{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:47:59Z","timestamp":1760150879932,"version":"build-2065373602"},"reference-count":48,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2023,12,29]],"date-time":"2023-12-29T00:00:00Z","timestamp":1703808000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Science and Technology Council, Taiwan, ROC","award":["109-2313-B-005-018-MY3"],"award-info":[{"award-number":["109-2313-B-005-018-MY3"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicates its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q\/S387L exhibits an approximately 34-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. The increased specificity of E380Q\/S387L not only enhances its ability to break down pro-immunogenic peptides but also positions this enzyme variant as a promising candidate for oral therapy for celiac disease.<\/jats:p>","DOI":"10.3390\/ijms25010505","type":"journal-article","created":{"date-parts":[[2023,12,29]],"date-time":"2023-12-29T11:19:21Z","timestamp":1703848761000},"page":"505","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Specificity Enhancement of Glutenase Bga1903 toward Celiac Disease-Eliciting Pro-Immunogenic Peptides via Active-Site Modification"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0887-4047","authenticated-orcid":false,"given":"Yu-You","family":"Liu","sequence":"first","affiliation":[{"name":"Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 40227, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rui-Ling","family":"Ye","sequence":"additional","affiliation":[{"name":"Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 40227, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0179-432X","authenticated-orcid":false,"given":"Menghsiao","family":"Meng","sequence":"additional","affiliation":[{"name":"Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 40227, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,12,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S19","DOI":"10.1053\/j.gastro.2005.02.010","article-title":"Clinical features and diagnosis of celiac disease","volume":"128","author":"Dewar","year":"2005","journal-title":"Gastroenterology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2226","DOI":"10.1172\/JCI27620","article-title":"HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease","volume":"116","author":"Tollefsen","year":"2006","journal-title":"J. Clin. Investig."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1539","DOI":"10.1038\/ajg.2015.296","article-title":"Risk of Celiac Disease in the First- and Second-Degree Relatives of Patients with Celiac Disease: A Systematic Review and Meta-Analysis","volume":"110","author":"Singh","year":"2015","journal-title":"Am. J. Gastroenterol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"8562","DOI":"10.3748\/wjg.v19.i46.8562","article-title":"Endoscopic tools for the diagnosis and evaluation of celiac disease","volume":"19","author":"Ianiro","year":"2013","journal-title":"World J. Gastroenterol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2370","DOI":"10.1373\/clinchem.2004.036111","article-title":"Serologic assay based on gliadin-related nonapeptides as a highly sensitive and specific diagnostic aid in celiac disease","volume":"50","author":"Schwertz","year":"2004","journal-title":"Clin. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/S0733-5210(86)80012-1","article-title":"The classification and nomenclature of wheat gluten proteins: A reassessment","volume":"4","author":"Shewry","year":"1986","journal-title":"J. Cereal Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2275","DOI":"10.1126\/science.1074129","article-title":"Structural basis for gluten intolerance in celiac sprue","volume":"297","author":"Shan","year":"2002","journal-title":"Science"},{"key":"ref_8","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_9","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1038\/nm0698-713","article-title":"Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease","volume":"4","author":"Molberg","year":"1998","journal-title":"Nat. Med."},{"key":"ref_10","first-page":"286","article-title":"IgA anti-gliadin antibodies in coeliac disease","volume":"46","author":"Unsworth","year":"1981","journal-title":"Clin. Exp. Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"204","DOI":"10.1016\/j.patbio.2004.07.022","article-title":"Tissue transglutaminase antibodies in celiac disease, comparison of an enzyme linked immunosorbent assay and a dot blot assay","volume":"53","author":"Mankai","year":"2005","journal-title":"Pathol. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/B978-0-12-394305-7.00001-X","article-title":"Cellular Functions of Tissue Transglutaminase","volume":"294","author":"Nurminskaya","year":"2012","journal-title":"Int. Rev. Cell Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1930","DOI":"10.1136\/gutjnl-2016-311964","article-title":"Intestinal cell damage and systemic immune activation in individuals reporting sensitivity to wheat in the absence of coeliac disease","volume":"65","author":"Uhde","year":"2016","journal-title":"Gut"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1111\/j.1365-277X.2011.01160.x","article-title":"Limited availability and higher cost of gluten-free foods","volume":"24","author":"Singh","year":"2011","journal-title":"J. Hum. Nutr. Diet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"792","DOI":"10.14309\/ajg.0000000000000202","article-title":"Detection of gluten in gluten-free labeled restaurant food: Analysis of crowd-sourced data","volume":"114","author":"Lerner","year":"2019","journal-title":"Am. J. Gastroenterol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"836","DOI":"10.1016\/j.molmed.2019.05.009","article-title":"Therapeutic and Diagnostic Implications of T Cell Scarring in Celiac Disease and Beyond","volume":"25","author":"Christophersen","year":"2019","journal-title":"Trends Mol. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.autrev.2009.05.002","article-title":"New therapeutic strategies for celiac disease","volume":"9","author":"Lerner","year":"2010","journal-title":"Autoimmun. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1016\/B978-0-12-416039-2.00013-6","article-title":"Oral enzyme therapy for celiac sprue","volume":"502","author":"Bethune","year":"2012","journal-title":"Methods Enzymol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.foodres.2016.11.021","article-title":"Novel approaches for enzymatic gluten degradation to create high-quality gluten-free products","volume":"110","author":"Scherf","year":"2018","journal-title":"Food Res. Int."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"174354","DOI":"10.4061\/2010\/174354","article-title":"Enzymatic strategies to detoxify gluten: Implications for celiac disease","volume":"2010","author":"Caputo","year":"2010","journal-title":"Enzym. Res."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Liu, Y.Y., Lee, C.C., Hsu, J.H., Leu, W.M., and Meng, M. (2021). Efficient hydrolysis of gluten-derived celiac disease-triggering immunogenic peptides by a bacterial serine protease from Burkholderia gladioli. Biomolecules, 11.","DOI":"10.3390\/biom11030451"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2258","DOI":"10.1016\/j.ijbiomac.2022.10.016","article-title":"Crystal structure of a Burkholderia peptidase and modification of the substrate-binding site for enhanced hydrolytic activity toward gluten-derived pro-immunogenic peptides","volume":"222","author":"Liu","year":"2022","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_23","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_24","doi-asserted-by":"crossref","first-page":"30980","DOI":"10.1038\/srep30980","article-title":"Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease","volume":"6","author":"Rey","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.plaphy.2022.04.027","article-title":"Neprosin belongs to a new family of glutamic peptidase based on in silico evidence","volume":"183","author":"Ting","year":"2022","journal-title":"Plant Physiol. Biochem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4446","DOI":"10.1038\/s41467-022-32215-1","article-title":"Molecular and in vivo studies of a glutamate-class prolyl-endopeptidase for coeliac disease therapy","volume":"13","author":"Mendes","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"17202","DOI":"10.1021\/acs.jafc.3c04750","article-title":"Hydrolysis of Gluten-Derived Celiac Disease-Triggering Peptides across a Broad pH Range by RmuAP1: A Novel Aspartic Peptidase Isolated from Rhodotorula mucilaginosa","volume":"71","author":"Zhang","year":"2023","journal-title":"J. Agric. Food Chem."},{"key":"ref_28","first-page":"91","article-title":"AN-PEP, Proline-Specific Endopeptidase, Degrades All Known Immunostimulatory Gluten Peptides in Beer Made from Barley Malt","volume":"74","author":"Akeroyd","year":"2018","journal-title":"J. Am. Soc. Brew. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Sestak, K., Thwin, H., Dufour, J., Liu, D.X., Alvarez, X., Laine, D., Clarke, A., Doyle, A., Aye, P.P., and Blanchard, J. (2016). Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques. Nutrients, 8.","DOI":"10.3390\/nu8070401"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1129","DOI":"10.1007\/s00217-015-2617-5","article-title":"Production of gluten-free beer by peptidase treatment","volume":"242","author":"Knorr","year":"2015","journal-title":"Eur. Food Res. Technol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"G571","DOI":"10.1152\/ajpgi.00185.2016","article-title":"Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease","volume":"311","author":"Wei","year":"2016","journal-title":"Am. J. Physiol. Gastrointest. Liver Physiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"8913","DOI":"10.1093\/nar\/13.24.8913","article-title":"Cloning sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis","volume":"13","author":"Jacobs","year":"1985","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"7505","DOI":"10.1038\/s41598-019-43837-9","article-title":"Pharmaceutically modified subtilisins withstand acidic conditions and effectively degrade gluten in vivo","volume":"9","author":"Darwish","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1016\/j.chembiol.2006.04.008","article-title":"Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease","volume":"13","author":"Bethune","year":"2006","journal-title":"Chem. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1178","DOI":"10.1124\/jpet.106.104315","article-title":"Effect of barley endoprotease EP-B2 on gluten digestion in the intact rat","volume":"318","author":"Gass","year":"2006","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_36","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_37","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1007\/s10620-011-1906-5","article-title":"Safety, tolerability, and activity of ALV003: Results from two phase 1 single, escalating-dose clinical trials","volume":"57","author":"Siegel","year":"2012","journal-title":"Dig. Dis. Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1649","DOI":"10.1053\/j.gastro.2014.02.031","article-title":"Glutenase ALV003 attenuates gluten-induced mucosal injury in patients with celiac disease","volume":"146","author":"Kaukinen","year":"2014","journal-title":"Gastroenterology"},{"key":"ref_39","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_40","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1053\/j.gastro.2016.11.004","article-title":"No Difference Between Latiglutenase and Placebo in Reducing Villous Atrophy or Improving Symptoms in Patients with Symptomatic Celiac Disease","volume":"152","author":"Murray","year":"2017","journal-title":"Gastroenterology"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1510","DOI":"10.1053\/j.gastro.2022.07.071","article-title":"Latiglutenase Protects the Mucosa and Attenuates Symptom Severity in Patients with Celiac Disease Exposed to a Gluten Challenge","volume":"163","author":"Murray","year":"2022","journal-title":"Gastroenterology"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"20513","DOI":"10.1021\/ja3094795","article-title":"Computational design of an alpha-gliadin peptidase","volume":"134","author":"Gordon","year":"2012","journal-title":"J. Am. Chem. Soc."},{"key":"ref_43","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_44","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1053\/j.gastro.2021.03.019","article-title":"Gluten Degradation, Pharmacokinetics, Safety, and Tolerability of TAK-062, an Engineered Enzyme to Treat Celiac Disease","volume":"161","author":"Pultz","year":"2021","journal-title":"Gastroenterology"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"W382","DOI":"10.1093\/nar\/gki387","article-title":"The FoldX web server: An online force field","volume":"33","author":"Schymkowitz","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Honorato, R.V., Koukos, P.I., Jim\u00e9nez-Garc\u00eda, B., Tsaregorodtsev, A., Verlato, M., Giachetti, A., Rosato, A., and Bonvin, A.M.J.J. (2021). Structural Biology in the Clouds: The WeNMR-EOSC Ecosystem. Front. Mol. Biosci., 8.","DOI":"10.3389\/fmolb.2021.729513"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1016\/j.jmb.2015.09.014","article-title":"The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes","volume":"428","author":"Rodrigues","year":"2016","journal-title":"J. Mol. Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2778","DOI":"10.1021\/ci200227u","article-title":"LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery","volume":"51","author":"Laskowski","year":"2011","journal-title":"J. Chem. Inf. Model."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/1\/505\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:44:33Z","timestamp":1760132673000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/1\/505"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,12,29]]},"references-count":48,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2024,1]]}},"alternative-id":["ijms25010505"],"URL":"https:\/\/doi.org\/10.3390\/ijms25010505","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2023,12,29]]}}}