysosomal Cathepsin L","volume":"140","author":"Dvoryakova","year":"2022","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"103488","DOI":"10.1016\/j.ibmb.2020.103488","article-title":"Cathepsins L and B in Dysdercus peruvianus, Rhodnius prolixus, and Mahanarva fimbriolata. Looking for Enzyme Adaptations to Digestion","volume":"127","author":"Pimentel","year":"2020","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Sparks, M.E., Bansal, R., Benoit, J.B., Blackburn, M.B., Chao, H., Chen, M., Cheng, S., Childers, C., Dinh, H., and Doddapaneni, H.V. (2020). Brown Marmorated Stink Bug, Halyomorpha halys (St\u00e5l), Genome: Putative Underpinnings of Polyphagy, Insecticide Resistance Potential and Biology of a Top Worldwide Pest. BMC Genom., 21.","DOI":"10.1186\/s12864-020-6510-7"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Martynov, A.G., Elpidina, E.N., Perkin, L., and Oppert, B. (2015). Functional Analysis of C1 Family Cysteine Peptidases in the Larval Gut of Tenebrio molitor and Tribolium castaneum. BMC Genom., 16.","DOI":"10.1186\/s12864-015-1306-x"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e1581","DOI":"10.7717\/peerj.1581","article-title":"Expression Patterns of Cysteine Peptidase Genes across the Tribolium Castaneum Life Cycle Provide Clues to Biological Function","volume":"4","author":"Perkin","year":"2016","journal-title":"PeerJ"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3889","DOI":"10.1021\/pr900168z","article-title":"Tribolium Castaneum Larval Gut Transcriptome and Proteome: A Resource for the Study of the Coleopteran Gut","volume":"8","author":"Morris","year":"2009","journal-title":"J. Proteome Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.cbpb.2011.10.005","article-title":"Cysteine Digestive Peptidases Function as Post-Glutamine Cleaving Enzymes in Tenebrionid Stored-Product Pests","volume":"161","author":"Goptar","year":"2012","journal-title":"Comp. Biochem. Physiol. Part B Biochem. Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1093\/jexbot\/53.370.947","article-title":"Cereal Seed Storage Proteins: Structures, Properties and Role in Grain Utilization","volume":"53","author":"Shewry","year":"2002","journal-title":"J. Exp. Bot."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1124\/jpet.104.068429","article-title":"Effect of Prolyl Endopeptidase on Digestive-Resistant Gliadin Peptides In Vivo","volume":"311","author":"Piper","year":"2004","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1042\/BJ20040907","article-title":"Comparative Biochemical Analysis of Three Bacterial Prolyl Endopeptidases: Implications for Coeliac Sprue","volume":"383","author":"Shan","year":"2004","journal-title":"Biochem. J."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1908","DOI":"10.1111\/j.1742-4658.2007.05749.x","article-title":"Gliadain, a Gibberellin-Inducible Cysteine Proteinase Occurring in Germinating Seeds of Wheat, Triticum aestivum L., Specifically Digests Gliadin and Is Regulated by Intrinsic Cystatins","volume":"274","author":"Kiyosaki","year":"2007","journal-title":"FEBS J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1111\/j.1472-765X.2008.02448.x","article-title":"Functionality of Lactic Acid Bacteria Peptidase Activities in the Hydrolysis of Gliadin-like Fragments","volume":"47","author":"Gerez","year":"2008","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.biocel.2015.03.001","article-title":"Glutenase and Collagenase Activities of Wheat Cysteine Protease Triticain-\u03b1: Feasibility for Enzymatic Therapy Assays","volume":"62","author":"Savvateeva","year":"2015","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"13106","DOI":"10.1021\/jacs.5b08325","article-title":"Engineering of Kuma030: A Gliadin Peptidase That Rapidly Degrades Immunogenic Gliadin Peptides in Gastric Conditions","volume":"137","author":"Wolf","year":"2015","journal-title":"J. Am. Chem. Soc."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2428","DOI":"10.1007\/s10620-017-4687-7","article-title":"Latiglutenase Improves Symptoms in Seropositive Celiac Disease Patients While on a Gluten-Free Diet","volume":"62","author":"Syage","year":"2017","journal-title":"Dig. Dis. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Martinez, M., G\u00f3mez-Cabellos, S., Gim\u00e9nez, M.J., Barro, F., Diaz, I., and Diaz-Mendoza, M. (2019). Plant Proteases: From Key Enzymes in Germination to Allies for Fighting Human Gluten-Related Disorders. Front. Plant Sci., 10.","DOI":"10.3389\/fpls.2019.00721"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Wei, G., Helmerhorst, E.J., Darwish, G., Blumenkranz, G., and Schuppan, D. (2020). Gluten Degrading Enzymes for Treatment of Celiac Disease. Nutrients, 12.","DOI":"10.3390\/nu12072095"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Dunaevsky, Y.E., Tereshchenkova, V.F., Belozersky, M.A., Filippova, I.Y., Oppert, B., and Elpidina, E.N. (2021). Effective Degradation of Gluten and Its Fragments by Gluten-Specific Peptidases: A Review on Application for the Treatment of Patients with Gluten Sensitivity. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13101603"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"810","DOI":"10.1016\/j.ijbiomac.2018.11.219","article-title":"Effect of C-Terminal His-Tag and Purification Routine on the Activity and Structure of the Metalloenzyme, l-Alanyl-d-Glutamate Peptidase of the Bacteriophage T5","volume":"124","author":"Kutyshenko","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1732","DOI":"10.1021\/pr050173t","article-title":"Identification and Analysis of Multivalent Proteolytically Resistant Peptides from Gluten: Implications for Celiac Sprue","volume":"4","author":"Shan","year":"2005","journal-title":"J. Proteome Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"6036","DOI":"10.3748\/wjg.v18.i42.6036","article-title":"Celiac Disease: Prevalence, Diagnosis, Pathogenesis and Treatment","volume":"18","author":"Gujral","year":"2012","journal-title":"World J. Gastroenterol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1016\/j.jcs.2014.02.012","article-title":"Production of Gluten-Free Wheat Starch by Peptidase Treatment","volume":"60","author":"Walter","year":"2014","journal-title":"J. Cereal Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/S1046-2023(03)00212-3","article-title":"Production and Activation of Recombinant Papain-like Cysteine Proteases","volume":"32","author":"Nallaseth","year":"2004","journal-title":"Methods"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/S0167-4838(99)00263-0","article-title":"Lysosomal Cysteine Proteases: More than Scavengers","volume":"1477","author":"Turk","year":"2000","journal-title":"Biochim. Biophys. Acta Protein Struct. Mol. Enzymol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4478","DOI":"10.1074\/jbc.273.8.4478","article-title":"Autocatalytic Processing of Recombinant Human Procathepsin L","volume":"273","author":"Carmona","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/0014-5793(94)00924-4","article-title":"Multi-Step Processing of Procathepsin L in Vitro","volume":"352","author":"Ishidoh","year":"1994","journal-title":"FEBS Lett."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"17038","DOI":"10.1074\/jbc.M308831200","article-title":"Cathepsin L1, the Major Protease Involved in Liver Fluke (Fasciola hepatica) Virulence","volume":"279","author":"Collins","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1021\/bi00052a046","article-title":"Kinetics of the PH-Induced Inactivation of Human Cathepsin L","volume":"32","author":"Turk","year":"1993","journal-title":"Biochemistry"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"ref_40","first-page":"403","article-title":"Tampone Universale Di Britton e Robinson a Forza Ionica Constante","volume":"87","author":"Frugoni","year":"1957","journal-title":"Gazz. Chim. Ital."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Filippova, I.Y., Dvoryakova, E.A., Sokolenko, N.I., Simonyan, T.R., Tereshchenkova, V.F., Zhiganov, N.I., Dunaevsky, Y.E., Belozersky, M.A., Oppert, B., and Elpidina, E.N. (2020). New Glutamine-Containing Substrates for the Assay of Cysteine Peptidases From the C1 Papain Family. Front. Mol. Biosci., 7.","DOI":"10.3389\/fmolb.2020.578758"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/0003-2697(82)90113-0","article-title":"Electrophoresis Buffers for Polyacrylamide Gels at Various PH","volume":"126","author":"McLellan","year":"1982","journal-title":"Anal. Biochem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.ab.2013.12.032","article-title":"Selective Chromogenic and Fluorogenic Peptide Substrates for the Assay of Cysteine Peptidases in Complex Mixtures","volume":"449","author":"Semashko","year":"2014","journal-title":"Anal. Biochem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.ab.2018.12.001","article-title":"Direct Detection of Cysteine Peptidases for MALDI-TOF MS Analysis Using Fluorogenic Substrates","volume":"567","author":"Elpidina","year":"2019","journal-title":"Anal. Biochem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1111\/j.1365-2583.2007.00740.x","article-title":"Sequence Analysis and Molecular Characterization of Larval Midgut CDNA Transcripts Encoding Peptidases from the Yellow Mealworm, Tenebrio molitor L","volume":"16","author":"Prabhakar","year":"2007","journal-title":"Insect Mol. Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"3551","DOI":"10.1002\/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2","article-title":"Probability-Based Protein Identification by Searching Sequence Databases Using Mass Spectrometry Data","volume":"20","author":"Perkins","year":"1999","journal-title":"Electrophoresis"},{"key":"ref_47","unstructured":"Stepanov, V.M., Lysogorskaya, E.N., Filippova, I.Y., Oksenoit, E.S., and Lyublinskaya, L.A. (1985). L-Pyroglutamyl-L-Phenylalanyl-L-Alanyne P-Nitroanilide\u2014A Chromogenic Substrate of Thiol Proteinases. (No. 1198082), Russian Inventor\u2019s Certificate, (In Russian)."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/13\/7001\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:38:55Z","timestamp":1760139535000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/13\/7001"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,23]]},"references-count":47,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2022,7]]}},"alternative-id":["ijms23137001"],"URL":"https:\/\/doi.org\/10.3390\/ijms23137001","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,23]]}}}